|
i

PLACE IN RETURN Box to remove this checkout from your record.

TO AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE

DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

5/08 K:lProj/Acc&Pres/CIRC/DaIeDue.indd

 

 

 

 

REPRODUCTIVE EFFECTS OF GESTATIONAL AND LACTATIONAL
EXPOSURE TO ESTROGENIC ENDOCRINE MODULATORS IN MICE

BY

Jiyou Han

A DISSERTATION

Submitted to
Michigan State University
In partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Animal Science
- Environmental Toxicology

2008

ABSTRACT
REPRODUCTIVE EFFECTS OF GESTATIONAL AND LACTATIONAL
EXPOSURE TO ESTROGENIC ENDOCRINE MODULATORS IN MICE
By

Jiyou Han

Our previous studies demonstrated decreased sperm production and
decreased in vitro sperm fertilizing ability in male offspring (F-1) after in utero and
lactational exposure to 1.0 and 10 pg diethylstilbestrol (DES)/kg maternal body
weight/day (bw/d). In contrast, fertilizing ability of sperm was increased
exposure to 0.1 pg DES/kg maternal bw. Genistein (GEN, an isoflavonoid)
increased after sperm production and fertilizing ability at a maternal dose as low
as 10 mg/kg bw. The aims of the present study were to examine the effects of
maternal exposure to 17a-ethynyl estradiol (EE2, a component of oral
contraceptives) on sperm production, sperm quality, and sperm fertilizing ability in
male Offspring (F-1) and effects on superovulation and egg fertilizing ability in
female offspring (F-1) maternally exposed to EE2, DES or GEN. Pregnant
C57BL/6 mice (F-O), bred with DEA/2 males, were gavaged daily with DES (0.1,
1.0, or 10 ug/kg maternal bw), EE2 (0.1, 1.0, or 10 ug/kg maternal bw) or GEN
(0.1, 0.5, 2.5, or 10 mg/kg maternal bw) from gestational day 12 to postnatal day
(PND) 20. Sperm production and quality were examined by using computer-
assisted sperm analysis (CASA). Fertilizing ability of sperm and eggs was
assessed using an in vitro fertilization (IVF) assay. Our results demonstrated

that in utero and lactational exposure to a high dose of EE2 (10 ug/kg maternal

bw) could decrease sperm production without Changing fertilizing ability and
sperm motion parameters in 45- to 48-week-old male offspring. On the other
hand, a significant increase in the number of ovulated eggs was observed as a
result of in utero and lactational exposure to 0.1 pg DES/kg maternal bw without
altering fertilizing ability of eggs. Fertilizing ability Of eggs was significantly
decreased in the 10 pg EE2/kg maternal BW animals at 3 to 6 weeks of age,
whereas GEN had no effect on the number of ovulated eggs and fertilizing ability

of eggs.

ACKNOWLEDGEMENTS

I grateful acknowledge Professor Karen Chou for encouraging and
providing me to learn science. She has contributed her time and hands in
helping me perform the research described in this dissertation. I would like to
express the deepest appreciation to my committee members, especially
Professor Steve Bursian and Professor Dale Rozeboom for their valuable input.
In addition, I thank Dr. Peter Saama and Professor Robert Tempelman, who
helped me analyze data. I also thank Raquel and Barb for their persistent help.

Many thanks go out to my family; Kun-Mo Han, Young-Hee Kim, Yongga
Chae, Hyongwoo Ham, Kyuhi Han, Anrakyugo, Kyong-A Han, Jinho Kim, Dong
Hun Han, and Sunjoo Lee. Especially, I thank my husband, Hyeongpil Ham, for
giving me endless support and love. I would like to thank my 11-month-old son,
Juwon Ham, for giving me strength to complete my doctoral degree and for

inspiring my sprits. Finally, I thank God who is always guiding me and loving me.

TABLE OF CONTENTS

LIST OF TABLES ................................................................................. viii
LIST OF FIGURES ................................................................................. x
ABBREVIATIONS ................................................................................. xi
CHAPTER 1
1.1 Rationale ................................................................................ 1
1.2 Hypothesis .............................................................................. 3
1.3 Objectives ............................................................................... 3
CHAPTER 2- Literature review I
2.1 Estrogen synthesis ................................................................... 4
2.1.1 Estrogen synthesis in females ............................................. 8
2.1.2 Estrogen synthesis in males .............................................. 12
2.4 Estrogen receptors .................................................................. 13
2.5 Modes of action of estrogen ...................................................... 16
2.5.1 Ligand-dependent mode .................................................... 16
2.5.2 Ligand-independent mode ................................................. 17
2.5.3 Estrogen response element-independent mode......... .........18
2.5.4 Nongenomic mode ........................................................... 20
2.6 Effects of estrogen on ovarian development, oogenesis, and
Folliculogenesis .................................................................... 21
2.6.1 Estrogen in ovarian development ........................................ 21
2.6.2 Estrogen in oogenesis and folliculogenesis ........................... 22
2.7 Effects of estrogen on testicular development and spennatogenesis.26
2.7.1 Estrogen in testicular development ..................................... 26
2.7.2 Estrogen in sperrnatogenesis ............................................. 31
2.8 Effects of estrogen on fertilization and implantation ........................ 38
2.9 Effects of estrogen on sex ratio of offspring .................................. 40
2.10 Effects of estrogen on anogenital distance .................................. 44
2.11 Effects of estrogen on puberty ................................................. 47
2.12 Effects of estrogen on body weight ........................................... 48
2.13 Bibliography ......................................................................... 52
CHAPTER 3- Literature review II
3.1 Definition of estrogenic endocrine modulators ............................... 77
3.2 Sources and exposure to EEMs ................................................. 78
3.3 Modes of action of EEMs ......................................................... 81
3.3.1 Altered sex steroid hormone synthesis ................................. 81

3.3.2 Altered sex steroid hormone storage and/or release...............83
3.3.3 Altered sex steroid hormone transport and Clearance...... ........83
3.3.4 Altered sex steroid hormone receptor recognition/binding........86
3.3.5 Altered sex steroid hormone postreceptor activation ............... 87

   

3.3.6 Altered gene expression without changing DNA sequence ....... 88

3.4 Phytoestrogen and mycotoxin ................................................... 91
3.4.1 Genistein ....................................................................... 91
3.4.2 Zearalenone .................................................................. 93

3.5 Pharmaceuticals .................................................................... 96

3.5.1 Diethylstilbestrol ............................................................. 96
3.5.2 Tamoxifen ..................................................................... 98
3.5.3 17o-ethynylestradiol ...................................................... 1 00

3.6 Pesticides ........................................................................... 103
3.6.1 Methoxychlor .............................................................. 103
3.6.2 Atrazine ..................................................................... 104

3.7 Industrial Chemicals ............................................................... 106
3.7.1 Bisphenol A ................................................................. 106
3.7.2 Phthalates .................................................................. 109

3.8 Environmental pollutants ........................................................ 112
3.8.1 Polychlorinated biphenyls .............................................. 112
3.8.2 Benzo[a]pyrene ........................................................... 114

3.9 Bibliography ......................................................................... 1 16

CHAPTER 4

Effects of In utero and Lactational Exposure to Diethylstilbestrol,

17a-Ethynyl estradiol, and Genistein on Ovulation
and In Vitro Fertilizing Ability of Eggs in Female Mouse Offspring

4.1 Abstract ............................................................................... 146

4.2 Introduction .......................................................................... 147

4.3 Materials and methods ........................................................... 150

4.4 Results ............................................................................... 155

4.5 Discussion .......................................................................... 165

4.6 Bibliography ......................................................................... 171
CHAPTER 5

Effects of In utero and Lactational Exposure to 17a—Ethynyl Estradiol
on Sperm Quality and In Vitro Fertilizing Ability in Male Mouse Offspring

5.1 Abstract ............................................................................... 178

5.2 Introduction .......................................................................... 1 79

5.3 Materials and methods ........................................................... 182

5.4 Results ............................................................................... 188

5.5 Discussion ........................................................................... 196

5.6 Bibliography ......................................................................... 203
CHAPTER 6

Effects of Gestational and Lactational Exposure of Female Mice to 17a-
Ethynyl Estradiol on Reproduction

6.1 Abstract ............................................................................... 212
6.2 Introduction .......................................................................... 213
6.3 Materials and methods ........................................................... 214

vi

6.4 Results ............................................................................... 217

6.5 Discussion .......................................................................... 224

6.6 Bibliography ........................................................................ 228
CHAPTER 7

7.1 Conclusion ............................................................................ 232

vii

LIST OF TABLES
CHAPTER 2
Table 2-1. Enzymes involved in steroidogenesis .......................................... 6

Table 2-2. Production rates and serum concentrations of estradiol during the
menstrual cycle in normal women ............................................ 11

Table 2-3. ERs and aromatase distribution in the rodent fetal testis ................ 29

Table 2-4. ERs and aromatase distribution in postnatal immature rodent
testis .................................................................................. 29

Table 2-5. ERs and aromatase distribution in the adult rodent testis ............... 29

Table 2-6. Reproductive phenotypes of ERK0 and ArKO male mouse
models ................................................................................ 32

CHAPTER 4

Table 4-1. Body weight of female offspring Of dams treated with DES, EE2, and
GEN from gestational day 12 through lactation ............................ 158

Table 4-2. Anogenital distance of female offspring Of dams treated with DES,
EE2, and GEN from gestational day 12 through lactation ............... 160

Table 4-3. Number of ovulated eggs of female offspring of dams treated with
DES, EE2, and GEN from gestational day 12 through lactation ...... 161

Table 4-4. In Vitro fertilizing ability of eggs from female offspring of dams treated
with DES, EE2, and GEN from gestational day 12 through
lactation .............................................................................. 162

Table 4-4a. Relative percentage of in Vitro fertilizing ability of eggs from female
offspring of dams treated with DES, EE2, and GEN from gestational
day 12 through lactation .......................................................... 163

Table 4-5. Percent of degenerated eggs from female offspring of dams treated

with DES, EE2, and GEN from gestational day 12 through
lactation .............................................................................. 164

viii

CHAPTER 5

Table 5-1. Organ weights of male offspring of dams treated with EE2 from
gestational day 12 through lactation ...................................... 190

Table 5-2. Anogenital distance of male offspring of dams treated with EE2 from
gestational day 12 through lactation ......................................... 192

Table 5-3. Epididymal sperm quality of male Offspring of dams treated with EE2
from gestational day 12 through lactation ................................... 193

Table 5-4. In Vitro fertilizing ability of epididymal sperm from male offspring of
dams treated with EE2 from gestational day 12 through lactation...194

Table 5-4a. Relative percentage of in Vitro fertilizing ability of epididymal sperm
from male offspring of dams treated with EE2 from gestational day 12
through lactation .............................................................................. 195

CHAPTER 6

Table 6-1. Reproductive performance of F-0 females gavaged with 17a-ethynyl
estradiol from gestational day 12 to lactation (postnatal day 20)....220

Table 6-2. Reproductive performance of F-O females gavaged with
diethylstilbestrol from gestational day 12 to lactation (postnatal day
20) .................................................................................... 222

Table 6-3. Reproductive performance of F-0 females gavaged with genistein
from gestational day 12 to lactation (postnatal day 20) ................. 223

LIST OF FIGURES

CHAPTER 2
Figure 2-1. Schematic presentation of steroidogenesis .................................. 5
Figure 2-2. Ovarian steroidogenesis ......................................................... 10
Figure 2-3. Schematic drawing of the mean serum levels of E1 and E2 in
relation to progesterone, luteinizing hormone, and follicular
stimulating hormone during pre- and postmenopause .................. 11
Figure 2-4. Schematic representative of the structural and functional domains of
ERor and ERB ...................................................................... 15
Figure 2-5. Schematic representation of modes Of action of estrogen .............. 19
Figure 2-6. Schematic representation of hormonal regulation of oogenesis in the
ovary .................................................................................. 25
Figure 2-7. Schematic representation of a cross-sectional view of the testis and
the seminiferous tubules ......................................................... 27
Figure 2-8. Schematic representation of hormonal regulation of
sperrnatogenesis ............................................................................... 35
CHAPTER 4
Figure 4-1. Schedule of maternal treatments and parameters measured in their
female offspring (F-1) ........................................................... 152
CHAPTER 5
Figure 5-1. Schedule of maternal treatments and parameters measured in their
male offspring (F-1) .............................................................. 184
CHAPTER 6
Figure 6-1. Schedule of treatments and parameters measured in females ...... 216

ABBREVIATIONS
ADI... ..Acceptable daily intake
AF-1 .....Activation function 1
AF-2. . . ..Activation function 2
AhR. . . ..Ary hydrocarbon receptor
AGD. . . .. Anogenital distance
ALH.....Amplitude of lateral head displacement
ANOVA ..... Analysis Of variance
AP-1 .. . ..Activator protein-1
AR.....Androgen receptor
ArKO. . . ..Aromatase knockout mouse
ATP... ..Adenosine tri-phosphate
B[a]P ..... Benzo[a]pyrene
BBP. . . ..Butyl benzyl phthalate
BG-1.....A human ovarian adenocarcinoma cell line
BSA... ..Bovine serum albumin
BPA ..... Bisphenol A
BW ..... Body weight
cAMP. . . ..Cyclic adenosine monophosphate
CASA.....Computer assisted sperm analysis
CCA. . . ..Cervical clear cell adenocarcinoma
CCI4. . . ..Carbon tetrachloride

C/EBP. . . ..CCAAT/enhancer binding protein

xi

CREB.....cAMP response element binding protein
CYP 19 ..... Cytochrome P450 19 (aromatase)
Cyp11a.....P450 side chain cleavage enzyme
DBCP. . . ..Dibromochloropropane

DBD. . . ..DNA binding domain

DBP ..... Dibutyl phthalate

DDE. . . ..Dichlorodiphenyl dichloroethene

DDT. . . ..1 , 1, 1-trichloro—2, 2,-bis (p-Chlorophenyl)ethane
DE2. . . ..(+)-Diol—epoxide-2

DES. . . ..Diethylstilbestrol

DEHP ..... Di-(2-ethylhexyl)-phthalate

DHD. . . ..(-)-Dihydrodiol

DHEA. . . ..And rostenedione

DH EAS. . . ..Dehyd roepiand rosterone sulfate

DHT. . . ..Dihyd rotestosterone

DINP. . . ..Diisononyl phthalate

DNA. . . ..Deoxyribonucleic acid

E1 .....Estrone

E2... ..17B-estradiol

E3... ..Estradiol

ECSCF ..... The European Commission’s Scientific Committee on Food
EE2. . . ..17or-ethynyl estradiol

EEMs. . . ..Estrogenic endocrine modulators

xii

EGF. . . ..Epiderrnal growth factor

ER... ..Estrogenic receptor

ERor. . . ..Estrogen receptor alpha

ERB. .. ..Estrogen receptor beta

ERE. . . ..Estrogen response element

ERKO. . . ..Estrogen receptor knockout mouse
orERKO.....Estrogen receptor alpha knockout mouse
BERKO.....Estrogen receptor beta knockout mouse
EU.....The European Union

FDA.....US Food and Drug Administration
ERR1 ..... Estrogen receptor-related receptor 1
FISH... ..Fluorescence in situ hybridization
FSH. . . ..Follicle stimulating hormone
GD.....Gestationa| day

GEN.....Genistein

GLUTs. . . ..Glucose transporter proteins
GnRH. . . ..Gonadotropin releasing hormone
HCB ..... Hexachlorobenzene

hCG ..... Human Chorionic gonadotropin

hERa ..... Human estrogen receptor a

HDL ..... High density Iipoprotein

Hoxa.....10 Homeobox A 10

HPTE. . . ..2,2-Bis(p—hydroxyphenyl)-1 ,1 ,1 -trichloroethane .

xiii

HSD. . . ..Hydroxy-steroid dehyd rogenase

3B-HSD ..... 3B-hydorxysteroid dehydrogenase
HSP70.....Heat shock protein 70 kDa
HSP90.....Heat shock protein 90 kDa

17B-HSR.. . ..17B-hydroxysteroid red uctase

lCl 164, 384 ..... Estra-1,3,5 (10)—triene-7-undecanamide
IGF-1 .....lnsulin-like growth factor-1

IL-1 .....lnterieukin-1

IVF. . . ..In Vitro fertilization

LBD ..... Ligand binding domain

LDso ..... Lethal dose to 50% of the experimental animals
LDL ..... Low density Iipoprotein

LH. . . ..Luteinizing hormone

LOAEL. . . ..The lowest Observed adverse effect level
LS-mean. . . ..Least squares mean

MAPK.....Mitogen activated protein kinase

mBP. . . ..Monobutyl phthalate

mBzP. . . ..Monobenzyl phthalate

MEHP ..... Mono-(2-ethylhexyl)-phthalate

mEP. . . ..Monoethyl phthalate

mEHP. . . ..Monoethylhexyl phthalate

mRNA ..... Messenger ribonucleic acid

MTD. . . ..Maximum tolerated dose

xiv

MXC. . . ..Methoxychlor

NADP.....Nicotinamide adenine dinucleotides phosphate

NF-xB.....Nuclear factor kappa B

NHANES.....The National Health and Nutrition Examination Survey

NGF.....Naturally occurring growth factor

NOAEL.....No observable adverse effect level

NOEL... ..No Observed effect levels

NR. . . ..Nuclear receptor

o,p-DDE. . . ..Ortho, para-dichlorodiphenyl dichloroethene

P ..... progesterone

p300/CBP.....A transcriptional CO-activating protein involved in G protein
signaling

P450scc ..... P450 side Chain cleavage enzyme

PCB... ..Polychlorinated blphenyl

PMSG ..... Pregnant mare’s serum gonadotropin

PND. . . ..Postnatal day

PPAR. . . ..Peroxisome proliferator-activated receptor

ppm.....Part per million

PR... ..Progesterone receptor

PVC... ..Polyvinyl chloride

RNA ..... Ribonucleic acid

SD... ..Standard deviation

SE.....Standard error

XV

SF-1 .....Steroidogenic factor 1

SHBG. . . ..Sex-steroid hormone-binding globulin
SMRT.....Silencing mediator for retinoids and thyroid hormone receptors
SOX—9.....SRY-box containing gene 9

SR-B1 .....Scavenger receptor B1

SRC-1.....Steroid receptor coactivator 1

StAR.....SterOidogenic acute regulatory protein

TCDD. . . ..2,3,7,8-Tetrachlordibenzo-p-dioxin

TDl ..... Tolerable daily intake

TEBG. . . ..Testosterone-estrogen-binding globulin

TGF. . . ..Transfonning growth factor

US. . . ..The United States

US EPA.....US Environmental Protection Agency

UK... ..The United Kingdom

Wnt-4.....Wingless-type MMTV integration site family, member 4
Wnt-7a ..... Wingless-type MMTV integration site family, member 7a

ZEA. . . ..Zearalenone

xvi

CHAPTER 1
RATIONALE, HYPOTHESIS AND OBJECTIVES
1.1 RATIONALE
Reproductive health is the most important fundamental factor for
. preservation of the species. The reproductive system is delicately maintained by
endogenous hormones such as the sex steroid hormones, estrogen and
androgen. Estrogen and androgen are mainly produced by female and male
gonads. They stimulate the development of female and male sex Characteristics
during organ developmental and puberty and the production of female and male
gametes by sexually matured gonads. Disruption of homeostasis by an
exogenous substance, which can act as antagonist or agonist of endogenous
hormones during the critical stage of development, may result in potential
adverse effects on reproductive health.
. Estrogen exerts its biological activities via binding to estrogen receptors
(ER) in estrogen target tissues. Laboratory in Vitro assays and in vivo models
demonstrated that selected xenobiotics and natural substances can bind to ER
and exert estrogenic responses. Thus, chemicals are called estrogenic
endocrine modulators (EEMs). There has been a great deal of attention and
debate regarding the hypothesis that exposure, particularly in utero exposure, to
EEMs might be capable of causing a spectrum of adverse reproductive and
developmental effects in human and animals. EEMs can alter development of the
endocrine system and of the organs that respond to endocrine signals in

organisms indirectly exposed during prenatal and/or early postnatal life. The

effects of exposure during development may be permanent and irreversible.
Although the exact modes of action of EEMs remain unclear, evidence
supporting the hypothesis that EEMs impact the endocrine system include: 1)
reproductive tract abnormalities and Clear cell adenocarcinoma (CCA) of the
vagina and cervix of human females exposed to the potent estrogenic drug
diethylstilbestrol in utero, 2) decreased sperm production and altered sperm
quality in laboratory animals exposed to EEMs, and 3) decreased fertility and
reproductive tract abnormalities in wildlife populations exposed to EEMs at high
concentration. Therefore, to assess the risk of exposure to EEMs on reproductive
and developmental health in humans, long-term health consequences of
exposure to EEMs during developmental periods need to be investigated.
Additionally, it is important to compare the potential effects and modes of
action between synthetic estrogenic compounds (e.g. diethylstilbestrol or DES
and 17a-ethynyl estradiol or EE2) and naturally occurring phytoestrogens (e.g.
genistein or GEN) for better assessing health risk. Therefore, in this study, EE2
(a major estrogenic component of oral contraceptives) and GEN (a
phytoestrogen formed in soybeans) were selected to examine the potential
adverse effects of EEMs on reproductive health. DES was selected as
estrogenic positive control since its estrogenic effects on reproductive health
have been well documented. To examine sperm production and quality,
computer assisted semen analysis (CASA) was used. Egg and sperm fertilizing

abilities were investigated by using an in-vitro fertilization assay.

1.2 HYPOTHESIS

Gestational and lactational exposure Of adult female mice to estrogenic
endocrine disruptors cause long-term alterations in ovulation and egg fertilizing
ability in female offspring and alterations in testicular development and sperm

quality in male offspring.

1.3 OBJECTIVES
. Determine the effects of weak (GEN) and potent (DES and EE2) EEMs on
murine ovulation and egg fertilizing ability following gestational and
lactational exposure.
0 Determine the effects of weak (GEN) and potent (DES and EE2) EEMs on
murine testicular development and sperm quality following gestational and

lactational exposure.

CHAPTER 2
LITERATURE REVIEW I.
2.1 ESTROGEN SYNTHESIS

There are three endogenous estrogens, estrone (E1), 178-estradiol (E2),
and estriol (E3). Estrogens are mainly synthesized in the gonad and also
produced in many other tissues such as the adrenal cortex, adipose tissue, brain,
and skin (1). Figure 2-1 illustrates the general scheme for synthesis of estrogens.
The synthesis of estrogens requires Cholesterol and they can be synthesized de
novo from intra—cellular lipid stores acquired from serum lipoproteins through the
scavenger receptor B1 (SR-B1). Steroidogenesis is the passage of Cholesterol
from the outer to the inner mitochondrial membrane, which is facilitated by
steroidogenic acute regulatory protein (StAR). Within the inner mitochondrial
membrane, cholesterol is cleaved into pregnenolone by the action of the side-
Chain-Cleavage enzyme (P450scc). Pregnenolone enters the smooth
endoplasmic reticulum (ER) where the remaining steroidogenic enzymes
responsible for testosterone synthesis are located (2). Aromatase (CYP19)
converts androstenedione or testosterone to E1 or E2.

Steroid hormone biosynthesis is catalyzed by two major types of enzymes:
cytochrome P450 (CYPs) and hydroxysteroid dehydrogenase (e.g. 3B—HSD and
17B—HSD) (3). Table 2-1 presents the major catalytic enzymes that are involved
in steroidogenesis. Thus, biosynthesis of specific steroids is depended on
quantitative expression of these two steroidogenic enzymes and their activity in

specific cells comprising to the specific endocrine tissue. Furthermore, these two

.Nm. .o E 9 95.90909 .o 05.09.906.20
9.02.00 8E>Ov 0090Eo.< 09000. 0.0 0.09.930 9.990909 .2 0.9.9.0000. 00E>Nc0 0.como.0.o.90 9.....0E0. 9:
0.9.3 Em. E23030. 0.Emm_aouc0 £095 9.. 0.9...0 9.0.8059... .Aoomomvav 9.5.9.0 wag—020-509.0003 9.. Co c0300 9: >0
05.9.0390 9:. 0050.0 0. 6.98.9.0 0:05:05. .m_.ucoc09.E .09.. 9: :.£.>> .Efiwv E90... .5530. 9:00 0.580.990
.3 00.9...09 .9.m.nE0E .m..9.9.09.E .09.. 9.. 9 .9:o 9.. E0... 8.93.9.0 .0 03009. 9.. 0. 0.39.09.06.90 .0 090 95......
.90. 9.... .Cmiwv Fm .9800. 09.0500 9: 59.0.... 9:90.09... E200 E9. 09.300... 00.90 0.0.. 8.2.0009... E2. o>oc 0.0
03.00550 9. :00 0.... 9.0 6.900.050 09.3.00. 00.990 .0 0.00550 9.... 0.8582990 .0 9.0.9509... 0..0E0...0m TN 0.39“.

 

 

 

        

 

 

             

 

 

 

 

 

 

 

 

     
  

o fl 0 u 0 fl 0 fl o a“ nu fl 0 fl 0 fl 0 D D U n "v u U a U o 0 o u 0 fl 0 fl 0 “v 0 fl 9 fl 0 “v o n 0 fl 0 D a ”U
a 0019 r a
o .o.00..wm 0c0..mm. _ m
m 00m: ..
a a
m 8 Hi0 ..
a Owflhw a
a a
m _ 0:90.08005535 m.
a g a
m 0:0.0.mo.0c0.000.0>c00 N wax/0 oémm
o o ...
fl . 0:0.OE0E w on
H mm LHOOEmU 0 fl 0 fl 0 fl 0 U a “- 0 fl 0 fl o "v 0 “v 0 fl 0 a” 0 U 0 fl 0 fl 0 fl 0 "v 0 flaw 0 fl 0 U . AU 0 U 0m
0 .w G
o o
n m
o 0:0.OE0E (00.2006. Edam IIIIiIIlIlv 6.0.00.0ch m
0on Beccozooeu. fr 923 .
6.900.050

 

   

L

 

0:0.OE0E =00

 

Table 2-1. Enzymes involved in steroidogenesis
Enzyme Gene Reaction

P450 type

 

Cholesterol side chain cleavage

P450800 CYP1 1A (3 succesive monooxygenations)
C-11 hydroxylation
P450c11 CYP1 1 B1 018 hydroxylation
C-18 hydroxylation
P450018 CYP11BZ C-18 oxidation
1 7a-hyd roxylation
P450017 CYP1 7 C17-C20 bond Cleavage
P450c21 CYP21 C-21 hydroxylation
Steroid ring-A aromatization
P450arom CYP19 (3 succesive monooxygenations)
Dehydrogenase type
_ 3B-hydroxy-steroid dehydrogenation
3B HSD H3038 5-ene-4—ene isomerization
17B-HSD HSD17B 17B-hydroxy-steroid dehydrogenation

(1 7-reductase) 1 7-keto-steroid reduction

Catalyzes testosterone to

sa-reductase SRD5A2 dihydrotestosterone

 

enzymes are expressed not only in reproductive tissues (e.g. gonads and
reproductive tract) but also in many other tissues (e.g., adipose tissue, liver, brain
etc) that have the ability to convert Circulating precursors (e.g. androstenedione)
into active hormones (e.g. testosterone or E2) (4-6). Variations in a certain
enzyme activities are generally dependent on gene expression of an enzyme
with three Characteristics (1) basal expression of the enzyme in the cell; (2)

hormonal signal regulated expression; (3) tissue- and cell-specific expression.

Cytochrome P450 constitutes a large superfamily of membrane bound
oxygenases. P450 families 1, 2, 3 and 4 have function in detoxification of
xenobiotic Chemicals by modifying substrates to become more polar forms prior
to excretion and the metabolism of natural endogenous steroids. They are not
substrate specific. Cytochrome P450 families 11, 17, 19, 21 and 27 are involved
in the synthesis of steroid hormones and they are substrate specific. Among
these P450 families, P450arom coded in Cyp19 gene irreversibly converts
testosterone or androstenedione into E2. P450arom is a microsomal enzymatic
complex composed of two proteins: a ubiquitous NADPH-cytochrome p450
reductase and a cytochrome P450 aromatase, which contains the heme and the
steroid-binding site.

Animal and epidemiological studies indicate that estrogens play important
roles in development and function of several target tissues involved in
cardiovascular performance, bone maintenance, homeostasis, and behavior by
affecting cell proliferation and cell differentiation (7-11). Specifically, numerous
studies and reviews have evaluated the effect Of estrogens on proper
development and function of human and animal reproductive tracts (12-15).

This chapter will review the general biological role of estrogen in
reproduction. More specifically, estrogen synthesis in females and males,
estrogen receptors, and modes of activation of estrogen will be discussed. Then,
the effects of estrogen on normal ovarian and testicular development and
function, spermatogenesis, oogenesis, fertilization, implantation, sex ratio,

anogenital distance (AGD), puberty, and body weight will be reviewed.

2.1.1 ESTROGEN SYNTHESIS IN FEMALES

In females, estrogens are mainly produced in the ovary and thus have
been thought of as female sex steroid hormones. Around the ninth week of
gestation in the human, estrogen synthesis occurs in the primitive ovary of the
female fetus (16). The ovarian steroidogenesis pathway is presented in Figure 2-
2. Estradiol is mainly synthesized by ovarian granulosa cells Of the growing
follicle (17). In the theca cells of the preovulatory follicle Of the ovaries,
luteinizing hormone (LH) stimulates the theca cell via the adenylyl cyclase
pathway, and low density lipoprotein (LDL) and high density lipoprotein (HDL)
across the cell membrane of theca cells due to an increase in the expression of
LDL and HDL receptors (SR-B1) on the cell membrane. With facilitation of StAR
protein, intracellular cholesterol passes from the outer to the inner mitochondrial
membrane. Cytochrome P450scc enzyme cleaves Cholesterol, which is
transported within the inner mitochondrial membrane, into pregnenolone. Thus,
the stimulated theca cell increases its synthesis of androstenedione. The
androstenedione synthesized in the theca cells freely diffuses to the granulosa
cells, whose aromatase activity has been stimulated by follicular stimulating
hormone (FSH). FSH, also acting via the adenylyl cyclase pathway, stimulates
the granulosa cell to prOduce aromatase. Aromatase converts androstenedione
to E1 and then 17B-hydroxy steroid dehydrogenase (17B-HSD) converts E1 to E2
(18, 19).

Production rates and serum concentrations of E2 vary dowsing the

different stages of the menstrual cycle. During the last five days before ovulation

in humans, E2 concentration rapidly increases from 30 to 40 up to 300 to 400
pg/ml and then decreases to approximately 100 pg/ml by the sixth day after
ovulation (20). The E2 concentration increases to 200 pg/ml during the luteal
phase, corresponding to the increase in progesterone, but it is not as high as
during the follicular phase (21). During the menstrual cycle, E2 production varies
cyclically, with the highest production rates and serum concentrations in the
preovulatory phase (Table 2-2) (22). In addition to ovarian production of E1, E2
is converted to E1 by 17B-HSD in the liver and E1 sulfate by aromatase in the
adrenal gland. During early pregnancy, maternal estrogen is synthesized in the
maternal ovaries, but by seven weeks of gestation, the placenta produces
estrogen (23, 24). With progression of pregnancy, maternal serum
concentrations of E1 and E2 increase from 50 to 100 pg/ml up to 30,000 pg/ml
and serum concentrations of E3 increase from 1.3 to 3.2 ng/ml at 20 weeks of
gestation to 6.0 to 19.5 ng/ml at 42 weeks of gestation (20).

In perimenopausal or menopausal women (the mean age of menopause is
51 years in Europe), the serum concentrations of E2 are frequently below 20
pg/ml since depletion of ovarian follicles leads to a steady decline in ovarian E2
production (25) (Figure 2-3). Because the negative feedback effects of E2 on
pituitary gonadotropin secretion are lost, semm FSH and LH concentrations are
significantly increased. Peripheral aromatization increases 3- to 4-fold and
becomes the main source of estrogen in menopausal women (26). In several

studies, aromatase activity has also been detected in adipose-containing tissues

..omm. ”00.30w 0000.50 60.00.93. ...... 0.0.0.000: 5.0000 .50. ...n... 00000690000 0.0.0.0085? .Qw... 009050 60..0.3E..0
0.0...0. .Iwn. 900000000000. 00.000000 2.9.0 .0526 90000000... 00.000000 .n....< 000.05 330000 .O< .Nm. 0. .m 000.000
690.005 00000690300 0.0.0.0 90.0.3-0: 000. 000 :m. o. 000.005.00.000 0...0>000 000.9005 00 .r .000.0E0.0 00300.0

9:00 000.3006 9.. 090.3800 $030.00 000.0:0 ......0000 00. 0.> 60:00 00.0 .19. .Iwu. >0 00.0.3E..0 0000 000 >..>..00 000.0E0.0
00003 .250 000.3006 00. 0. 003.00 000.. 0. 0:00 009.. 00. 0. 000000.00 000.005.00.000 9.. 000300 ..00 0000. 00. 0. 00900.90
.0 0.00.. 002000909000 .0 0.000.000 0.. 00000.00. ..00 0000. 00. 090.3600 030 .. 0005000690 0.0. 00900.06 .0..000000..E
.000. 00. 0.0....) 00.50009. 0. 00.0.5 5.900.000 00>00.0 000.000 000...va 00 0. 00900.08 .0..000000..0. .000. 00. 0. .930

00. E0.. 0630.0. 000000 5.900.000 .0.3..000..0. 0.0.0.0 02$ .0 00..0....00. 0.05 .0090E0E ..00 00. 00 Swim. 0.0.0000. ..o...
000 ..n... .0 5.00900 00000.00. 0. 030 0:00 0000. .0 00900.00 ..00 00. 0.0. 000.0 00... 000 ..n... 000 $030.00 000.05 ...:0000
00. 0.> ..00 0000. 00. 00.0.3E..0 I.. 00005 00. .0 0.2.5. 00.0.3590 00. .0 0:00 0000. 00. 0. .0.0000600.0.90 00..0>O .N-N 0.36.“.

     

 

 

         

 

 

 

 

 

 

 

Imu-
. =00 .000.-3:000 i, jil :00 0005.0 i
_0_Uw._u0m_ iilil Il\ 00.0.0u00u00...
M0 \\\.Q /
DWI: 000.0050?! fi\\ .J@.
0000:: P \ /«\\.
\i/ 0:0.u0w \\ /
U< . 0C0..0u00u00.r
(Hibii/ / N \\ 0C0..00C0u00..uc<
A.\ 005“ [V 009.000.0010. CWT—I \ \\ 00
//Al\i 8020 F \\ a
““5303... u \ . \ 0:00.0«000000.
/ l\\.. 0:0.UOC/0u000UC< kl IOIGSQENI r
,i .
.i A/ QF<iU 0C0.0u00®0.0 0C000000m00n.
.. / a .\I
a 0.51
7 GEO—0:00.000.”—
14. V1 0CO.0C0CO0.Q 0k .
\III/ II /
. M a , 0<
H n n:>_<u /[..
Dr fi/ill\
.000u00_00.0 a
C .000u00_0£0
lemw

 

10

Table 2-2. Production rates and serum concentrations of estradiol during the
menstrual cycle in normal women.

Estradiol (E2)

 

Phase Serum concentration Daily production (pg)
(pg/ml)
Follicular 30-200 60-1 50
Preovulatory 250-500 200-400
Luteal 1 00-200 1 50-300
Premenstrual 30—50 50-70
Postmenstrual <20 5-25

 

Source: (20, 22, 27)
(Le. breast, muscle, brain, and skin) as well as in osteoblasts and osteoclasts in

bone (28-36).

 

160

140

120 \
\ \
100 ‘

 

 

 

 

 

 

 

 

 

 

Serum estrogen concentration (pg/ml)

 

 

 

 

80
60
40
20 /‘--.---—----
0 l I L I
44 48 52 56 60
Age (years)

 

 

F

FSH .:.;.;.;.;.;,-__;,;.;.;.;.;.;.;.;.;.;LH - - P _ (E1 —E2

Figure 2-3. Schematic drawing of the mean serum levels of E1 and E2 in
relation to progesterone (P), LH, and FSH during pre- and postmenopause
[modified according to (20)].

11

2.1.2 ESTROGEN SYNTHESIS IN MALES

Estrogens have been considered the predominant female sex steroid
hormones but many studies have demonstrated that estrogens are produced in a
variety Of tissues in mammalian males such as the brain, liver, adrenal glands,
testes, mammary glands, and adipose tissue (6, 26, 37-39). Among these
various male tissues, the testis is the important reproductive organ in estrogen
synthesis. The testis is composed of the seminiferous tubules and the interstitial
space between the tubules. In these two compartments, there are three major
cell types; the germinal cells, Leydig cells that synthesize testosterone and are
present only in the interstitial space and Sertoli cells that nurture germ cells
within the seminiferous tubules. Leydig cells have been considered the main
source of estrogen. However, both Leydig cells and Sertoli cells produce
estrogen in Vitro (40-42). Aromatase activity has been detected in Leydig cells,
Sertoli cells, round and elongated spennatids, spermatozoa, and the epididymis
throughout spen'natogenesis (14, 43-47). The main source of estrogen in
rodents is the Sertoli cells during fetal life (as eariy as 17 days of gestation),
whereas it is the Leydig cells during adulthood (48, 49).

Serum concentrations of estrogens vary in different species. The testes
account for approximately 15% of circulating estrogens in the man (50), whereas
the adult rat testis synthesizes approximately 10 to 25% of Circulating estrogens
(51, 52). Estrogen concentration in the rat rete testis fluid was nearly 250 pg/ml
(53), whereas the serum concentrations of estrogens are about 40 pg/ml in adult

male rats (54), 20 to 40 pg/ml in human males (55), and 2 to 180 pg/ml in other

12

species (56, 57). Overall, these concentrations of estrogens suggest that
estrogen production in man could have important biological roles in reproductive

heaflh.

2.4. ESTROGEN RECEPTORS

The biological effects of estrogens occur as a result Of binding to the
estrogen receptor (ER), which is a steroid nuclear-receptor superfamily that
functions as a ligand-inducible DNA-binding transcription factor. There are two
isofonns of the ER, termed ERa, which is mapped to the long arm of
Chromosome 6 and ERB, which is mapped to chromosome 14 (58-60). A
schematic representative Of the structural and functional domains of ERot and
ERB are shown in Figure 2-4. All nuclear receptors share a similar molecular
structure. There are six modular domains of the ER designated A to F from the
amino terminal start site to the carboxy terminal end Of the protein (60). The A/B
region contains the highly variable ligand-independent activation function (AF-1).
Studies showed that AF-1 activates transcription in a cell- and gene-specific
manner via interaction with coactivators or corepressors (58, 61, 62). The C
domain contains two zinc-fingers and functions in dimerization of ERs,
translocation of the dimerized ER-ligand complex to the nucleus, and
reorganization of the estrogen response element (ERE) on DNA. The D domain
is a link between the DNA binding domain (DBD) and the ligand binding domain
(LBD). Both domains E and F contain the LBD and function in nuclear translation,

hsp90 binding, and dimerization. Through interaction with coactivators or

13

corepressors, the E and F domains also have the ligand—dependent activation
function (AF-2) that modulates transcription (58). AF-1 and AF-2 activate
transcription independently or synergistically and act in a promoter-specific and
cell-specific manner (63). The F domain probably plays a role in distinguishing

estrogen agonists versus antagonists via interaction with cell-specific
coactivators but the function is not Clear (64-66). Comparing ERor and ERB, ERB
(530 amino acids) is slightly shorter than the Classical ERor,(595 amino acids).
The regions of greatest homology between them are the DBD (96%) and the
LBD (58%). In contrast, the N-terminal region of ERB has very poor sequence
homology to that of ERa (28%) and is well conserved between different species
such as rat, mouse, and human, which would imply functional importance. The
potential biological roles of ERa and ERB in the reproductive system have been
investigated by using knockout mouse models that lack either ERor (orERKO) or
ERB (BERKO) or both (aBERKO) (67-69). Characterization of ER-null mice (ERor,
ERB, or both) showed that each subtype has similarities such as a comparable
life span to their wild-type litterrnates and normal development in early age.
However, several unique abnormal phenotypes were observed in each ERKO
model as adults. Details of female and male phenotypes Of ER knockout mice
will be discussed in the following sections. With the cloning of two isoforrns of
the ER, and the generation of antibodies, it has been possible to detect the
ontogeny and cell-specific expression patterns of both ER isoforrns within
different tissues in a variety of species. ERa is predominant in the anterior

pituitary, uterus, vagina, testis, liver and kidney, while ERB is predominant in the

14

thyroid, ovary, prostate, skin, bladder, lungs, gastrointestinal tract, cartilage and
bone (70). Furthermore, a cell-specific localization for ER isoforrns has been
observed. For example, ERa is localized in the stroma and theca cells and ERB

is localized in granulosa cells in the maturing follicles (71 ). A study demonstrated

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

ER alpha 595
‘ if?
N ' “B C D E F F- COOH
ER beta 530
N " 28% 96% 17 530/, 18 f1“- COOH
% %
Transcriptional A” 3::
activation
..... . "...-....-." Nuclea
trmslocation
Dimerization ....
DNA bindhg
llsp90 binding ..
.. .... Coactivator
binding
Compressor ...—......
binding

Figure 2-4. Schematic representative of the structural and functional domains of
ERor and ERB.

15

that the ERB inhibited ERor transcriptional activity and decreased overall cellular
sensitivity to E2 due to the formation Of heterodimers between ERor and ERB
(72). Therefore, in areas of a target cell where both ERor and ERB are

expressed, the relative expression levels of the two isoforrns would be a key

determinant of cellular responses to agonist and antagonists.

2.5 MODES OF ACTION OF ESTROGEN
2.5.1 LIGAND-DEPENDENT MODE

Estrogen can pass through the cell membrane by simple diffusion
because Of its lipophilic nature (Figure 2-5). Within the target cells, estrogen
binds to the ER, which is complexed with Chaperone protein (e.g. heat shock
protein 90 (HSP90)). HSP70, HSP90 and other associated proteins help the
ERs to fold and prevent aggregation. The ligand-bound ER undergoes
conformational changes to an activated form, presumably by dissociation Of
associated Chaperone proteins. This activated form of the ERs dimerizes into
either homodimers (ERor/ERor, ERB/ERB) or heterodimers (ERor/ERB) (59, 73).
After ER dimerization occurs, the ligand-ER dimers are translocated into the
nucleus and they bind to specific DNA sequences in the vicinity of target genes,
the EREs (5’-GGTCAnnnTGACC-3’), to stimulate transcription of target genes.
The EREs vary in their sequence and usually consist of 15-20 base pairs (73).
When the ligand-ER complex binds to an ERE. Chromatin initiates remodeling,
and interacts between its carboxy-tenninal, DBD and CO-activators (e.g.

p160/SRC; steroid receptor coactivators and p300/CBP; CAMP response element

16

binding protein [CREB], which synergistically increases ERor-induced
transcription in Vitro) or CO-repressors (e.g. SMRT; silencing mediator for
retinoids and thyroid hormone receptors) with the target genes transcription
machinery (66, 74). Estrogen target genes are expressed and cellular and

physiological responses occur.

2.5.2 LIGAND-INDEPENDENT MODE

Many studies demonstrated that ERs phosphorylated in Vitro by different
kinases exert their biological action in the absence of ligand binding. The ability
of polypeptide growth factors such as epidermal growth factor (EGF) and insulin-
like growth factor-1 (IGF-1) bind to CAMP on the plasma membrane and induce
signaling transduction via activating intracellular kinases cascades (Figure 2-5)
(75). Activated kinases transfer the terminal phosphate group of ATP to serine
residues 104, 106, 118, and 167 in the N-terminal Of ERs, which do not have
estrogenic ligand binding. The activated form of the ER is dimerized and it binds
to ERE-Containing promoters on DNA leading to transcription and translation of
estrogen target genes e.g. Sp1, B-actin, and calcium binding protein (76, 77).
Therefore, modification of ER phosphorylation by different intracellular kinases
may serve as an important mechanism of ligand-independent ER action. A study
demonstrated that EGF or lGF treatment induced phosphrylation of AF-1
domains of ERor via the MAPK pathways, and phosphorylated ERa interacts with
coactivators p68 leading to target gene transcription (78). The MAPK pathway

also involves the activity of the ERB via stimulating the recruitment Of SRC-1(79).

17

Furthermore, transcriptional coactivators, p160/SRO and p300/CBP, can interact

with the amino-terminal (A/B) domain of ERs in the absence of ligand (80).

2.5.3 ERE-INDEPENDENT MODE

Beside ERE-mediated gene expression, the ligand-ER dimers can
regulate gene expression through protein-protein interactions of ERs with other
DNA-bound transcription factors (Fos/Jun) at alternative ERE-containing
promoters on DNA (Figure 2-5). The alternative ERE site consists of AP-1
responsive elements corresponding to the Fos/Jun complex for gene
transcription (81, 82). It has been known that ERoc activation of lGF-1 is
mediated through interaction of the receptor with Fos/Jun at AP-1 binding sites,
whereas an ERor-Sp1 complex activates genes containing GC-rich binding sites.
Additionally, E2-ERor activation through binding at activator protein-1 (AP-1)
responsive elements on DNA requires AF-1 and AF-2 domains of ERs to induce
the activity of the p160 coactivator leading to recruitment of Fos/Jun. ERB may
modulate ERa—mediated transcriptional activation by altering the recruitment of
transcriptional factors, Fos and Jun (83). It indicates that the distinct
physiological actions of the two different ERs are due to the regulation of unique
subsets of genes.

As shown in ligand-dependent ER activation and ligand-independent
pathways, HSPs, corepressors, and/or coactivators have important roles in the

modes of action of estrogen. Studies demonstrated that estrogen induces

18

 

 

 

 

 

 

  

 

 

\ Cellllz 01d physiological

@/ W

 

 

 

Figure 2-5. Schematic representation of modes of action of estrogen. Estrogen can pass
through the cell membrane by simple diffusion because of its lipophilic nature. Within the
target cells, estrogen binds to ER which is complexed to chaperones, HSP90. HSP70,
HSP90 and other associated proteins help ERs to fold and prevent aggregation. Ligand
bound ER undergoes conformational Changes to an activated form, presumably by
dissociation of associated Chaperone proteins. This activated form of the ER dimerizes
either into homodimers (ERor/ERot, ERB/ERB) or heterodimers (ERor/ERB). After ER
dimerization occurs, the ligand-ER dimers are translocated into the nucleus and they
bind to specific DNA sequences in the vicinity of target genes, the EREs (5’-
GGTCAnnnTGACC—3’), to stimulate transcription of target genes and physiological
responses. In a ligand-independent pathway, GP or CAMP activates intracellular kinase
pathways. In an ERE-independent pathway, E2-ER complexes bind to alternative
response elements such as AP-1 through association with other DNA-bound
transcription factors (Fos/Jun), and alter transcription of genes. In a non-genomic
pathway, E2 binds to a putative membrane-associated binding receptor or site. E2 alters
intracellular signal transduction pathways that generate rapid tissue responses. ER,
estrogen receptor; HSP90, heat shock protein 90; ERE, estrogen response element; GF,
growth factors; CAMP, cyclic adenosine monophosphate; P, phosphorylation; E2,
17beta-estradiol. Modified from [81].

19

expression of HSPs (i.e. HSP90) and heat shock cognate protein (i.e. HSP73) in
the murine uterus and hypothalamus (84). Voss et al. demonstrated that there
were gender difference in expressions of HSPs by E2 treatment (85). In
conclusion, this may suggest that ER modulators can specifically act in males or

females in different tissues.

2.5.4 NONGENOMIC MODE

It has been suggested that estrogen can bind to plasma membrane
receptors, including ERs that are localized on the plasma membrane, and thus
can induce biological activity without binding to EREs on DNA (86).
Characteristic effects of this nongenomic pathway are (1) rapid responses
(effects occur within seconds or minutes); (2) resistance to the inhibitors of
estrogen target gene expression; (3) different properties compared to Classical
nuclear receptors (typically not blocked by the antagonists of nuclear receptors);
(4) effects of estrogen coupled with high-molecular weight substances (e.g., p-3-
[O-Carboxymethy]oxime: bovine serum albumin [BSA] coupled with estrogen
increases acrosome reaction) that do not pass across the plasma membrane
(87-92).

Furthermore, a nongenomic mode of action of estrogen has been
suggested for synthesized estrogens in the brain, that can locally exert their
biological activity without dependence on gonadal secretion by binding to cellular
membrane ERs and activating G-protein-couples receptor signaling resulting in

both short- and long-term Changes in neuronal excitability (93). Overall, these

20

rapid effects of estrogen mediated by nongenomic pathways would be critical for

reproduction, sexual behavior, learning, memory, motor control, and mood.

2.6 EFFECTS OF ESTROGEN ON OVARIAN DEVELOPMENT, OOGENESIS
AND FOLLICULOGENESIS
2.6.1 ESTROGEN IN OVARIAN DEVELOPMENT

Exposure to estrogen during fetal life plays a key role in regulating ovarian
development and function, which in turn facilitates the female reproductive
process. During the fetal developmental period, estrogen exerts its effects
directly on the fetal non-human primate ovary via binding to ERs and indirectly
acting on the fetal hypothalamiC-pituitary axis (94). A study showed that the
expression of the steroidogenic enzyme, 17B-HSD, in the human fetal ovary was
detected as early as 17 weeks of age, which is the earliest time point evaluated
(95).

Corresponding to maternal serum concentrations of estrogen and
expression of steroidogenic enzyme in the fetal ovary, expression of ERs in the
developing human fetal ovary was observed from 20 weeks of gestational age,
and the highest expression of ERs was observed at the beginning of the last
trimester in the granulosa cells of growing follicles and in the oocytes (95). ERB
predominates throughout, which that ERot is localized in theca and interstitial
cells in the developing fetal human ovary (96). However, there are different
expression patterns of ERs between the fetal mouse ovary and fetal human

ovary. Unlike in the human fetal ovary, expression of ERor predominates in fetal

21

mouse ovary, whereas expression of ERB is negligible (97). Taken together, the
differences in different isoforrns of ER expression among species may result in
different biological roles Of estrogen in fetal ovarian development.

Currently, the biological roles of estrogen in ovarian development and
reproductive function have been studied using ERKO animal models (68, 98-100).
Female aERKO and aBERKO mice are infertile or exhibit reduced fertility with a
reduced number of pups per litter when compared with wild-type mice (69). All
ERKO female mice have normal ovarian morphology during prenatal
development and at birth. In contrast, mature aERKO females have hemorrhagic
ovaries and cystic follicles (101), while BERKO females have a normal gross
morphology (68, 100). orBERKO adult female mice are infertile and have the
most abnormal ovarian phenotype. For example, their ovaries had structures
resembling seminiferous tubules of the testis with Sertoli-like cells as well as
production of Miillerian inhibiting substance (69, 99). These results indicate that
ERor and ERB are required for the normal development of oocytes and somatic

cells in the postnatal ovary.

2.6.2 ESTROGEN IN OOGENESIS AND FOLLICULOGENESIS

Oocytogenesis consists of two major components; (1) oogenesis is the
creation of an ovum in the ovarian follicle of the ovary before birth and (2)
folliculogenesis is the maturation of ovarian follicles, which is the progression of a
number of small primordial follicles inside Of a densely-packed shell of somatic

cells into large preovulatory follicles that are released during the menstrual cycle.

22

Oocytes are a permanent cell population generated before birth.
Therefore, the total number of oocytes present in the mature ovary originates
from a definite number of primordial germ cells, which migrate into the
developing ovary and begin to differentiate into oogonia (102). In mammals,
oogonia are transformed into diploid primary oocytes after several rounds of
mitotic divisions, which occur until either shortly before or after birth (103). The
further development of diploid primary oocytes is arrested during prophase l of
meiosis, and the initiation of meiosis in the oocytes coincides with the onset of
puberty.

On day one of the menstrual cycle at puberty, rising concentration of
gonadotropin-releasing hormone (GnRH) from the hypothalamus stimulate the
anterior pituitary to release FSH and LH leading to rapid growth of follicular cells.
As the follicles are growing, estrogen concentration from granulosa cells of
follicles increase and inhibit FSH and LH via a negative feedback effect on the
hypothalamus (Figure 2-6). After serum estrogen concentrations reach
approximately 800 pg/ml, there is a sudden release Of LH from the pituitary at
about day 14. It triggers the completion Of the first meiotic division of the oocyte
and leads to ovulation.

During ovulation, serum concentrations of estrogen decline but LH
stimulates the ruptured follicle to form the corpus luteum. Diploid primary
Oocytes undergo the first meiotic division. During prophase, homologous
Chromosomes pair and split from one another during anaphase to become

haploid secondary oocytes and the first polar body. Development of the

23

secondary oocytes are arrested and ovulated from the ovary during this stage in
many species. It is known that follicles grow independently of gonadotropins until
the late preantral stage. The second meiotic division of the ovum starts when
sperm penetrates the plasma membrane of the ovum. The ovum divides in
meiosis II and produces the second polar body. As a result of nucleus fusion of
two haploid nuclei of sperm and ovum, fertilization is completed. Human
Chorionic gonadotropin (hCG) is released from the embryo and then eventually it
is produced by the placenta.

Oocyte maturation and ovulation are regulated by interaction between
gonadotropins and estrogen. ERKO studies demonstrated that ERa-mediated
estrogen action plays a minimal role in granulosa cell differentiation and ovulation,
whereas ERB-meditated estrogen actions are critical to somatic cell
differentiation and organization. This includes FSH-induced granulosa cell
differentiation via LH receptor signaling pathway to respond to a gonadotropin
surge. A healthy oocyte is expelled and the antrum and cumulus oocyte complex
form in preovulatory follicles (104). As a consequence of failing to ovulate,
ERKO female mice are infertile. Furthermore, reduced aromatase activity,
reduced E2 synthesis from granulosa cells, and decreased expression Of the LH
receptor were Observed in the BERKO female mice (105). On the other hand,
adult aromatase knockout (ArKO) female mice are anovulatory with ovaries that
contain progressively hemorrhagic cystic follicles (106). Young ArKO females (4

or 7 weeks of age) have apparently healthy ovaries. However, when ArKO

24

female mice were superovulated without hCG injection, and their oocytes were

manually recovered from ovaries at 4 or 7 weeks of age, maturation, fertilization,

 

Hypothalam us 34,
GnRH

   

 

Feminization of
the female
reproductive Oogenesis
system

Figure 2-6. Schematic representation of hormonal regulation of oogenesis in the

ovary.
and development their oocytes to the blastocysts stage did not significantly differ
from those of wild-type and heterozygote litterrnates. Therefore, estrogen is not
necessary for the maturation, fertilization, and development of oocytes, but it is
required for continued follicle growth.

In conclusion, results from ERK0 and ArKO mouse models suggest that

estrogen is required for normal follicle growth beyond the antral stage, for

formation of the female phenotype of the somatic cells within the ovaries and for

25

feedback regulation by hypothalamus-pituitary axis. However, oocyte maturation

and fertilization do not required estrogen.

2.7 EFFECTS OF ESTROGEN ON TESTICULAR DEVELOPMENT AND
SPERMATOGENESIS
2.7.1 ESTROGEN IN TESTICULAR DEVELOPEMENT

Steroidogenesis and sperrnatogenesis are two major biological functions
of the testis. Normal testicular function is important to male fetal sexual
differentiation and production of progeny by the mature adult. During 3 to 5
weeks of gestation in humans, primordial germ cells initially develop in the
primitive gut and migrate to the undifferentiated gonadal ridges to form
undifferentiated gonads that contain the primitive germ cells and stromal cells.
During the 7th week of gestation in humans, the undifferentiated gonad begins to
differentiate into a testis. From PND 10 to PND 26, Leydig cells and Sertoli cells
in the immature rat are dividing and undergoing functional maturation (107).
Sertoli cells in the human male gonadal ridge begin to synthesize Miillerian
inhibiting substance and aggregate into early seminiferous tubules at 8 to 9
weeks of gestation. In the developing male fetus at approximately 9 weeks of
gestation, testosterone is secreted from the Leydig cells with unknown causes.
Some studies suggest that the cells are being stimulated by placental
gonadotropins (108), other studies suggest that Leydig cells are responding to

the fetal pituitary-hypothalamic axis (109).

26

1. Head or upper
pole of testis

2. Tunlca abuglnea

3. Testicula septa

4. Anterior margh

5 lateral suface

6. Tail or lower pole
of testis

7. Testiculu lobules

8. Semlnlferous
tubule

9. Efferent ductules

10. Rete testis

11. Posterior magh

12. Epididymls

 

     

Leydig cell \ '1
Spermatozoa

 
 
        
   

Sertoli cell nuclei \ do,“
,,/ umhal compatment
Semlnll'erom twule Lunen Lymphatic system

A‘
Basalcormatment \ R E »
Jmctional complexes

Figure 2-7. A cross-sectional view of the testis and the seminiferous tubules. Within
the seminiferous tubules, Sertoli cells extend from the basal lamina to the luminal
surface and form basal and adluminal compartments by virtue of tight junctions.

Within the seminiferous tubules, Sertoli cells extend from the basal lamina
to the luminal surface (Figure 2-7). Testosterone, secreted by Leydig cells,
induces the Woiffian duct to differentiate into the epididymis, vas, ejaculatory

duct, and seminal vesicle. The internal reproductive development is completed

27

by the third month of gestation, with the degeneration of the Mtillerian structures
and the differentiation of the Wolffian ducts.

Possible biological roles of estrogen have been suggested by the cell-
specific expression pattern of ERa and ERB within the testis and other
components of the male reproductive system in different species (110-112).
Tables 2-3, 2-4 and 2-5 present a summary Of the expression of ERs and
aromatase in the different cell types of fetal, immature, and adult rodent testes
from several studies. ERs and aromatase are found at all stages of testicular
development in the rodent (113, 114). The expression of ERs and aromatase
mRNA and protein was detected in the undifferentiated gonad of the mouse as
early as day 10 and day 11.5 of gestation, a time when expression of the
androgen receptor was not detectable (115-119). This suggests that estrogen
may be important in the testicular differentiation process. However, due to
differences in species, strain, antibodies or methods Of detection, these
differences in mRNA or protein levels of the two isoforrns of ERs in the testis are
difficult to compare among studies for establishing a solid conclusion.

ERa expression was detected in the Leydig cells and in the epithelium of
the rete testis and efferent ducts in fetal/neonatal/adult rodent but not in the
epididymis and Sertoli cells (43, 111, 120). Efferent ductules were the first male
mouse reproductive structures to express ERs beginning on GD16 (120).
Epithelial ERs began to express in the initial segment of the epididymis on GD19,

soon after its differentiation. ERs started to be expressed in the epithelium of the

28

seminal vesicle on PND 6. Further studies are needed to confirm whether ERor

are expressed in adult rodent germ cells.

Table 2-3. ERs and aromatase distribution in the fetal rodent testis

 

 

ERa ERB Aromatase
Leydig cells + + +
Sertoli cells - + +
Germ cells - + -
Seminiferous tubules +/- + +
Efferent ducts +/. + -

 

Source: (113, 114)

Table 2-4. ERs and aromatase distribution in postnatal rodent testis

 

 

ERa ERB Aromatase
Leydig cells + + +
Sertoli cells - + +
Germ cells - + -
Seminiferous tubules - + +
Efferent ducts + + Not available

 

Source: (113, 114)

Table 2-5. ERs and aromatase distribution in the adult rodent testis

 

 

ERor ERB Aromatase
Leydig cells + +/- +
Sertoli cells - + +
Germ cells +/- + +
Spennatogonia - + +
Spennatocytes +/- + +
Round spennatids +/- + +
spermatozoa + + +
Efferent ductules + + -

 

Source: (113, 114)

ERB expression was detected as eariy as day 16 of gestation in the Leydig,
Sertoli, and germ cells in the adult rodent and primate (118, 121-123). Even

considering the variation in results of immunohistochemical studies, there has

29

been considerable evidence that presence of ERB and aromatase are present in
adult rodent germ cells. ERB was also expressed in rat associated ducts, where
sperm become reproductively mature, throughout all ages. Due to dominant
expression of ERB in male testis and the reproductive system throughout life,
ERB may have a more important biological role in testicular development and
spennatogenesis than ERa.

Aromatase is expressed in both Leydig cells and Sertoli cells in the rodent
fetal testis but not in germ cells and seminal tract by postnatal day 19 (124). In
the adult rodent, higher aromatase activity in Leydig cells was observed,
compared to every other age and Sertoli cells. Aromatase is expressed in germ
cells throughout all ages and it appears to increase as germ cells mature (116,
125, 126). This suggests that germ cells may be a major source of estrogen in
adult rodent testes.

Testes of male CERKO mice are smaller with degenerated and atrophiC
seminiferous tubules and epididymis sperm having low motility (Table 2-5) (69).
These deteriorations appear at around 20 days of age and advance with age
(127). These mice had dilated rete testis and efferent ductules in the head of the
epididymis resulted in accumulation of fluid in the seminiferous tubules and
dilution of sperm (44). Unlike phenotypes of aERKO, BERKO mice showed no
apparent reproductive tract abnormalities. Therefore, the biological functions of
ERB are unknown, even through the expression of ERB was Observed in the
testis. ArKO mice also showed histologically normal testes at 9 weeks Of age but

developed Leydig cell hypertrophy/hyperplasia (128).

30

In conclusion, expression of ERs and aromatase in developing fetal testis
imply that estrogen has important roles in the process of differentiation and

maturation of developing rodent and human testis.

2.7.2 ESTROGEN IN SPERMATOGENESIS

The primordial germ cells migrate into the gonad during embryogenesis
and become immature germ cells or spermatogonia, which can divide many
times by mitosis in a process called spennatocytogenesis. Beginning at puberty,
these spennatogonia reside on the basement membrane of the stratified
seminiferous epithelium. Spennatogenesis, which takes place within the
seminiferous tubules of the testis, is the meiotic process to produce diploid germ
cells that develop into haploid spermatozoa. In this process, round sperrnatids
elongate into spermatozoa, in a process called sperrniogenesis. The
spermatozoa are Characterized by compacted DNA, a flagellum for motility, and
an acrosome for oocyte penetration. After this gametogenesis, from the
spennatogonia to the spermatozoa, the spermatozoa are released into the lumen
of the seminiferous tubule and travel to the epididymis, where they become more
concentrated and matures obtaining the ability to fertilize an egg. Compared to
oogenesis in the female (mitotic division occurs before birth and produce only
one mature ovum), sperrnatogonia proliferate only after puberty and the meiotic
divisions of primary spermatocytes produce four mature spermatozoa. The
duration of sperrnatogenesis varies in different species (74 days in the human,

26 to 35 days in the mouse, and 48 to 53 days in the rat) (129). The life span of

31

human germ cells is 16 to 18 days for spermatogonia, 23 days for primary
spermatocytes, 1 day for secondary spermatocytes, and approximately 23 days
for spennatids (130).

Although ER expression in the somatic cells of the male reproductive tract
is required for fertility, the role of estrogen in spennatogenesis has been unclear
until ERKO mouse model was established. A number of ERKO mouse studies
demonstrated that testicular aromatase activity has an important role in
sperrnatogenesis and intratesticular estrogen synthesis has been well
documented. Table 2-6 presents a summary of reproductive Characteristics of

ERKO male mouse models.

Table 2-6. Reproductive pheynotypes of ERKO and ArKO male mouse
models

 

 

aERKO BERKO aBERKO ArKO
Infertility (127) Normal Infertility (101) Normal fertility in young
fertility (68) mice, infertility with

advancing age (between
4.5 months and a year)

(128,131)
Normal FSH - - Normal FSH
Increased LH - - Increased LH
Increased T - - Increased T
Increased E2 - - Undetectable E2
Germ cell Normal Testicular Histology of the testis is
deprivation testicular histology disrupted with advancing
with dilated histology similar to age (Leydig cell
seminiferous aERKO mice hyperplasia/hypertrophy)
tubules Decreased sperm
maturation

 

Both male aERKO and aBERKO mice are infertile throughout their life with

swollen seminiferous tubules as a consequence of pressure from failure of fluid

32

transport across the epithelium of the head of the epididymis (43). However,
BERKO male have normal fertility compared to wild-type mice (69).

Male ArKO mice develop a progressive infertility and by one year of age,
most male ArKO mice are infertile (132). Round spen'natids in ArKO mice have
acrosomal dysgenesis and the immunolocalization of aromatase and ERB in the
Golgi complex, which is required for acrosome biogenesis, of the developing
spennatid (128, 133). Spennatogenesis at around 20 to 23 weeks of age was
arrested at early round sperrnatid differentiation with Leydig cell hyperplasia
resulting from the increased LH concentrations, and there were no mature
sperrnatids observed in male ArKO mice. However, there were no significant
Changes in the semen volume of seminiferous tubule lumen.

There have been increasing concerns about significantly decreased sperm
concentrations and increased cancers of the human male reproductive system
during the past several decades due to fetal and neonatal exposure to EEMs.
This hypothesis was formulated by Sharpe and Skakkebak (134) and it has been
extensively investigated due to controversial results from several studies. As
early as 1927, Macomber and Sanders reported the normal sperm density to be
100 x 106/ml in the US based on the sperm counts of 294 individuals without
regard to fertility status (135). In 1992, Carlsen et al. reported a significant
decrease in human sperm concentration from 113 x 106/ml in 1940 to 55 x 106/ml
in 1990. However, this meta-analysis of 61 studies was criticized due to
methodological variation in counting sperm. Additionally, the average sperm

concentration in humans varied with geographical location. For example, sperm

33

count was 72.7 x 106/ml in California, 100.8 x 106/ml in Minnesota, and 131.5 x
106/ml in New York (136, 137). Despite the limitations, Sharpe and Skakkebak’s
hypothesis has gained support. During this period (1940 to 1990), the incidence
of testicular cancer has increased 2 to 4% per year and incidences of
undescended testes (cryptorchidism) and male genital malformations
(hypospadias) also appears to be increasing (138, 139).

It has been known that prenatal or neonatal exposure to estrogen affects
normal development and function of the male reproductive tract such as
decreased testis weights, sperm maturation, and impairment of sperrnatogenesis.
It suggests that there is the possibility of a direct action Of estrogen on germ cells
during various stages of germ cell development (Figure 2-8). Studies
demonstrated that estrogen has a predominantly stimulatory effect on germ cells
and on differentiating spermatogonia, even though it has inhibitory effects on
Leydig and Sertoli cell via negative feedback through hypothalamus-pituitary-
gonad axis (140, 141). In a study by Li et al (141) in Vitro differentiation of
sperrnatogonia was stimulated by a 1 pM dose of E2 but not by higher doses of
E2. Additionally, the stimulatory effect was blocked by the estrogen antagonist
ICI 164, 384.

Sertoli cells provide a structural scaffold for developing sperm and secrete
a number of proteins and nutrients that regulate the initiation and progression of
sperrnatogenesis. For example, FSH-stimulated Sertoli cells produced

androgen-binding protein, which helps keep local testosterone concentrations

34

high; aromatase, which converts testosterone to estradiol; growth factors

including

 

   
 
 
  

 

Estrogen .—

 

 

 

Spennatogenesis “"7 Masculinization of the
male reproductive
Testosterone (T} 4' system

Figure 2-8. Schematic representation of hormonal regulation of sperrnatogenesis in
the testis.

lGF-1, transforming growth factor-0 (TGF-a), TGF-B, naturally occurring growth
factor (NGF), and interleukin-1 (IL-1), which stimulate differentiation of sperm
(130, 142). Additionally, tight juctional complexes between Sertoli cells on the
basal membrane of seminiferous tubules provide a blood-testis barrier to protect
germ cells from immunological attack (143). Studies indicates that aromatase
activity in the Sertoli cells is high during the neonatal period when germ cells are

proliferating and differentiating into spermatogonia, which contain ERB (144, 145)

35

(Table 2-3). Therefore, a direct paracrine action of estrogen in stimulating
precursor germ cell mitosis may be possible.

It has been suggested that ER-mediated estrogen may act as a germ cell
survival factor in the human testis in Vitro study (146). When segments of human
seminiferous tubules were incubated with 10'9 or 10'10 mon of E2, germ cell
apoptosis was inhibited. Furthermore, both ERa and ERI3 expression were
detected in early meiotic sperrnatocytes and elongated sperrnatids of the human
testis. In agreement with this finding, significant decreases in round and
elongated sperrnatids were observed in 18-week-old ArKO mice (128). Estrogen
deficiency following by inhibiting aromatase activity decreased conversion of
round sperrnatids to elongated spennatids and sperm production in adult male
primates (147) and increased round sperrnatid degeneration in adult male rats
between 1 and 4.5 years (128, 148).

The fully developed sperrnatids released from the Sertoli cells are
transported into the rete testis, where estrogen concentration is relative high.
ERu regulates the expression of the sodium/hydrogen exchanger-3 in the
efferent duct (149). This gene is associated with fluid reabsorption in the efferent
ducts. Therefore, estrogen has an important role in dilution of cauda epididymal
sperm, disruption of sperm morphology, inhibition of sodium transport and
subsequent water reabsorption, increased secretion of Cl', and eventual
decreased fertility (14, 43, 44, 150). Correspondingly, the role of ERa in
reabsorption of fluid for concentrating sperm in the efferent ducts and head of the

epididymis has been established. Disruption of the ERO, either in the knockout

36

or by treatment with 1 mglkg of ICI 182,780 (an antiestrogen) for 3 days, before
removal of the efferent ductules in adult mice, results in increased diameter of
the lumen of ligated efferent ductules in Vitro after incubation (8). A possible
mechanism of increased testicular mass would be that testicular secretions
accumulate in the lumen of the seminiferous tubule and back-pressure of the
accumulated fluid on epithelium of efferent ductules causes long-term atrophy of
testes.

Furthermore, aromatase activity was detected in the cytoplasmic droplet
that remains attached to the flagellum as the sperm make their way through the
epididymis, suggesting that mature human spermatozoa may synthesize their
own estrogen as they traverse the efferent ducts (151, 152). The ability to
synthesize estrogen gradually decreases as long as the droplet slowly moves to
the end of the tail during move to the epididymis. It suggests that the sperm itseif
may control the concentration of estrogen in the luminal fluid and modulate the
reabsorption of fluid from the efferent ductules. Overall, these results suggest
that estrogen converted by aromatase may be required for sperm transportation
through ductules and acrosome biogenesis.

In conclustion, the initiation of sperrnatogenesis and the full sperrnatogenic
potential of the adult require normal proliferation, differentiation, and functional
maturation of Sertoli cells. Considering the duration of spemiatogenesis and life
span of germ cells, neonatal exposure to potential EEMs leads to permanent
defects in germ cells due to permanent defects in Sertoli cell function rather than

on germ cells (153).

37

2.8 EFFECTS OF ESTROGEN ON FERTILIZATION AND IMPLANTATION

Rapid responses of sperm capacitation and acrosome reaction to
estrogen have been shown to involve an increase in CAMP levels, intracellular
calcium [Caz‘] concentrations, protein kinase activity, phospholipase C and A via
binding to plasma membrane (154-156). Protein kinases and phosphatases are
involved in regulatory roles in sperm capacitation and these enzymes are known
to be inhibited by genistein (157). On the contrary, uncapaciated human sperm
suspensions incubated in the presence of genistein (100 nmoI/l), which is an
phytoestrogen, increased sperm capacitation and acrosomal reaction by 30%
(156). Correspondingly, Adeoya-Osiguwa et al. (158) observed significantly
stimulated sperm capacitation and acrosome reactions in uncapacitated mouse
sperm incubated with 2 1 pmol/l of E2 or _>_ 0.001 pmol/I of genistein for 30 min.
In the same study, sperm fertilizing ability was significantly increased in the 10
pmollL E2 treatment, compared with untreated control sperm (76 vs. 36%,
respectively). Furthermore, the influence exerted by estrogen on maturation of
sperm has been investigated in ERKO mice and was found to decrease motility
and fertilizing potential (101 ).

Estrogen has an important role in the implantation of the blastocysts in the
uterus and its action is mediated by the progesterone receptor (PR) and growth
factors at the local sites. In the wild-type mouse uterus, where ERor predominates
estradiol treatment induces both mRNA and protein levels of the progesterone
receptor in the stroma, and down-regulates PR expression in the uterine

epithelium. In the aERKO females, up-regulation of the PR in the uterine stroma

38

occurred (159). This suggests that estrogen has important roles in implantation
by regulating the progesterone receptor in the uterus.

The successful growth and development of the fetus are dependent on the
balance of endocrinological events after implantation. Approximately 8 to 12% of
all pregnancy losses in humans are the result of endocrine factors (160). Prior to
four weeks of gestation, the main source of estrogen in maternal Circulation is the
maternal ovaries but around seven weeks of gestation, the main source of both
maternal and fetal estrogen is the placenta (23). During pregnancy in humans
and primates, maternal serum estrogen concentration increase from 50 to 100
pg/ml to 30,000 pg/ml at birth (20). The major role of estrogen is to maintains the
endometrium by enhancing uteroplacental blood flow and to regulate the
production of progesterone throughout the pregnancy by stimulating receptor-
mediated uptake of LDL increase the activity of placental mitochondrial
Cholesterol side-chain Cleavage enzyme (P450scc) (161-163). A study
demonstrated that estrogen administration (5 mglanimal by interamuscular
injection on gestational day 9 and 10), before the conceptus secretes estrogen
on gestational day 12, alters the pattern of IGF gene expression through the
nuclear factor kappa B (NF-KB) system desynchronizing the uterine environment
for conceptus implantation resulting in later embryonic loss in pigs (164). These
studies indicate that estrogen serves as the signal for maternal recognition and
maintenance of pregnancy but at the same time, timing of endometrial exposure

to estrogen is critical to embryonic survival.

39

2.9 EFFECTS OF ESTROGEN ON SEX RATIO OF OFFSPRING

It has been hypothesized that the mammalian sex ratio at birth (male
births/total births) is influenced by biological factors (e.g., high level of parental
sex steroid hormones at the time Of conception, excision of accessory sex glands
in rodents) and environmental factors (e.g., exposure to dioxin) (165-169).
Review studies by James indicated that high concentrations of LH at the time of
conception may result in more female Offspring, while high levels of estrogen,
testosterone and progesterone may be associated with male offspring (168, 170-
172). Furthermore, he suggested that the same hormones may affect the sex
ratio in opposite directions in different species and more male offspring are
produced as a result of suboptimal health of the parents (173). Based on
James’s hypothesis, EEMs may have potential effects in altering the sex ratio in
both humans and wildlife species. However, there are no Clear answers for why
different hormone concentrations cause variations of the sex ratio and the
possible mechanisms underlying his hypothesis (174).

In addition to biological factors, epidemiologic studies demonstrated that
some environmental contaminants such as 2,3,7,8-tetraChlorochlibenzo-p-dioxin
(TCDD), hexachlorobenzene (HCB), dibromochloropropane (DBCP) and
Clomiphene Citrate may be associated with the decline in the sex ratio in humans
that has been observed in some western countries (166, 169, 175-177). It is
known that these compounds have estrogenic and antiestrogenic properties, thus
altering the sex ratio via Changing parental biological factors. For example, high

serum concentrations of TCDD in fathers exposed during the Seveso accident in

40

1976, were significantly associated with a decline in sex ratio up to eight years
after the incident in two areas having the highest levels of contamination (Meda
and Seveso) (P = 0.008) (178). The effect of TCDD on the sex ratio was
detected at serum concentration lower than 20 ng/kg body weight in the fathers
who were exposed to TCDD (166). Furthermore, studies suggest that exposure
to high concentration of TCDD may decrease male offspring for up to 10 years,
but this effect diminished over time. On the other hand, some studies related to
dioxin-like Chemicals conducted in Taiwan and in European countries did not find
any significant association between dioxin-like Chemicals and altered sex ratio or
high concentration of pesticide or industrial pollution and the proportion of male
births (177, 179, 180).

Hexachlorobenzene is a fungicide used to control Tilletia tritiCi, a wheat
based fungus. It is resistant to metabolic degradation and has an undetermined
haIf-life in humans because of its intense lipophilicity (181). An epidemiological
study in Turkey reported that women exposed to high concentrations of HCB
between 1955 and 1957 had a significantly lower proportion of males than those
exposed before 1950 or later 1970 (181). Based on in Vitro cytochrome P4501A
induction, the potency of HCB is approximately 10,000 times less than dioxin.
Furthermore, HCB binds to the aryl hydrocarbon receptor (AhR) with 10,000
times less affinity compared to dioxin (182). This suggests that HCB may act via
the same mechanism Of action as TCDD to decrease the sex ratio. However, the

mechanisms of TCDD and HCB in altering sex ratio need further investigation.

41

Dibromochloropropane, a pesticide, was used to control worms that cause
damage to crops and other plants. It was banned in the US in 1977 because it
was found to lower the sex ratio in DBCP-exposed male workers. For example,
an epidemiological study in Israel reported that the decreased sex ratio was
significantly associated with paternal DBCP exposure (P < 0.025)(183). Studies
confirmed that DBCP causes infertility (e.g., azosperrnia), spontaneous abortion,
and it has potential to cause tumors in the breast, lung and other organs in
laboratory animals (184-187). Rats subcutaneously injected with DBCP had
decreased testes weights and increased infertility with degenerated seminiferous
tubules. Dibromochloropropane significantly increased gonadotrophin levels in
DBCP-exposed agricultural workers who had azosperrnia and in infertile DBCP-
treated laboratory rats (188, 189). However, serum testosterone concentrations
were not different in either exposed humans or laboratory species, compared to
control groups (183, 189). Corresponding to James’s hypothesis, high
concentrations of gonadotrophin induced by exposure to DBCP may cause a
decreased sex ratio in humans and laboratory rats (174). However, the
mechanism of this phenomenon is also unknown.

A potential mechanism of action of EEMs in altering sex-ratio could be
through an alteration of the uptake of glucose in the female reproduction tract or
in sperm after fertilization has occurred. It has been postulated that high
concentrations of glucose in the reproductive tissues of both parents may
increase the sex ratio. For example, the presence of glucose (5.56 mmol/l) in the

culture medium when bovine embryos were cultured for 24 to 48 h after

42

insemination, lead to a higher percentage of Cleavage among the male embryos
(190). In addition to increased male embryo Cleavage, glucose (5.56 mmOl/l) in
the culture medium increased the number of acrosome reactions and sperm
motility after 18 hours of capacitation of human sperm, compared to media
containing no glucose (191). Furthermore, sperm motility in fertilization medium
can be sustained by least 2.78 mM of glucose. Chemically induced diabetes in
pregnant female mice resulted in maternal hyperglycemia and a significantly
decreased sex ratio in their live offspring (192). Corresponding to these results,
a meta-analysis study demonstrated that excess glucose in the maternal
circulation around conception favors the development of male blastocysts during
early embryo cell division (193).

The other possible mode of EEMs in decreasing sex ratio may be via
altering the maturation of the oocytes. A study investigated the effects of an in
Vitro maturation culture period for bovine oocytes on the sex ratio of in Vitro
produced blastocysts (194). An increase in the proportion of male blastocysts
with an increase in maturation culture period (34h vs. 16 and 22h) Of cumulus-
oocyte complexes was Observed (194). Studies demonstrated that the heads Of
Y-bearing spermatozoa and their length were significantly smaller and their
necks and tails were shorter than those of X-bearing ones (195). Therefore,
differential migration of the Y- and X-bearing spermatozoa was suggested (196).

However, published studies addressing possible association or effects of
EEMs on mammalian sex ratios have still produced contradictory results because

of unknown mechanisms. Further studies are needed to confirm the effects of

43

hormones on the sex-selection and to investigate biological mechanisms of the

sex-selection.

2.10 EFFECTS OF ESTROGEN ON ANOGENITAL DISTANCE (AGD)
Anogenital distance (AGD) is the distance between the anus and the base
Of the external genitals. The AGD in males is approximately twice as long
compared to females because testosterone produced by the gonads increases
the length of the perineum separating the anus from the testicles via stimulation
of cell proliferation (197, 198). The use of AGD has been explored as an
indicator of prior androgen exposure due to intrauterine position (199).
Corresponding to this, recent studies demonstrated that AGD is a sensitive
indicator of demasculinization of male genitalia exposed to exogenous estrogen
or antiandrogenic Chemicals during the prenatal developmental period (197, 200-
202). The reason is that the reproductive system is Changing rapidly at both the
organ and molecular levels via hormonal stimulation during the prenatal period.
Therefore, the consequences of hormonal effects are established for the duration
of the individual’s life span. For example, testicular testosterone synthesis in
humans begins approximately 65 days after fertilization and influences
differentiation of the Wolffian duct system into the epididymis, vas deferens, and
seminal vesicles (203). In the male rat, the growth rate of AGD in male rats is
increased around gestational day 15, when testosterone synthesis begins (204).
Clark et al. (205) confirmed that the growth rate of the AGD was controlled by

dihydroxytestosterone rather than by testosterone. Consequently, EEMs that

44

interfere with the synthesis of dihydroxytestosterone or decrease its bioactivity
can decrease the growth rate of AGD. Evan et al. (206) reported that GEN
inhibits the activity of Soc-reductase in genital skin fibroblasts.

A number of studies demonstrated that prenatal and postnatal exposure to
EEM decreases the male AGD (12, 207-210). For example, a study
demonstrated that estrogen inhibited human fetal penile smooth muscle cell
proliferation in a dose-dependent manner from 10'12 mol/l, whereas tamoxifen
acted as an antiestrogen to reverse the estrogen effect, in an in Vitro cellular
model (211). Another well-known example of EEMs that decrease male AGD in
laboratory animals and humans is phthalate esters, which are among the most
extensively produced industrial Chemicals and used principally as plasticizers. to
improve flexibility and durability in soft vinyl plastic toys, shampoos, soaps, nail
polish, and vinyl flooring. The first reported evidence of their antiandrogenic
effects on male AGD was Sohoni and Sumpter’s paper (212). In their study, rats
were orally administered high doses Of phthalate esters (250, 500, and 750
mglkg bw/d) throughout gestation to postnatal day 20. Further epidemiologic
studies confirmed that certain phthalates are significantly associated with
reduced AGD in human male infants (207, 213-215). Among these
epidemiologic sttidies, a study in the United State reported that the estimated
median and 95th percentile of daily exposure to phthalates and its metabolites
[di-n-butyl phthalate (DnBP), diethyl phthalate (DEP), butylbenzyl phthalate
(BBzP), diisobutyl phthalate (DiBP), di-2-ethylhexyl phthalate (DEHP)] are

approximately 9.45 and 126.77 pg/kg bw/d (213). These values are substantially

45

lower than the US EPA reference doses of 100 pg/kg bw/d for DBP, 800 pg/kg
bw/d for DEP, 200 pg/kg bw/d for BBzP, and 20 pg/kg bw/d for DEHP. It
suggests that phthalates have antiandrogenic effects on the AGD at low doses
during the prenatal developmental period and that further risk assessments for
these Chemicals are needed.

Conversely, a known natural mycotoxin EEM, zearalenone, increases
AGD in both male and female rat offspring maternally exposed to zearalenone
(oral daily doses of 1 mg zearalenone/kg bw/d from gestational day 6 to 19) (216).
This suggests that zearalenone has androgenic effects during prenatal
development or has specific effects on the AGD.

Additionally, bisphenol A, used to produce polycarbonate plastic and
epoxy resins, is a known EEM in both in vivo and in Vitro assays. However,
AGDs in female and male rat offspring exposed to bisphenol A (4 to 40 mglkg
bw/d gestational day 6 to postnatal day 20) were not different, compared to the
control groups (217). Our previous study also demonstrated that gestational and
lactational exposure to the phytoestrogen, genistein, did not Change AGD in male
and female mouse Offspring on postnatal day 21 (218) (female data shown in
Chapter 4). Overall, these results suggest how complex the hormonal balance in
developing animal fetuses is. Taken together, the AGD can be used as a
sensitive biomarker of effects of EEMs altering the intrauterine androgen
environment but several confounding factors should be considered including

species differences, doses of EEMs, and the timing of exposure.

46

2.11 EFFECTS OF ESTROGEN ON PUBERTY

Puberty is initiated by the activity of the hypothalamic pulse. In infancy
and Childhood, the activity of the hypothalamic pulse doest not occur, and it
increases slowly as reflected by the increase of the number and amplitude of LH
and FSH pulses between the sixth and ninth year of life (219). The serum levels
of estrogen in humans remain less than 12 pg/ml until the onset of puberty
(approximately 7 years Of age), and increase up to 392 pg/ml in girls and 86
pg/ml in boys (20, 220).

The activity Of the hypothalamic pulse during puberty is initiated by
unknown mechanisms (221). Estrogen has been thought of as an activator of
the GnRH pulse generator but studies demonstrated that estrogens are not
activators Of the GnRH pulse generator but rather have modulating effects on
gonadotropin release (222, 223). As a result of increased GnRH secretion from
the hypothalamus, FSH from anterior pituitary increases intracellular CAMP
concentrations via a G-protein-coupled receptor and it leads to an induction of
the aromatase for estrogen synthesis (220). Under the influence of increased
estrogen from gonads, ovaries, uterus, the fallopian tube, vagina, and breast
glands grow and mature in females. The first menstrual bleeding, called
menarche, is initiated by a temporary slight drop in estrogen concentrations,
which leads to an estrogen withdrawal hemorrhage.

In the pubertal boy, estrogen is produced in testicular tissue, and
increased estrogen concentrations are much higher than those found before

puberty (12 vs. 86 pg/ml) (20). Epidemiological studies reported that estrogen

47

deficiency due to mutations in the aromatase gene and estrogen resistance due
to dismptive mutations in the estrogen receptor gene have no effect on normal
male sexual maturation at puberty (224).

There has been concern about precocious puberty. Even though there
are several causes of this, including improved nutrition, exposure to EEMs could
partly be associated with precocious puberty. Relationships between pubertal
development and perinatal and postnatal exposure to EEMs (i.e., polychlorinated
biphenyls, 1,1,1-trichloro-2,2-bis(p-Chlorophenyl)ethane, phthalate esters, furans
and the pesticide endosulfan) have been reported (225). However, further study
is needed to understand the mechanisms that link pubertal development and

EEMs.

2.12 EFFECTS OF ESTROGEN ON BODY WEIGHT

The increasing prevalence of obesity is a growing concern woridwide.
The incidence Of Obesity has significantly increased in the US over the past two
to three decades (226). According to the National Health and Nutrition
Examination Survey (NHANES)1 in 1999 to 2002, 31% of adults aged at least 20
years were Obese and 5% were extremely obese with respect to children aged 6
through 19 years, 16% were overweight (227, 228). Since overweight

adolescents have a 70% chance of becoming overweight or obese adults (80% if

 

‘ The National Health and Nutrition Examination Survey (NHANES) have been monitoring the
national prevalence Of overweight and Obesity. Since 1960, NHANES data on measured height
and weight have been used to determine obesity levels in the US. Body mass index (BMI) is
calculated as weight in kilograms divided by the square of height in meters.

48

one parent is obese or overweight), the prognosis for the future health of
Americans is declining (229).

Based on dramatically increased obesity after the 19803 and an important
role of estrogen in natural weight-control mechanisms, a hypothesis has been
suggested that EEMs alter the body's natural weight-control mechanisms
resulting in interference of the normal developmental and homeostatic controls
over adipogenesis and energy balance.

Epidemiological evidences has indicated that EEMs have a positive
association with increased body weight in humans and laboratory animals (230,
231). The ERa (aERKO) and ERB (BERKO) knockout mice as well as the
double ER knockout (orBERKO) mouse and the aromatase knockout (ArKO)
mouse, have shown that lipid and carbohydrate phenotypes involve increased
adiposity with insulin resistance, hyperiipidemia and hyperleptinemia (232-235).
ERa knockout mice have a significantly increased body fat content with a 10-fold
increase in E2 (235, 236). This suggests that EEMs, which can increase
signaling ERB or block signaling ERa, may contribute to obesity in ERor knockout
mice.

A cross-sectional epidemiological study of two single-nucleotide
polymorphisms (a T to C (Pvull) and an A to G (Xbal)) in the first intron of the
ERor gene showed that there is no association between the ERor gene
polymorphisms and body fat distribution in men, but women with the Xbal
polymorphism may be predisposed to the development of upper-body obesity in

middle-aged (40 to 59 years) individuals. However, older aged (68 to 69 years)

49

women with the Xbal polymorphism had decreased whole-body and abdominal
fat tissue (237). These results indicate that the ERor has an active role in female
upper-body obesity in an age-related manner.

Generally, EEMs at high levels of exposure decrease body weight of
laboratory animals but at much lower levels of exposure, they may increase body
weight with no adverse effects (238). Low levels of EEMs have been widely
measured since they have been used as pharmaceuticals ingredients (e.g., EE2),
organochlorine pesticides (e.g., DDT), plasticizers (e.g., phthalate), and growth
promoting hormones (e.g. zearalenone) in humans and livestock (239-242).
Laboratory animal studies demonstrated that concentrations as low as 0.5 pg E2
caused approximately an approximate doubling of uterine wet weight in mice
(243). Therefore, it is important to consider increased body weight as a low-dose
effect of EEMs, which can be a potential contributor of Obesity.

Most EEMs have lipophilic properties. Therefore, they can be
bioaccumulated in body fat in relatively high concentrations and transfer across
the fetal-matemal blood barrier. Consequently, the current obesity epidemic may
be significantly associated with the level of EEM exposure in early life and in
utero (226, 238). Suggested mechanisms of altering the natural body weight-
control in early life and in utero are: (1) disruption of the major weight controlling
hormones, such as thyroid hormones, estrogen, testosterone, corticosteroids,
insulin, growth hormone, and leptin (2) alteration of the levels of, and sensitivity
to, neurotransmitters (in particular dopamine, noradrenaline, and serotonin) (3)

interference with many metabolic processes (4) induction of widespread damage

50

to body tissues (nerve and muscle tissue in particular), often at concentrations
that human beings are currently exposed to.

However, the extent to which each individual is affected could also be
significantly related to a given individual’s genetic ability to deal with exposure to
EEMs. Furthermore, it is known that decreased endogenous estrogen
concentrations in postmenopausal women and in ovariectomized laboratory
rodents cause increased adipose tissues in females (244, 245). Additionally,
studies demonstrated that estrogen has direct effects on adipocytes to inhibit
lipogenesis and on food consumption and energy expenditure that contribute to
its inhibitory effect on adipose deposition in both females and males during the
developmental period and adulthood (246, 247). Taken together, further
research is needed to investigate which of the environmental estrogenic
compounds is probably causing the greatest damage to the natural weight-

Control system.

51

2.1 3 BIBLIOGRAPHY

1.

10.

Nelson LR, Bultm SE 2001 Estrogen production and action. J Am Acad
Derrnatol 45:81 16-124

Fisher JS 2004 Are all EDC effects mediated via steroid hormone
receptors? Toxicology 205:33—41

Sanderson JT 2006 The steroid hormone biosynthesis pathway as a
target for endocrine-disrupting Chemicals. Toxicol Sci 94:3—21

Meseguer A, Puche C, Cabero A 2002 Sex steroid biosynthesis in white
adipose tissue. Horm Metab Res 34:731-736

Kershaw EE, Flier JS 2004 Adipose tissue as an endocrine organ. J Clin
Endocrinol Metab 89:2548-2556

Roselli CE, Resko JA, Stormshak F 2003 Estrogen synthesis in fetal
sheep brain: effect of maternal treatment with an aromatase inhibitor. Biol
Reprod 682370-374

Dubey RK, Tofovic SP, and Jackson EK. 2004 Cardiovascular
pharmacology of estradiol metabolites. J Phannacol Exp Ther 308:403-
409.

Hess RA, Bunick D, Lee KH, Bahr J, Taylor JA, Korach KS, and
Lubahn DB. 1997 A role for oestrogens in the male reproductive system.
Nature 390:509-512.

Jones ME, Thorbum AW, Britt KL, Hewitt KN, Wreford NG, Proietto J,
Oz OK, Leury BJ, Robertson KM, Yao S, and Simpson ER. 2000
Aromatase-deficient (ArKO) mice have a phenotype Of increased
adiposity. PrOC Natl Acad Sci U S A 97:12735-12740.

Jones ME, Thorbum AW, Britt KL, Hewitt KN, Misso ML, Wreford NG,
Proietto J, Oz OK, Leury BJ, Robertson KM, Yao S, and Simpson ER.
2001 Aromatase-deficient (ArKO) mice accumulate excess adipose tissue.
J Steroid Biochem Mol Biol 79:39

52

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

Migliaccio S, Newbold RR, Bullock BC, Jefferson WJ, Sutton FG Jr,
McLachIan JA, and Korach KS. 1996 Alterations of maternal estrogen
levels during gestation affect the skeleton of female offspring.
Endocrinology 137:21 18-2125.

Mylchreest E, Cattley RC, Foster PM 1998 Male reproductive tract
maiforrnations in rats following gestational and lactational exposure to
Di(n-butyl) phthalate: an antiandrogenic mechanism? Toxicol Sci 43:47-60

Carreau S, Genissel C, Bilinska B, Levallet J 1999 Sources of
oestrogen in the testis and reproductive tract of the male. Int J Androl
22:211-223.

Hess RA, Bunick D, Bahr J 2001 Oestrogen, its receptors and function in
the male reproductive tract - a review. Mol Cell Endocrinol 178229-38.

Yin Y, Ma L 2005 Development of the mammalian female reproductive
tract. J Biochem (Tokyo) 137:677-683

Jones EJ, DeChemey AH 2003 The female reproductive system. In:
Boron WF, Boulpaep EL eds. Medical physiology. Philadelphia: Saunders;
1141-1208

Dubey RK, Tofovic SP, Jackson EK 2004 Cardiovascular pharmacology
of estradiol metabolites. J Pharmacol Exp Ther 308:403-409.

Hillier SG, Whitelaw PF, Smyth CD 1994 Follicular oestrogen synthesis:
the 'two-cell, two-gonadotrophin' model revisited. Mol Cell Endocrinol
100251-54

Erickson GF 1978 Normal ovarian function. Clin Obstet Gynecol 21 :31-52

Dotsch J, Dorr HG, Wildt L 2001 Exposure to endogenous estrogens
during lifetime. In: Hutzinger Oe, Metzler M eds. Endocrine disruptors part
I. New York: Springer-Verlag Berlin Heidelberg New York; 83-98

Lenton EA 1988 Pituitary and ovarian hormones in implantation and early
pregnancy. In: Chapman M, Grudzinskas G, Chard T eds. Implantation.
Heidelberg: Springer; 17

53

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

Baird DT, Fraser IS 1974 Blood production and ovarian secretion rates Of
estradiol-17 beta and estrone in women throughout the menstrual cycle. J
Clin Endocrinol Metab 38: 1 009—1 01 7

Siiteri PK, MacDonald PC 1966 Placental estrogen biosynthesis during
human pregnancy. J Clin Endocrinol Metab 26:751-761

MacDonald PC, Siiter PK 1965 Origin of estrogen in women pregnant
withan anencephalic fetus. J Clin Invest 44:465-474

Dotsch J, Dorr HG, Wildt L 2001 Exposure to endogenous estrogens
during lifetime. In: Metzler M ed. Endocrine disruptors part I. New York:
Springer; 81-99

Brodie AM 1979 Recent advances in studies on estrogen biosynthesis. J
Endocrinol Invest 2:445-460

Grow DR 2002 Metabolism of endogenous and exogenous reproductive
hormones. Obstet Gynecol Clin North Am 29:425-436

Simpson ER, Zhao Y, Agarwal VR, Michael MD, Bulun SE,
Hinshelwood MM, Graham-Lorence S, Sun T, Fisher CR, Qin K,
Mendelson CR 1997 Aromatase expression in health and disease.
Recent Prog Horm Res 52:185-213.

Simpson ER 2004 Aromatase: biologic relevance of tissue-specific
expression. Semin Reprod Med 22:11-23.

Jakob F, Homann D, Seufert J, Schneider D, Kohrle J 1995 Expression
and regulation of aromatase cytochrome P450 in THP 1 human myeloid
leukaemia cells. Mol Cell Endocrinol 110:27-33

Naftolin F, Ryan KJ, Davies IJ, Petro Z, Kuhn M 1975 The formation
and metabolism of estrogens in brain tissues. Adv Biosci 15:105-121

Simpson ER 2003 Sources of estrogen and their importance. J Steroid
Biochem Mol Biol 86:225-230

54

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

Simpson ER 2003 Sources of estrogen and their importance. J Steroid
Biochem Mol Biol 862225-230

Miller WR 1991 Aromatase activity in breast tissue. J Steroid Biochem
Mol Biol 39:783—790

Matsumine H, Hirato K, Yanaihara T, Tamada T, Yoshida M 1986
Aromatization by skeletal muscle. J Clin Endocrinol Metab 632717-720

Miller WR 1991 Aromatase activity in breast tissue. J Steroid Biochem
Mol Biol 392783-790

Schlinger BA, Arnold AP 1991 Brain is the major site of estrogen
synthesis in a male songbird. Proc Natl Acad Sci 88:4191-4194

Patchev AV, Gotz F, Rohde W 2004 Differential role of estrogen receptor
isoforrns in sex-specific brain organization. The FASEB Journal Express
Article

Kula K, Walczak-Jedrzejowska R, SIowikowska-Hilczer J, Wranicz JK,
Kula P, Oszukowska E, Marchlewska K 2005 Important functions of
estrogens in mew-breakthrough in contemporary medicine. Przegl Lek
622908-915

Payne AH, Kelch RP, Muslch SS, Halpem ME 1976 lntratesticular site
of aromatization in the human. J Clin Endocrinol Metab 42:1081-1087

Carreau S 1996 Paracrine control of human Leydig cell and Sertoli cell
functions. Folia Histochem Cytobiol 34:111-119

Pomerantz DK 1979 Effects of in vivo gonadotropin treatment on
estrogen levels in the testis Of the immature rat. Biol Reprod 2121247-1255

Hess RA, Bunick D, Lee KH, Bahr J, Taylor JA, Korach KS, Lubahn
DB 1997 A role for oestrogens in the male reproductive system. Nature
390:509-512

55

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

Hess RA 2003 Estrogen in the adult male reproductive tract: A review.
Reprod Biol Endocrinol 1252.

Pepe GJ, Albrecht ED 1995 Actions of placental and fetal adrenal steroid
hormones in primate pregnancy. Endocr Rev 162608-648

Janulis L, Bahr JM, Hess RA, Janssen S, Osawa Y, Bunick D 1998 Rat
testicular germ cells and epididymal sperm contain active P450
aromatase. J Androl 19:65-71.

Lambard S, Carreau S 2005 Aromatase and oestrogens in human male
germ cells. Int J Androl 282254-259

Weniger JP 1993 Estrogen production by fetal rat gonads. J Steroid
Biochem Mol Biol 44:459-462

Delbes G, Levacher C, Habert R 2006 Estrogen effects on fetal and
neonatal testicular development. Reproduction 132:527-538

Gennari L, Nuti R, Bilezikian JP 2004 Aromatase activity and bone
homeostasis in men. J Clin Endocrinol Metab 89:5898-5907

Levine AC, Kirschenbaum A, Gabrilove JL 1997 The role of sex
steroids in the pathogenesis and maintenance of benign prostatiC
hyperplasia. Mt Sinai J Med 64220-25

Dorrington JH, Fritz IB, Armstrong DT 1978 Control of testicular
estrogen synthesis. Biol Reprod 18:55-64

Free MJ, Jaffe RA 1979 Collection of rete testis fluid from rats without
previous efferent duct ligation. Biol Reprod 20:269-278

Brewster ME, Anderson WR, Pop E 1997 Effect of sustained estradiol
release in the intact male rat: correlation of estradiol serum levels with
actions on body weight, serum testosterone, and peripheral androgen-
dependent tissues. Physiol Behav 61 :225-229

56

55.

56.

57.

58.

59.

60.

61.

62.

63.

Nagata C, Takatsuka N, Shimizu H, Hayashi H, Akamatsu T, Murase K
2001 Effect of soymilk consumption on serum estrogen and androgen
concentrations in Japanese men. Cancer Epidemiol Biomarkers Prev
10:179-184

Weinstein RL, Kelch RP, Jenner MR, Kaplan SL, Grumbach MM 1974
Secretion of unconjugated androgens and estrogens by the normal and
abnormal human testis before and after human Chorionic gonadotropin. J
Clin Invest 53:1-6

Akingbemi BT 2005 Estrogen regulation of testicular function. Reprod
Biol Endocrinol 3:51

Klinge CM 2000 Estrogen receptor interaction with co-activators and CO-
repressors. Steroids 652227-251

Pettersson K, Grandien K, Kuiper GG, Gustafsson JA 1997 Mouse
estrogen receptor beta forms estrogen response element-binding
heterodimers with estrogen receptor alpha. Mol Endocrinol 1121486-1496

Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA
1996 Cloning of a novel estrogen receptor expressed in rat prostate and
ovary. Proc Natl Acad Sci 93:5925-2930

Kurebayashi J, Otsuki T, Kunisue H, Tanaka K, Yamamoto S, Sonoo
H 2000 Expression levels of estrogen receptor-alpha, estrogen receptor-
beta, coactivators, and corepressors in breast cancer. Clin Cancer Res
6:512-518

Glaros S, Atanaskova N, Zhao C, Skafar DF, Reddy KB 2006 Activation
Function -1 domain Of estrogen receptor regulates the agonistic and
antagonistic actions of Tamoxifen. MOI Endocrinolepub ahead of print

Kobayashi Y, Kitamoto T, Masuhiro Y, Watanabe M, Kase T, Metzger
D, Yanagisawa J, Kato S 2000 p300 mediates functional synergism
between AF-1 and AF-2 of estrogen receptor alpha and beta by interacting
directly with the N-terminal A/B domains. J Biol Chem 275:15645-15651

57

64.

65.

66.

67.

68.

69.

70.

71.

72.

73.

Osborne CK, Zhao H, Fuqua SA 2000 Selective estrogen receptor
modulators: structure, function, and Clinical use. J Clin Oncol 1823172-
3186

Enmark E, Gustafsson JA 1999 Oestrogen receptors - an overview. J
lntem Med 246:133-138

Pettersson K, Gustafsson JA 2001 Role of estrogen receptor beta in
estrogen action. Annu Rev Physiol 63:165—192

Lubahn DB, Moyer JS, Golding TS, Couse JF, Korach KS, Smithies O
1993 Alteration of reproductive function but not prenatal sexual
development after insertional disruption of the mouse estrogen receptor
gene. Proc Natl Acad Sci U S A 90:11162-11166

Krege JH, Hodgin JB, Couse JF, Enmark E, Warner M, Mahler JF, Sar
M, Korach KS, Gustafsson JA, Smithies O 1998 Generation and
reproductive phenotypes Of mice lacking estrogen receptor beta. Proc Natl
Acad Sci U S A 95:15677-15682

Dupont S, Krust A, Gansmuller A, Dierich A, Chambon P, Mark M
2000 Effect of single and compound knockouts of estrogen receptors
alpha (ERaIpha) and beta (ERbeta) on mouse reproductive phenotypes.
Development 127:4277—4291

Pelletier G 2000 Localization of androgen and estrogen receptors in rat
and primate tissues. Histol Histopathol 15:1261-1270

Fitzpatrick SL, Funkhouser JM, Sindoni DM, Stevis PE, Deecher DC,
Bapat AR, Merchenthaler I, Frail DE 1999 Expression of estrogen
receptor-beta protein in rodent ovary. Endocrinology 140:2581-2591

Hall J, McDonnell DP 1999 The estrogen receptor beta-isoforrn (ERbeta)
of the human estrogen receptor modulates ERalpha transcriptional activity
and is a key regulator of the cellular response tO estrogens and
antiestrogens. Endocrinology 140:5566-5578.

Beato M, Klug J 2000 Steroid hormone receptors: an update. Hum
Reprod Update 6:225-236

58

74.

75.

76.

77.

78.

79.

80.

81.

82.

83.

Suen CS, Berrodin TJ, Mastroeni R, Cheskis RJ, Lyttle R, Frail DE
1998 A transcriptional coactivator, steroid receptor coactivator-3,
selectively augments steroid receptor transcriptional activity. The Journal
Of Biological Chemistry 273:27645—27653

Smith CL 1998 Cross-talk between peptide growth factor and estrogen
receptor signaling pathways. Biol Reprod 58:627—632

Hall JM, Couse JF, Korach KS 2001 The multifaceted mechanisms of
estradiol and estrogen receptor signaling. J Biol Chem 276:36869-36872

Ohmichi M, Tasaka K, Kurachi H, Murata Y 2005 Molecular mechanism
of action of selective estrogen receptor modulator in target tissues. Endocr
J 52:161-167

Kato S 2001 Estrogen receptor-mediated cross-talk with growth factor
signaling pathways. Breast Cancer 8:3—9

Tremblay A, Tremblay GB, Labrie F, Giguere V 1999 Ligand-
independent recruitment of SRC—1 to estrogen receptor beta through
phosphorylation Of activation function AF-1. Mol Cell 3:51 3-519

Dutertre M, Smith CL 2003 Ligand-independent interactions Of
p160/steroid receptor coactivators and CREB-binding protein (CBP) with
estrogen receptor-alpha: regulation by phosphorylation sites in the NB
region depends on other receptor domains. Mol Endocrinol 17:1296-1314

Paech K, Webb P, Kuiper GG, Nilsson S, Gustafsson J, Kushner PJ,
Scanlan TS 1997 Differential ligand activation of estrogen receptors
ERalpha and ERbeta at AP1 sites. Science 277:1508-1510

Cheung E, Acevedo ML, Cole PA, Kraus WL 2005 Altered
pharmacology and distinct coactivator usage for estrogen receptor-
dependent transcription through activating protein-1. Proc Natl Acad Sci U
S A 102:559-564

Matthews J, Wihlen B, Tujague M, Wan J, Strom A, Gustafsson JA
2005 Estrogen receptor (ER) beta modulates ERalpha-mediated
transcriptional activation by altering the recruitment of C-Fos and C-Jun to
estrogen-responsive promoters. Mol Endocrinol 20:534-543

59

84.

85.

86.

87.

88.

89.

90.

91.

92.

93.

Krebs CJ, Jarvis ED, Pfaff DW 1999 The 70-kDa heat shock cognate
protein (Hsc73) gene is enhanced by ovarian hormones in the
ventromedial hypothalamus. Proc Natl Acad Sci U S A 96:1686—1691

Voss MR, Stallone JN, Li M, Comelussen RN, Knuefermann P,
Knowlton AA 2003 Gender differences in the expression of heat shock
proteins: the effect of estrogen. Am J Physiol Heart Circ Physiol
285:H687-692

Nelson J, Clarke R, Murphy RF 1986 The unoccupied estrogen receptor:
some comments on localization. Steroids 482121-124

Falkenstein E, Wehling M 2000 Nongenomically initiated steroid actions.
Eur J Clin Invest 30:51-54

Falkenstein E, Tillmann HC, Christ M, Feuring M, Wehling M 2000
Multiple actions of steroid hormones—a focus on rapid, nongenomic
effects. Pharmacol Rev 522513-556

Falkenstein E, Norman AW, Wehling M 2000 Mannheim Classification of
nongenomically initiated (rapid) steroid action(s). J Clin Endocrinol Metab
85:2072-2075

Revelli A, Massobrio M, Tesarik J 1998 Nongenomic actions of steroid
hormones in reproductive tissues. Endocr Rev 1923-17

Luconi M, Francavilla F, Porazzi I, Macerola B, Forti G, Baldi E 2004
Human spermatozoa as a model for studying membrane receptors
mediating rapid nongenomic effects of progesterone and estrogens.
Steroids 692553-559

Moats RKn, Ramirez VD 1998 Rapid uptake and binding of estradiol-
17beta-6-(O-carboxymethyl)oxime:125l-labeled BSA by female rat liver.
Biol Reprod 582531-538

Boulware MI, Mermelstein PG 2005 The influence of estradiol on
nervous system function. Drug News Perspect 182631 -637

60

94.

95.

96.

97.

98.

99.

100.

101.

102.

Pepe GJ, Billiar RB, Leavltt MG, Zachos NC, Gustafsson JA, Albrecht
ED 2002 Expression of estrogen receptors alpha and beta in the baboon
fetal ovary. Biol Reprod 66:1054-1060

Vaskivuo TE, Maentausta M, Torn S, Oduwole O, Lonnberg A, Herva
R, Isomaa V, Tapanainen JS 2005 Estrogen receptors and estrogen-
metabolizing enzymes in human ovaries during fetal development. J Clin
Endocrinol Metab 90:3752-3756

Brandenberger AW, Tee MK, Jaffe RB 1998 Estrogen receptor alpha
(ER-alpha) and beta (ER-beta) mRNAs in normal ovary, ovarian serous
cystadenocarcinoma and ovarian cancer cell lines: down-regulation of ER-
beta in neoplastic tissues. J Clin Endocrinol Metab 83:1025-1028

Okada A, Ohta Y, Buchanan DL, Sato T, Inoue S, Hiroi H, Muramatsu
M, Iguchi T 2002 Changes in ontogenetic expression of estrogen receptor
alpha and not of estrogen receptor beta in the female rat reproductive
tract. J Mol Endocrinol 28:87-97

Lubahn DB, Moyer JS, Golding TS, Couse JF, Korach KS, Smithies O
1993 Alteration of reproductive function but not prenatal sexual
development after insertional disruption of the mouse estrogen receptor
gene. PrOC Natl Acad Sci USA 90:11162-11166

Couse JF, Hewitt SC, Bunch DO, Sar M, Walker VR, Davis BJ, Korach
KS 1999 Postnatal sex reversal of the ovaries in mice lacking estrogen
receptors alpha and beta. Science 286:2328-2331

Britt KL, Findlay JK 2002 Estrogen actions in the ovary revisited. J
Endocrinol 175:269-276

Couse JF, Korach KS 1999 Estrogen receptor null mice: what have we
Ieamed and where will they lead us? Endocr Rev 20:358—41 7

Byskov AG 1994 Embryology of mammalian gonads and ducts. In: Knobil
E, Neill JD eds. The physiology of reproduction. New York: Raven Press;
487-540

61

103.

104.

105.

106.

107.

108.

109.

110.

111.

112.

Gosden RG, Bownes M 1995 Cellular and molecular aspects of oocyte
development. In: Grudzinskas JG, YoviCh JL eds. The oocyte. Cambridge:
Cambridge reviews in human reproduction; 25-53

Couse JF, Yates MM, Deroo BJ, Korach KS 2005 Estrogen receptor-
beta is critical to granulosa cell differentiation and the ovulatory response
to gonadotropins. Endocrinology 146:3247-3262

Drummond AE 2006 The role Of steroids in follicular growth. Reprod Biol
Endocrinol 4216

Huynh K, Jones G, Thouas G, Britt KL, Simpson ER, Jones ME 2004
Estrogen is not directly required for oocyte developmental competence.
Biol Reprod 70:1263—1269.

de Kretser DM, Kerr JB 1988 The cytology of the testis. In: Knobil E NJ
ed. The Physiology of Reproduction. New York: Raven Press Ltd; 837-932

Byskov AG 1986 Differentiation of mammalian embryonic gonad. Physiol
Rev 66:71-117

Winter JSD, Faiman C, Reyes Fl 1977 Sex steroid production by the
human fetus: its role in morphogenesis and control by gonadotropins. In:
Blandau RJ, Bergsma D eds. Morphogenesis and Malformation of the
Genital System. New York, NY: AR Liss; 41 -58

Sar M, Welsch F 2000 Oestrogen receptor alpha and beta in rat prostate
and epididymis. Andrologia 32:295-301

Fisher JS, Millar MR, Majdic G, Saunders PT, Fraser HM, Sharpe RM
1997 lmmunolocalisation of oestrogen receptor-alpha within the testis and
excurrent ducts of the rat and marmoset monkey from perinatal life to
adulthood. J Endocrinol 153:485-495

Yamashita S 2004 Localization of estrogen and androgen receptors in
male reproductive tissues of mice and rats. Anat Rec A Discov Mol Cell
Evol Biol 279:768-778

62

113.

114.

115.

116.

117.

118.

119.

120.

121.

122.

Hughes lA, Coleman N, Faisal Ahmed S, Ng KL, Cheng A, Lim HN,
Hawkins JR 1999 Sexual dimorphism in the neonatal gonad. Acta
Paediatr Suppl 88:23-30

Gustafson ML, Donahoe PK 1994 Male sex determination: current
concepts of male sexual differentiation. Annu Rev Med 45:505—524

Greco TL, Furlow JD, Duello TM, Gorski J 1992 lmmunodetection of
estrogen receptors in fetal and neonatal male mouse reproductive tracts.
Endocrinology 1 30:421-429

Carreau S, Lambard S, Delalande C, Denis-Galeraud l, Bilinska B,
Bourguiba S 2003 Aromatase expression and role of estrogens in male
gonad : a review. Reprod Biol Endocrinol 11:35.

O'Donnell L, Robertson KM, Jones ME, Simpson ER 2001 Estrogen
and sperrnatogenesis. Endocr Rev 222289-318

Nielsen M, Bjomsdottir S, Hoyer PE, Byskov AG 2000 Ontogeny of
oestrogen receptor alpha in gonads and sex ducts of fetal and newborn
mice. J Reprod Fertil 1 18:195-204

Majdic G, Millar MR, Saunders PT 1995 lmmunolocalisation of androgen
receptor to interstitial cells in fetal rat testes and to mesenchymal and
epithelial cells of associated ducts. J Endocrinol 147:285-293

Cooke PS, Young P, Hess RA, Cunha GR 1991 Estrogen receptor
expression in developing epididymis, efferent ductules, and other male
reproductive organs. Endocrinology 128:2874-2879

Shughrue PJ, Lane MV, Scrimo PJ, Merchenthaler I 1998 Comparative
distribution of estrogen receptor-alpha (ER-alpha) and beta (ER-beta)
mRNA in the rat pituitary, gonad, and reproductive tract. Steroids 63:498-
504

Pelletier G, Labrie C, Labrie F 2000 Localization of oestrogen receptor
alpha, oestrogen receptor beta and androgen receptors in the rat
reproductive organs. J Endocrinol 165:359-370

63

123.

124.

125.

126.

127.

128.

129.

130.

131.

132.

van Pelt AM, do Rooij DG, van der Burg B, van der Saag PT,
Gustafsson JA, Kuiper GG 1999 Ontogeny of estrogen receptor-beta
expression in rat testis. Endocrinology 140:478-483

Weniger JP, Zeis A 1987 Oestrogen synthesis by the foetal rat testis in
organ culture. J Steroid Biochem 28:307-310

Lambard S, Silandre D, Delalande C, Denis-Galeraud I, Bourguiba S,
Carreau S 2005 Aromatase in testis: expression and role in male
reproduction. J Steroid Biochem Mol Biol 95:63-69

Janulis L, Bahr JM, Hess RA, Bunick D 1996 P450 aromatase
messenger ribonucleic acid expression in male rat germ cells: detection by
reverse transcription-polymerase chain reaction amplification. J Androl
17:651-658.

Eddy EM, Washbum TF, Bunch DO, Goulding EH, Gladen BC,
Lubahn DB, Korach KS 1996 Targeted disruption of the estrogen
receptor gene in male mice causes alteration of sperrnatogenesis and
infertility. Endocrinology 137:4796-4805

Robertson KM, O'Donnell L, Jones ME, Meachem SJ, Boon WC,
Fisher CR, Graves KH, McLachIan RI, Simpson ER 1999 Impairment of
sperrnatogenesis in mice lacking a functional aromatase (cyp 19) gene.
Proc Natl Acad Sci U S A 96:7986-7991.

Derelanko MJ 2000 Toxicologists's pocket handbook. In. Boca Raton,
Florida: CRC Press; 85-122

Jones EE, DeChemey AH 2003 The male reproductive system. In: Boron
WF, Boulpaep EL eds. Medical physiology. Philadelphia: Saunders; 1122-
1 140

Fisher CR, Graves KH, Parlow AF, Simpson ER 1998 Characterization
of mice deficient in aromatase (ArKO) because of targeted disruption of
the cyp19 gene. Proc Natl Acad Sci U S A 95:6965-6970

Robertson KM, O'Donnell L, Jones ME, Meachem SJ, Boon WC,
Fisher CR, Graves KH, McLachIan RI, Simpson ER 1999 Impairment of

64

133.

134.

135.

136.

137.

138.

139.

140.

141.

sperrnatogenesis in mice lacking a functional aromatase (cyp 19) gene.
Proc Natl Acad Sci U S A 96:7986-7991

Nitta H, Bunick D, Hess RA, Janulis L, Newton SC, Millette CF, Osawa
Y, Shizuta Y, Toda K, Bahr JM 1993 Germ cells of the mouse testis
express P450 aromatase. Endocrinology 132:1396-1401

Sharpe RM, Skakkebaek NE 1993 Are oestrogens involved in falling
sperm counts and disorders of the male reproductive tract? Lancet
341 :1392-1395

Macomber D, Sanders M 1929 The spermatozoa count: Its value in the
diagnosis, prognosis, and treatment of sterility. N Engl J Med 200:981-984

Eskenazi B, Wyrobek AJ, Sloter E, Kidd SA, Moore L, Young S,
Moore D 2003 The association of age and semen quality in healthy men.
Hum Reprod 18:447-454

Fisch H, Goluboff ET, Olson JH, Feldshuh J, Broder SJ, Barad DH
1996 Semen analyses in 1,283 men from the United States over a 25-year
period: no decline in quality. Fertil Steril 65:1009-1014

Giwercman A, Carlsen E, Keiding N, Skakkebaek NE 1993 Evidence
for increasing incidence of abnormalities of the human testis: a review.
Environ Health Perspect 101 :65-71

Toppari J, Skakkebaek NE 1998 Sexual differentiation and
environmental endocrine disrupters. Baillieres Clin Endocrinol Metab
12:143-156

Kula K 1988 Induction of precocious maturation of sperrnatogenesis in
infant rats by human menopausal gonadotropin and inhibition by
simultaneous administration of gonadotropins and testosterone.
Endocrinology 122:34-39

Li H, Papadopoulos V, Vidic B, Dym M, Culty M 1997 Regulation of rat
testis gonocyte proliferation by platelet-derived growth factor and estradiol:
identification of signaling mechanisms involved. Endocrinology 138:1289-
1298

65

142.

143.

144.

145.

146.

147.

148.

149.

150.

151.

Mullaney BP, Skinner MK 1991 Growth factors as mediators of testicular
cell-cell interactions. Baillieres Clin Endocrinol Metab 52771-790

Fijak M, Meinhardt A 2006 The testis in immune privilege. Immunol Rev
213:66-81

Jefferson WN, Couse JF, Banks EP, Korach KS, Newbold RR 2000
Expression of estrogen receptor ?is developmentally regulated in
reproductive tissues of male and female mice. Biol Reprod 622310-317

Saunders PT, Fisher JS, Sharpe RM, Millar MR 1998 Expression of
oestrogen receptor beta (ER beta) occurs in multiple cell types, including
some germ cells, in the rat testis. J Endocrinol 156:R13-17

Pentikainen V, Erkkila K, Suomalainen L, Parvinen M, Dunkel L 2000
Estradiol acts as a germ cell survival factor in the human testis in vitro. J
Clin Endocrinol Metab 85:2057-2067

Shetty G, Krishnamurthy H, Krishnamurthy HN, Bhatnagar S,
Moudgal RN 1997 Effect of estrogen deprivation on the reproductive
physiology of male and female primates. J Steroid Biochem Mol Biol
61:157-166

Tsutsumi l, Fugimori K, Nakamura RM, Mather JP, Ono T, diZerega
GS 1987 Disruption of seminiferous epithelial function in the rat by ovarian
protein. Biol Reprod 362451-461 '

Zhou Q, Clarke L, Nie R, Carnes K, Lai LW, Lien YH, Verkman A,
Lubahn D, Fisher JS, Katzenellenbogen BS, and Hess RA. 2001
Estrogen action and male fertility: roles of the sodium/hydrogen
exchanger-3 and fluid reabsorption in reproductive tract function. Proc Natl
Acad Sci U S A 98:14132-14137.

Hess RA, Zhou Q, Nie R, Oliveira C, Cho H, Nakaia M, Carnes K 2001
Estrogens and epididymal function. Reprod Fertil Dev 13:273-283.

Rago V, Bilinska B, Palma A, Ando S, Carpino A 2003 Evidence of
aromatase localization in cytoplasmic droplet of human immature
ejaculated spermatozoa. Folia Histochem Cytobiol 41:23-27

66

152.

153.

154.

155.

156.

157.

158.

159.

160.

161.

Aquila S, Sisci D, Gentile M, Middea E, Siciliano L, Ando S 2002
Human ejaculated spermatozoa contain active P450 aromatase. J Clin
Endocrinol Metab 87:3385-3390

Sharpe RM, Atanassova N, McKinneII C, Parte P, Turner KJ, Fisher
JS, Kerr JB, Groome NP, Macpherson S, Millar MR, Saunders PT
1998 Abnormalities in functional development of the Sertoli cells in rats
treated neonatally with diethylstilbestrol: a possible role for estrogens in
Sertoli cell development. Biol Reprod 59:1084-1094.

Kelly MJ, Levin ER 2001 Rapid actions of plasma membrane estrogen
receptors. Trends Endocrinol Metab 122152-156

Luconi M, Muratori M, Forti G, Baldi E 1999 Identification and
characterization of a novel functional estrogen receptor on human sperm
membrane that interferes with progesterone effects. J Clin Endocrinol
Metab 84:1670-1678

Fraser LR, Beyret E, Milligan SR, Adeoya-Osiguwa SA 2006 Effects of
estrogenic xenobiotics on human and mouse spermatozoa. Hum Reprod
21:1184—1193

Furuya S, Endo Y, Oba M, Nozawa S, Suzuki S 1992 Effects of
modulators of protein kinases and phosphatases on mouse sperm
capacitation. J Assist Reprod Genet 9:391-399

Adeoya-Osiguwa SA, Markoulaki S, Pocock V, Milligan SR, Fraser LR
2003 17beta-Estradiol and environmental estrogens significantly affect
mammalian sperm function. Hum Reprod 182100-107

Emmen JM, Korach KS 2003 Estrogen receptor knockout mice:
phenotypes in the female reproductive tract. Gynecol Endocrinol 17:169-
176

Arredondo F, Noble LS 2006 Endocrinology of recurrent pregnancy loss.
Semin Reprod Med 24:33-39

Babischkin JS, Pepe GJ, Albrecht ED 1989 Regulation of progesterone
biosynthesis by estrogen during baboon pregnancy: placental
mitochondrial cholesterol side-chain cleavage activity in antiestrogen

67

162.

163.

164.

.165.

166.

167.

168.

169.

170.

(ethamoxytriphetol, MER—25)—treated baboons. Endocrinology 124:1638-
1645

Babischkin JS, Pepe GJ, Albrecht ED 1997 Estrogen regulation of
placental P-450 cholesterol side-chain cleavage enzyme messenger
ribonucleic acid levels and activity during baboon pregnancy.
Endocrinology 138:452-459

Baggia S, Albrecht ED, Pepe GJ 1990 Regulation of 11 beta-
hydroxysteroid dehydrogenase activity in the baboon placenta by
estrogen. Endocrinology 126:2742-2748

Geisert RD, Ross JW, Ashworth MD, White FJ, Johnson GA, DeSiIva
U 2006 Maternal recognition of pregnancy signal or endocrine disruptor.
the two faces of oestrogen during establishment of pregnancy in the pig.
Reprod Suppl 62:131-145

James WH 1996 Evidence that mammalian sex ratios at birth are partially
controlled by parental hormone levels at the time of conception. J Theor
Biol 1802271 -286

Mocarelli P, Gerthoux PM, Ferrari E, Patterson DGJ, Kieszak SM,
Brambilla P, Vincoli N, Signorini S, Tramacere P, Carreri V, Sampson
EJ, Turner WE, Needham LL 2000 Paternal concentrations of dioxin and
sex ratio of offspring. Lancet 355:1858-1863

Pergament E, Todydemir PB, Fiddler M 2002 Sex ratio: a biological
perspective of 'Sex and the City'. Reprod Biomed OnIine 5:43-46

James WH 1976 Timing of fertilization and sex ratio of offspring—a review.
Ann Hum Biol 32549-556

Davis DL, Gottlieb MB, Stampnitzky JR 1998 Reduced ratio of male to
female births in several industrial countries: a sentinel health indicator?
JAMA 279:1018-1023

James WH 1980 Time of fertilisation and sex of infants. Lancet 121124-
1126

68

171.

172.

173.

174.

175.

176.

. 177.

178.

179.

180.

181.

James WH 1985 Sex ratio, dominance status and maternal hormone
levels at the time of conception. J Theor Biol 1 142505-510

James WH 1986 Hormonal control of sex ratio. J Theor Biol 118:427-441

James WH 2006 Possible constraints on adaptive variation in sex ratio at
birth in humans and other primates. J Theor Biol 238:383-394

James WH 1989 Parental hormone levels and mammalian sex ratios at
birth. J Theor Biol 139259-67

Ryan JJ, Amirova Z, Carrier G 2002 Sex ratios of children of Russian
pesticide producers exposed to dioxin. Environ Health Perspect 1102A699-
701

Martuzzi M, Di Tanno ND, Bertollini R 2001 Declining trends of male
proportion at birth in Europe. Arch Environ Health 56:358-364

Vartiainen T, Kartovaara L, Tuomisto J 1999 Environmental chemicals
and changes in sex ratio: analysis over 250 years in finland. Environ
Health Perspect 107:813-815

Figa-Talamanca l, Tarquini M, Lauria L 2003 Is it possible to use sex
ratio at birth as indicator of the presence of endocrine disrupters in
environmental pollution?]. G Ital Med Lav Ergon 25:52-53

Rogan WJ, Gladen BC, Guo YL, Hsu CC 1999 Sex ratio after exposure
to dioxin-like chemicals in Taiwan. Lancet 353:206—207

Figa-Talamanca I, Carbone P, Lauria L, Spinelli A, Ulizzi L 2003
Environmental factors and the proportion of males at birth in Italy. Arch
Environ Health 58:1 19-124

Jarrell JF, Gocmen A, Akyol D, Brant R 2002 Hexachlorobenzene
exposure and the proportion of male births in Turkey 1935-1990. Reprod
Toxicol 16:65-70

69

182.

183.

184.

185.

186.

187.

188.

189.

190.

191.

van Birgelen AP 1998 Hexachlorobenzene as a possible major
contributor to the dioxin activity of human milk. Environ Health Perspect
106:683-688

Potashnik G, Porath A 1995 Dibromochloropropane (DBCP): a 17-year
reassessment of testicular function and reproductive performance. J
Occup Environ Med 37:1287-1292

Slutsky M, Levin JL, Levy BS 1999 Azoospennia and oligosperrnia
among a large cohort of DBCP applicators in 12 countries. Int J Occup
Environ Health 5:116-122

Thrupp LA 1991 Sterilization of workers from pesticide exposure: the
causes and consequences of DBCP-induced damage in Costa Rica and
beyond. Int J Health Serv 212731-757

Wesseling C, Ahlbom A, Antich D, Rodriguez AC, Castro R 1996
Cancer in banana plantation workers in Costa Rica. Int J Epidemiol
25:1125-1131

Meistrich ML, Wilson G, Shuttlesworth GA, Porter KL 2003
Dibromochloropropane inhibits sperrnatogonial development in rats.
Reprod Toxicol 17:263—271

Goldsmith JR 1997 Dibromochloropropane: epidemiological findings and
current questions. Ann N Y Acad Sci 837:300-306

Sod-Moriah UA, Sror U, Shemi D, Potashnik G, Chayoth R, Shaked I,
Kaplanski J 1988 Long term effects of dibromochloropropane (DBCP) on
male rats' reproductive system. Andrologia 20:60-66

Bredbacka K, Bredbacka P 1996 Glucose controls sex-related growth
rate differences of bovine embryos produced in vitro. J Reprod Fertil
1062169-172

Williams AC, Ford WC 2001 The role of glucose in supporting motility
and capacitation in human spermatozoa. J Androl 22:680-695

70

192.

193.

194.

195.

196.

197.

198.

199.

200.

201.

Machado AF, Zimmerman EF, Hovland DNJ, Weiss R, Collins MD
2001 Diabetic embryopathy in C57BL/6Jmice. Altered fetal sex ratio and
impact of the splotch allele. Diabetes 50:1193-1199

Cameron E2 2004 Facultative adjustment of mammalian sex ratios in
support of the Trivers-Willard hypothesis: evidence for a mechanism. Proc
Biol Sci 271 :1 549

Agung B, Otoi T, Wongsrikeao P, Taniguchi M, Shimizu R, Watari H,
Nagai T 2006 Effect of maturation culture period of oocytes on the sex
ratio of in vitro fertilized bovine embryos. J Reprod Dev 522123-127

Cui KH 1997 Size differences between human X and Y spermatozoa and
prefertilization diagnosis. Mol Hum Reprod 3:61-67

Broer KH, Winkhaus I, Sombroek H, Kaiser R 1976 Frequency of Y-
chromatin bearing spermatozoa in intracervical and intrauterine postcoital
tests. Int J Fertil 212181-185

Hotchkiss AK, Vandenbergh JG 2005 The anogenital distance index of
mice (Mus musculus domesticus): an analysis. Contemp Top Lab Anim
Sci 44246-48

Salazar-Martinez E, Romano-Riquer P, Yanez-Marquez E, Longnecker
MP, Hemandez-Avila M 2004 Anogenital distance in human male and
female newborns: a descriptive, cross-sectional study. Environ Health 328

Vandenbergh JG, Huggett CL 1995 The anogenital distance index, a
predictor of the intrauterine position effects on reproduction in female
house mice. Lab Anim Sci 45:567-573

Kerin TK, Vogler GP, Blizard DA, Stout JT, McCleam GE,
Vandenbergh DJ 2003 Anogenital distance measured at weaning is
correlated with measures of blood chemistry and behaviors in 450-day-old
female mice. Physiol Behav 78:697-702

Marois G 1968 Action of progesterone, testosterone and estradiol on the
anogenital distance and somatic sexual differentiation in rats. Biol Med
(Paris) 57:44-90

71

202.

203.

204.

205.

206.

207.

208.

209.

210.

Swan SH, Main KM, Liu F, Stewart SL, Kruse RL, Calafat AM, Mao CS,
Redmon JB, Temand CL, Sullivan S, Teague JL 2005 Decrease in
anogenital distance among male infants with prenatal phthalate exposure.
Environ Health Perspect 113:1056-1061

Kelce WR, Earl Gray L, Jr. 1999 Environmental antiandrogens as
endocrine disruptors. In: Naaz A ed. Endocrine dismptors: effects on male
and female reproductive systems. Florida: CRC Press; 247-277

Habert R, Picon R 1984 Testosterone, dihydrotestosterone and estradiol-
17 beta levels in maternal and fetal plasma and in fetal testes in the rat. J
Steroid Biochem 21:193-198

Clark RL, Antonello JM, Grossman SJ, Wise LD, Anderson C, Bagdon
WJ, Prahalada S, MacDonald JS, Robertson RT 1990 External genitalia
abnormalities in male rats exposed in utero to finasteride, a 5 alpha-
reductase inhibitor. Teratology 42291-100

Evans BA, Griffiths K, Morton MS 1995 Inhibition of 5 alpha-reductase V
in genital skin fibroblasts and prostate tissue by dietary lignans and
isoflavonoids. J Endocrinol 147:295-302

Swan SH 2006 Prenatal phthalate exposure and anogenital distance in
male infants. Environ Health Perspect 1142A88-89

Gallavan RH, Jr., Holson JF, Stump DG, Knapp JF, Reynolds VL 1999
Interpreting the toxicologic significance of alterations in anogenital
distance: potential for confounding effects of progeny body weights.
Reproductive Toxicology 13:383-390.

Levy JR, Faber KA, Ayyash L, Hughes CL, Jr. 1995 The effect of
prenatal exposure to the phytoestrogen genistein on sexual differentiation
in rats. Proc Soc Exp Biol Med 208260-66

McIntyre BS, Barlow NJ, Foster PMD 2001 Androgen-mediated
development in male rat offspring exposed to flutamide in utero:
permanence and correlation of early postnatal changes in anogenital
distance and nipple retention with malformations in androgen-dependent
tissues. Toxicol Sci 62:236-249

72

211.

212.

213.

214.

215.

216.

217.

218.

219.

Crescioli C, Maggi M, Vannelli GB, Ferruzzi P, Granchi S, Mancina R,
Muratori M, Forti G, Serio M, Luconi M 2003 Expression of functional
estrogen receptors in human fetal male external genitalia. J Clin
Endocrinol Metab 88:1815-1824

Sohoni P, Sumpter JP 1998 Several environmental oestrogens are also
anti-androgens. J Endocrinol 158:327-339

Marsee K, Woodruff TJ, Axelrad DA, Calafat AM, Swan SH 2006
Estimated daily phthalate exposures in a population of mothers of male
infants exhibiting reduced anogenital distance. Environ Health Perspect
114:805-809

Borch J, Axelstad M, Vinggaard AM, Dalgaard M 2006 Diisobutyl
phthalate has comparable anti-androgenic effects to di-n-butyl phthalate in
fetal rat testis. Toxicol Lett 1 63:183-190

Blount BC, Silva MJ, Caudill SP, Needham LL, Pirkle JL, Sampson
EJ, Lucier GW, Jackson RJ, Brock JW 2000 Levels of seven urinary
phthalate metabolites in a human reference population. Environ Health
Perspect 1082 979- 982 ~

Collins TF, Sprando RL, Black TN, Olejnik N, Eppley RM, Alam HZ,
Rorie J, Ruggles DI 2006 Effects of zearalenone on in utero development
in rats. Food Chem Toxicol 44:1455-1465

' Kebayashi K, Miyagawa M, Wang RS, Sekiguchi S, Suda M, Honma T

2002 Effects of in utero and lactational exposure to bisphenol A on
somatic growth and anogenital distance in F1 rat offspring. Ind Health
402375-381

Fielden MR, Samy SM, Chou KC, Zacharewski TR 2003 Effect of
human dietary exposure levels of genistein during gestation and lactation
on Iong-terrn reproductive development and sperm quality in mice. Food
Chem Toxicol 41:447-454.

Bidlingmaier F, Butenandt O, Knorr D 1977 Plasma gonadotropins and
estrogens in girls with idiopathic precocious puberty. Pediatr Res 11:91—94

73

220.

221.

222.

223.

224.

225.

226.

227.

228.

229.

Tanner JM 1973 Trend towards earlier menarche in London, Olso,
Copenhagen, the Netherlands and Hungary. Nature 243295-96

Ruf KB 1973 How does the brain control the process of puberty? Z Neurol
204296-105

Ordog T, Knobil E 1995 Estradiol and the inhibition of hypothalamic
gonadotropin-releasing hormone pulse generator activity in the rhesus
monkey. Proc Natl Acad Sci U S A 92:5813-5816

O'Byrne KT, Chen MD, Nishihara M, Williams CL, Thalabard JC,
Hotchkiss J, Knobil E 1993 Ovarian control of gonadotropin horrnone-
releasing hormone pulse generator activity in the rhesus monkey: duration
of the associated hypothalamic signal. Neuroendocrinology 572588-592

MacGillivray MH, Morishima A, Conte F, Grumbach M, Smith EP 1998
Pediatric endocrinology update: an overview. The essential roles of
estrogens in pubertal growth, epiphyseal fusion and bone turnover:
lessons from mutations in the genes for aromatase and the estrogen
receptor. Horm Res; 49:2-8

Schoeters G, Den Hond E, Dhooge W, van Larebeke N, Leijs M 2008
Endocrine dismptors and abnormalities of pubertal development. Basic &
clinical pharmacology & toxicology 102:168-175

Oken E, Gillman MW 2003 Fetal origins of obesity. Obes Res 112496-506

Hedley AA, Ogden CL, Johnson CL, Carroll MD, Curtin LR, Flegal KM
2004 Prevalence of ovenrveight and obesity among US children,
adolescents, and adults, 1999-2002. JAMA 291 :2847-2850

Flegal KM, Carroll MD, Ogden CL, Johnson CL 2002 Prevalence and
trends in obesity among US adults, 1999-2000. JAMA 288:1723—1727

Heindel JJ 2003 Endocrine disruptors and the obesity epidemic. Toxicol
Sci 76:247-249

74

230.

231 .

232.

233.

234.

235.

236.

237.

238.

Schildkraut JM, Demark-Wahnefried W, DeVoto E, Hughes C, Laseter
JL, Newman B 1999 Environmental contaminants and body fat
distribution. Cancer Epidemiol Biomarkers Prev 8:179-183

Wolff MS, Anderson HA 1999 Environmental contaminants and body fat
distribution. Cancer Epidemiol Biomarkers Prev 82951-952

Jones ME, Thorbum AW, Britt KL, Hewitt KN, Wreford NG, Proietto J,
Oz OK, Leury BJ, Robertson KM, Yao S, Simpson ER 2000
Aromatase-deficient (ArKO) mice have a phenotype of increased
adiposity. Proc Natl Acad Sci U S A 97:12735—12740.

Jones ME, A.W. T, Britt KL, Hewitt KN, Misso ML, Wreford NG,
Proietto J, Oz OK, Leury B, Robertson KM, Yao S, Simpson ER 2001
Aromatase-deficient (ArKO) mice accumulate excess adipose tissue. J
Steroid Biochem Mol Biol 7923-9.

Naaz A, Zakroczymski M, Heine P, Taylor J, Saunders P, Lubahn D,
Cooke PS 2002 Effect of ovariectomy on adipose tissue of mice in the
absence of estrogen receptor alpha (ERalpha): a potential role for
estrogen receptor beta (ERbeta). Horm Metab Res 342758-763.

Heine PA, Taylor JA, lwamoto GA, Lubahn DB, Cooke PS 2000
Increased adipose tissue in male and female estrogen receptor-alpha
knockout mice. Proc Natl Acad Sci USA 97:12729-12734

Naaz A, Zakroczymski M, Heine P, Taylor J, Saunders P, Lubahn D,
Cooke PS 2002 Effect of ovariectomy on adipose tissue of mice in the
absence of estrogen receptor alpha (ERalpha): a potential role for
estrogen receptor beta (ERbeta). Horm Metab Res 34:758-763

Okura T, Koda M, Ando F, Niino N, Ohta S, Shimokata H 2003
Association of polymorphisms in the estrogen receptor alpha gene with
body fat distribution. Int J Obes Relat Metab Disord 27:1020-1027

Baillie-Hamilton PF 2002 Chemical toxins: a hypothesis to explain the
global obesity epidemic. J Altem Complement Med 8:185-192

75

239.

240.

241.

242.

243.

244.

245.

246.

247.

Sweeney T 2002 Is exposure to endocrine disrupting compounds during
fetal/post-natal development affecting the reproductive potential of farm
animals? Domest Anim Endocrinol 232203-209

Ankley GT, Jensen KM, Makynen EA, Kahl MD, Korte JJ, Homung
MW, Henry TR, Denny JS, Leino RL, Wilson VS, Cardon MC, Hartig
PC, Gray LE 2003 Effects of the androgenic growth promoter 17-beta-
trenbolone on fecundity and reproductive endocrinology of the fathead
minnow. Environ Toxicol Chem 22:1350—1360

Le Guevel R, Pakdel F 2001 Assessment of oestrogenic potency of
chemicals used as growth promoter by in-vitro methods. Hum Reprod
16:1030-1036

Barbini DA, Vanni F, Pelosi P, Generali T, Amendola G, Stefanelli P,
Girolimetti S, Di Muccio A, Mantovani A, Spera G, Silvestroni L 2004
Low levels of organochlorine pesticides in subjects with metabolic
disturbances: a survey taken in Rome in 2001-2002. Bull Environ Contam
Toxicol 732219-226

Mizejewski GJ, Vonnegut M, Jacobson HI 1983 Estradiol-activated
alpha-fetoprotein suppresses the uterotropic response to estrogens. Proc
Natl Acad Sci U S A 80:27.33—2737

Szafran H, Smielak-Koromberl W 1998 The role of estrogens in
hormonal regulation of lipid metabolism in women. Przel-Lek 55:266-270.

Simpson ER, Clyne C, Rubin G, Boon WC, Robertson K, Britt K,
Speed C, Jones M 2002 Aromatase—a brief overview. Annu Rev Physiol
64293-127.

Cooke PS, Naaz A 2004 Role of estrogens in adipocyte development and
function. Exp Biol Med (Maywood) 229:1 127-1 135

Cooke PS, Heine PA, Taylor JA, Lubahn DB 2001 The role of estrogen
and estrogen receptor-alpha in male adipose tissue. Mol Cell Endocrinol
178:147-154

76

CHAPTER 3

LITERATURE REVIEW II.
3.1 DEFINITION OF ESTROGENIC ENDOCRINE MODULATORS

Wildlife and laboratory animals exposed to exogenous estrogenic
chemicals during in utero and postnatal development have adverse effects on
reproductive tract development and fertility (1-3). As described in Chapter 2,
endogenous estrogen has important roles in the reproductive system, and
alteration in its biological function by interfering exogenous estrogenic chemicals
via binding to receptors, secretion, synthesis, transport, or elimination of
endogenous estrogens necessary for normal function and homeostasis of
reproduction, development and/or behavior could lead to functional and
morphological adverse effects on reproduction. These endocrine disrupting
chemicals, both naturally occurring as well as man-made chemicals, are known
by a variety of terms (e.g., environmental estrogens, environmental hormones,
endocrine disrupting chemicals, endocrine modulators, endocrine disrupters, and
endocrine disruptors) to describe the phenomenon that can mimic endogenous
estrogen in the body. The US EPA adopted the definition established by Kavlock
et al.(4) that endocrine disrupters interfere with the synthesis, secretion, transport,
binding, action, or elimination of endogenous estrogen that is responsible for the
maintenance of homeostasis, reproduction, development, and/or behavior (5). In
1997, the European Commission defined an endocrine disrupter as an
exogenous substance that causes adverse health effects in an intact organism,

or its progeny, secondary to changes in endocrine function; a potential endocrine

77

disruptor is a substance that possesses properties that might be expected to lead
to endocrine disruption in an intact organism (6).

However, transient effects of some endocrine disrupters may be
considered adverse effects such as a temporary reduction in sperm production
that may lead to infertility but not sterility. Therefore, Dickerson et al. (7) gave
two definitions of endocrine disruptor and endocrine modulator. Based on these
definitions, an endocrine disruptor is a compound that causes irreversible or
adverse effects on endocrine function. An endocrine modulator is a compound
that causes transient and reversible alterations of endocrine function within the
physiological (or homeostatic) range, resulting in effects that normally would not
be considered adverse.

In this paper, the term estrogenic endocrine modulators (EEMs) will be
used because 1) this paper will focus on estrogenic chemicals, 2) it is difficult to
distinguish transient, non-adverse effects and permanent, adverse effects on the
endocrine system, and 3) there is no agreement as to what constitutes low-dose

effects of endocrine disrupting chemicals.

3.2 SOURCES AND EXPOSURE OF EEMs

Estrogen endocrine modulators are ubiquitous in our environment (i.e., air,
water, and soil) because they have been produced naturally or synthetically.
Therefore, both humans and animals can be exposed to EEMs through drinking
contaminated water, breathing contaminated air, ingesting food, or contacting

contaminated soil. According to the US EPA, approximately 87,000 industrial

78

and agricultural chemicals are used in the US alone. Since the mid-1940s, the
US plastics industry has grown at the rate of 6 to 12% per year with an annual
production of 85 billion pounds in 1996, whereas plastics production in
developing countries is expanding at the rate of 40% per year (8). Among these
chemicals, only a few chemicals have been tested for endocrine disrupter activity.
Currently approximately 60 chemicals have been identified as EEMs (9). Some
of them have long environmental half-lives (e.g., 15 years for DDT and PCB) due
to their lipophilicity. An increase in the environmental concentrations of
chemicals can lead to bioaccumulation in adipose tissue and biomagnification in
species at the top of the food chain. Increased concentrations of EEMs in the
body of the higher predator can exert toxic effects on the reproductive system.
High concentrations of EEMs are often measured in sewage sludge from
industrial, agricultural and domestic origin (10-12). The contaminated sewage
sludge can be used as fertilizer for farm animals or land application (13, 14). If
these EEMs are ingested with food and drinking water by food-producing animals
then there is potential for adverse effects in humans via consuming food-
produ'cing animals or animal products. Furthermore, low body burden of EEMs,
so called “low-dose effects of EEMs” have been debated because the range of
endogenous steroid hormones exert their bioactivities is nmol or pmol range (15,
16). Thus, further research and risk assessment for low-dose effects of EEMs
are needed to confirm whether low-dose effects of EEMs have potential risk to

reproductive health.

79

There have been concerns regarding the deleterious effects of
phytoestrogens on reproduction and development in humans and animals (17-
20). Most natural EEMs are generally plant-produced estrogens. Many plants
(e.g., legumes, fruits, and vegetables) produce phytoestrogen (e.g., genistein
and daidzein). For example, a study demonstrated a detectable concentrations
of genistein and daidzein in currants and raisins (2,350 mg and 1,840 mglkg of
wet weight of food) among 80 different fruits and nuts (21). Concentrations of
phytoestrogens vary in plants but are mainly formed in legumes (genistein and
daidzein occur up to 3.8 x 106 mglkg wet weight of soybean) (17, 22, 23).

The , other source of natural EEMs is mycotoxins. A mycotoxin,
zearalenone, is synthesized by several species of Fusarium (F. graminearum, F.
culmorum, F. croolwellense, F. sambucinum and F. equisetr) commonly
contaminating poorly stored agricultural products and foodstuffs. Zearalenone is
a non-steroidal mycotoxin with estrogen-like activity (24).

Synthetically produced EEMs have been used in pharmaceuticals (e.g.,
diethylstilbestrol, tamoxifen, and 17a-ethynyl estradiol), pesticides (e.g.,
methoxychlor and atrazine), industrial chemicals (e.g., bisphenol A and
phthalate), and occur as environmental pollutants (e.g., polychlorinated biphenyl
and benzo[a]pyrene). Details of each EEM mentioned above will be discussed

later in this chapter.

80

3.3 MODES OF ACTION OF EEMs
3.3.1 ALTERED SEX STEROID HORMONE SYNTHESIS

It has been known that some EEMs affect the reproductive system
through altering endogenous estrogen and androgen synthesis via interfering
P450 enzymes (Table 2-1 and Figure 2-1). For example, mono-(2-ethylhexyl)—
phthalate (MEHP), which is an active metabolite of a plasticizer, di-(2-ethylhexyl)-
phthalate (DEHP), may interfere with estrogen synthesis by decreasing
transcription of the aromatase enzyme in cultured rat granulosa cells (25, 26).
Additionally, Andrade et al. (27) demonstrated that maternal exposure to DEHP
during the gestational and lactational periods alters aromatase activity in the
hypothalamus with J-shaped dose-response curve, which means that aromatase
activity was inhibited by low doses of DEHP (0.135 and 0.405 mglkg bw/d) and
stimulated by high doses (15, 45 and 405 mglkg bw/d) in male rats on postnatal
day 1. Corresponding to the results of laboratory animal studies, it has been
hypothesized that in utero and early postnatal exposure to phthalates may be
responsible for testis abnormalities (e.g. cryptorchidism and hypospadias) in
human males via stimulation of aromatase activity and suppression of androgen
production from fetal Leydig cells (28-30).

Unlike aromatase, which is encoded by a single gene in the human and
rodent, hydroxysteroid dehydrogenases are encoded by at least 2 to 3
homologous genes. Human 3B-HSD shares 80 to 94% amino acid sequence
identity with rat (31) and human 17a-HSD shares 77, 70 and 72% amino acid

sequence identity with mouse type 5 17B-HSD, 3a-HSD and 200-HSD

81

respectively (32). Inhibitory effects of phytoestrogens on 36- or 17B-HSD have
been reported (33). Some studies demonstrated that the number of
hydroxylations of carbon molecules in A ring and B ring (Figure 3-1) of
phytoestrogens is positively correlated with the inhibitory effects on the activity of
313- or 17B-HSD type 1 and 5 in human placental microsomes or adrenocortical
H295R cells (34, 35). These results suggest that phytoestrogens are potent
inhibitors of human placental estrogen biosynthesis.

The biological role of Sci-reductase is to catalyze the conversion of
testosterone to Soc-dihydrotestosterone (DHT). Both testosterone and DHT can
bind to the androgen receptor to promote androgen-dependent gene transcription,
which plays important roles for sperrnatogenesis and accessory sex gland
function. It is known that DHT is a more potent androgen than testosterone (e.g.
2.4 times more potent at maintaining rat prostate weight) (36, 37). An in vivo
study also confirmed that maternal exposure to 3 or 30 ug/kg bw/d of 3,3',4,4’,5-
pentachlorobiphenyl (PCB126) and 3,3’4,4’,5,5’-hexachlorobiphenyl (PCB169)
increases the relative activity of 3B—HSD, and decreased the relative activity of
SOL-reductase at 15 weeks in the testis of male rat offspring (38). In this study,
plasma testosterone concentrations in maternally exposed PCB169 male
offspring were decreased at 3 weeks (14 vs. 1 ng/dl, respectively) but were
increased at 15 weeks (120 vs. 266 ng/dl, respectively). It suggests that fetal
exposure to PCB126 or PCB169 has long-term effects on steroidogenic enzymes
activities leading to alter action of the concentrations of steroid hormones, which

may suppress sperrnatogenesis. Collectively, future studies need to consider

82

effects of EEMs on the reproductive system via through alteration of

steroidogenic enzymes.

3.3.2 ALTERED STEROID HORMONE STORAGE AND/OR RELEASE

Steroid hormones do not appear to be stored intracellularly within
membranous secretory granules (39). They are more likely synthesized and
released instantly after gonadotrophin stimulates the gonads. For example,
testosterone is rapidly synthesized by the Leydig cells of the testis upon
stimulation by LH from the hypothalamus. Catecholamine hormones such as
norepinephrine are stored in granular vesicles and thus they can be released
quickly on demand (40). Norepinephrine is critical for the preovulatory increase
in the pulsatile release of GnRH and the subsequent ovulatory surge of LH. For
example, insecticides such as chlordimeforrn and amitraz have been reported. to
block norepinephrine binding to the alpha2-adrenergic receptor, thus inhibiting
release of GnRH and production of progesterone in human and rodent granulosa
cells without affecting estrogen production (41, 42). It indicates that these
pesticides may disrupt in the timing of the LH surge, and alter the viability and the

quality of the oocytes (43).

3.3.3 ALTERED STEROID HORMONE TRANSPORT AND CLEARANCE
Hormones are transported from blood in the free (biologically active form)
or bound state. Steroid hormones and EEMs are mostly transported in the blood

by albumin and specialized transport proteins (carriers), known as sex-steroid

83

hormone-binding globulin (SHBG) or testosterone-estrogen-binding globulin
(T EBG). Therefore, these carrier proteins in blood are important in transporting
lipophilic endogenous hormones and EEMs to target organs and making
available free steroids to target cells. It suggests that EEM carrier proteins have
a role in the detoxification process by retarding the binding of EEMs to ERs,
which allows the inactivation and excretion of EEMs to occur without potential
adverse effects on reproduction.

Albumin is the major serum protein that binds various lipophilic
compounds such as steroid hormones, fatty acids, retinoids, and thyroid
hormone and prostaglandins as well as many lipophilic EEMs because of its high
serum concentration (approximately 45 mg/mL, 0.67 mM in humans) (44).
Corresponding to the high concentration of albumin in serum, 60% of E2 is
bound to albumin. Among the endogenous estrogens, E3 has 1/3 the affinity of
E2 for the ERs but in the presence of albumin, the affinity of E3 is twice that of
E2 for the ERs (44-47).

Sex-steroid hormone-binding globulin is the major binding protein for sex
steroid hormones in plasma, and mediates androgen and estrogen signaling at
the cell membrane through CAMP-induced pathways (48). It is known that
estrogen increases the plasma concentration of SHBG via increasing synthesis
of SHBG in the liver (49). Concentrations of sex-steroid hormone-binding
globulin are twice as high in women as they are in men due to relatively high
production of endogenous estrogen or synthetic estrogen derived from birth

control pills in women. When woman reach at the final menstrual period,

84

approximately 38 to 60% of E2 and 8% of E1 are bound to SHBG (50).
Considering E2 binding to both albumin and SHBG, 1 to 2% of total plasma E2
circulates as the free hormone in women (50, 51).

In addition to‘albumin and SHBG, estrogens in the amniotic fluid of
pregnant women and rodents are bound to alpha-fetoprotein (AFP) (52). Alpha-
fetoprotein in human fetal serum was recognized as a fetal component not
commonly found in adults (53). Oviductal fluids from rabbits, monkeys, and
women secrete a variety of proteins such as albumin and alpha-fetoprotein (54,
55). The production of albumin and AFP found in human oviductal fluid is under
hormonal control and concentrations of such protein secretions are positively
correlated with the estrogen peak of the menstrual cycle (56). Studies
demonstrated that AFP also can bind and transport several ligands, including
fatty acids, retinoids, steroids, heavy metals, dyes, phytoestrogens, dioxins, and
various organic drugs (57-59).

Regulation of the concentration of albumin, SHBG, and AFP in the serum
is of practical significance because they either increase or decrease the
clearance of endogenous hormones or access of EEMs to target receptors
resulting in altered bioavailability to the target organs. Therefore, humans and
animals can have a selective advantage in protection against the potential

adverse effects of EEMs.

85

3.3.4 ALTERED STEROID HORMONE RECEPTOR RECOGNITION/BINDING

Studies demonstrated that hormones elicit responses in their respective
target organs through direct interactions with either intracellular receptors or
membrane-bound receptors. Specific binding of the endogenous ligand to its
receptor is a critical step in hormone function. Intracellular (nuclear) receptors,
such as those for sex steroids, adrenal steroids, and thyroid hormones, regulate
gene transcription in a ligand-dependent manner through their interaction with
specific DNA sequences (response elements). A number of EEMs may alter this
process by mimicking the endogenous estrogen and acting as an agonist or by
inhibiting binding and acting as an antagonist. The best known examples are
methoxychlor, DDT, some PCBs and alkylphenols, which can compete with
endogenous estrogen to bind ERs. Interestingly, some EEMs may act through
one or more intracellular receptors. For example, o,p’-DDT has estrogenic and
androgenic effects became of binding to both ERs and AR, while, p,p’-DDT
inhibits androgen binding to the AR in vitro (60, 61). The pesticides dieldrin,
endosulfan, and fenarimol are also known to bind both ERs and AR. Additionally,
Kelce et al. (62) observed that chlordecone, which is a synthetic chlorinated
organic compound and has been used as an insecticide and fungicide, mainly
binds to ER but it also binds to AR at higher concentrations. Therefore, it is
difficult to categorize a compound as either estrogenic or androgenic endocrine
modulator.

It has been hypothesized that EEMs induce or reduce the degradation of

steroid hormone receptors resulting in altered physiological effects of

86

endogenous steroid hormones. Studies have demonstrated that the half-life of
ERs is approximately 1 to 5 hr in uterine tissues and in cultured cells by using
dense amino acid or [358]methionine/cysteine incorporation, or radiolabeled
tamoxifen aziridine binding (63-65). There have been controversial results
regarding the ERa degradation in transcriptional activation. Some studies
suggest that ERa degradation may be required to maintain ERa-mediated
transcription with turnover of a specific proteasome (e.g. 26$ proteasome) and
coactivators (e.g. Fos/Jun) (66), while other studies suggest that ERa
degradation has no direct effects on transcription in pituitary cells or in ERa-
positive breast cancer cells (67, 68).

Collectively, ligands including EEMs may form a stable complex with their
receptors, the ligand bound steroid hormone receptor complexes are required to
carry on transcription of target genes for extended periods of time. Therefore,
- degradation of the ligand bound receptor complex at transcription is important to
maintain transcription activation in response to rapid changes in hormone

concentration as well as physiological cellular response.

3.3.5 ALTERED STEROID HORMONE POSTRECEPTOR ACTIVATION

Once the endogenous ligands or EEMs bind to their receptors, a cascade
of events is initiated for the appropriate cellular response, which is necessary for
signal transduction across the membrane or the initiation of transcription and
protein synthesis via nuclear receptors (49). When hormones bind to their

receptors, transduction of a signal across the membrane is mediated by the

87

 

activation of second messenger systems. These may include (1) alterations in
G-protein/cAMP-dependent protein kinase A (e.g., after LH stimulation of the
Leydig cell); (2) phosphatidylinositol regulation of protein kinase C and inositol
triphosphate (e.g., after GnRH stimulation of gonadotrophs, thyrotropin releasing
hormone stimulation of thyrotrophs); (3) tyrosine kinase (e.g., after insulin binding
to the membrane receptor); and (4) calcium ion flux (e.g. the initiation of the
calcium/calmodulin-dependent cellular response). EEMs thus can disrupt signal
transduction of peptide hormones if they interfere with one or more of these
processes (69). For example, 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure
indirectly down regulates steroid hormone receptor activation (70, 71).
Consequently, because of the diverse known pathways of EEMs, any
assessment must consider the net result of all influences on hormone receptor

function and feedback regulation.

3.3.6 ALTERED GENE EXPRESSION WITH CHANGING DNA SEQUENCE
Epigenetics refers to the molecular phenomena that regulate gene
expression without alterations to the deoxyribonucleic acid (DNA) sequence,
which contains genomic information of the cell by encoding the sequence of the
amino acid residues in proteins. Genes in mammals are transcribed from
methylation-free promoters, while keeping noncoding DNA, containing introns
suppressed. Therefore, methylation can either increase or decrease the level of

transcription, depending on whether the methylation inactivates a positive or

88

 

negative regulatory element leading to long-term epigenetic silencing of imprinted
genes.

DNA methylation is specific to cytosines in CpG dinucleotides and
achieved by the transfer of a methyl group from S-adenosyl-methionine to the C5
position of cytosine. It is catalyzed by DNA methyltransferases (e.g., DNA
cytosine methyltransferases). Approximately, 70 percent of all cytosines in CpG
dinucleotides in the human genome are methylated (72). Two DNA methylation
patterns happen during the developmental period (73). The first one is the
lineage-specific DNA methylation pattern, which occurs during gastrulation, and
the second one is the germ-line DNA methylation pattern, which occurs during
gonadal development and sex determination. The lineage-specific DNA
methylation pattern is based on the genetic material transferred from the egg and
sperm, which can result in developmental defects or embryonic lethality by any
possible alterations of DNA methylation (73, 74). Unlike the lineage-specific
DNA methylation pattern, any potential alteration of the germ-line DNA
methylation pattern could be critical for imprinted genes in germ cellsand‘ result
in transgenerational effects (75-77). During gonadal development, primordial
germ cells in mouse embryos migrate down the genital ridge and a complete
DNA demethylation is achieved from embryonic day 10.5 to day 11.5 (78).
Thereafter, the germ cells undergo a remethylation involving a sex-specific
determination of the germ cells during sex determination in the gonad.

There is a hypothesis that EEMs can affect, through an epigenetic process,

the germ line by altering DNA methylation status in germ cells during the period

89

 

of sex determination in the developing gonad (79). Since the estrogen-regulated
genes are programmed during development, altered hormone response may be
transmitted to subsequent generations. An example of an epigenetic mechanism
of DES, such as an increased tumor incidence in the reproductive tract in
subsequent generations, has been well documented in human and laboratory
animals (76, 80, 81). Newbold et al. (82) observed that 31% of uterine tumor
incidence in 18 month-old F1 mice exposed to DES at a dose of 0.002 ug/pup
per day on PND 1 through 5, whereas the incidence was 11% in their DES
descendants. Corresponding to this increased tumor incidence in subsequent
generations, studies demonstrated that altered methylation patterns in uterine
genes can be passed to subsequent generations (83) and hypomethylated
estrogen-responsive proteins LF and c-fos were permanently up-regulated in the
uterus after developmental exposure to DES (84). Additionally, methoxychlor
caused transgenerational adverse effects in spennatogenic capacity including
cell number and viability, and increased male infertility in rats maternally exposed
to 100 or 200 mglkg bw/d from embryonic day 8 to 15 with epigenetic alteration
in the DNA hyper— and hypomethylation, which was observed in subsequent
generations (F1 to F4) (85).

Currently, many studies into transgenerational effects of other EEMs and
possible epigenetic mechanisms that govern these effects have been

investigated.

90

3.4 PHYTOESTROGEN AND MYCOTOXIN
3.4.1 GENISTEIN (GEN)

Genistein (4’,5,7-trihydrozyisoflavone or GEN) is a phytoestrogen, the
predominant isoflavonoids in leguminous plants. A study showed that plant
concentrations of GEN vary widely depending on genotype of the soybean,
location, and temperature. The average concentration of GEN in 31 different
genotypes of vegetable soybeans was 34 pg/g (86). GEN is found in fruits and
vegetables such as grapefnrit, licorice black, and licorice tea (27, 599, 26
jig/1009 of plant tissues, respectively) (87).

Since biochemical structure of GEN is similar to E2, it has been reported
that GEN exerts its estrogenic effects via binding to ERs as demonstrated in in
vitro and in vivo studies. The basic structural feature of GEN is the flavone
nucleus, which is composed of 2 benzene rings, A and B, linked through a
heterocyclic pyrane C ring (Figure 3-1). Compared to E2, its estrogenic potency
is 104 to 105 times less potent in the luciferase reporter gene assay and the E-
screen (88, 89). GEN has 20-fold higher binding affinity for ERB than ERor as
determined in an in vitro assay (90). Furthermore, the transactivational potency
of GEN at ERa and ERB is approximately 2-fold greater than a similar
concentration of E2 (91 ).

There have been great concerns of adverse effects on reproductive health
of GEN exposure during the developmental period because of its concentrations
measured in soy-based infant formula and its similarity of biochemical structure

with E2 (92, 93). Some soy-based formulas contains 40 mg/l of soy isoflavones

91

consisting in part of GEN and its glycosidic conjugates (94). The estimated daily
intake of total isoflavones in infants fed a soy-based infant formula is 5 to 8
mglkg bw/d, which is approximately 200 times higher compared to that in adults
who consumed soy-based products (0.025 to 0.068 mglkg bw/d) (23).
Furthermore, the plasma concentration of GEN measured in infants fed soy-
based formulas was approximately 684 ng/ml, which was a greater amount than
found in infants fed cow’s milk formula (3.2 ng/ml) or human breast milk (2.8
ng/ml) (95).

In laboratory animal studies, adverse effects of GEN in the reproductive
system have been reported. In utero exposure to 50 mg GEN lkg bw/d results in
significant increases in uterine adenocarcinomas in 18-month—old mice, which is
roughly middle age (20). Maternal exposure to GEN (100 mglkg of maternal
body weight) from GD 7 decreased average live pup weight at birth and offspring
body weight (96). Neonatal exposure to GEN (1, 10, or 100 ug/pup/day) for 5
days caused a dose-related multioocyte follicles and induced ERa expression in
granulosa cells in mice (97). Additionally, in the same study, an increase in the
number of ovulated oocytes was observed in the 1 pg treated mice, whereas a
decrease was observed in 10 and 100 pg GEN-treated mice. A dose- and age-
related prolonged estrous cyclicity in mice was also observed following neonatal
exposure to GEN at doses ranging from 0.5 to 50 mglkg bw/d from PND 1
through PND 5 (98). Fertility and the number of live pups at birth were also
decreased. Additionally, maternal exposure to GEN (15 mglkg maternal body

weight) increased the estrogen-regulated progesterone receptor in the rat, which

92

has important roles in uterine proliferation, implantation, and maintenance of
pregnancy (99).

However, a study demonstrated that GEN (0.1, 0.5, 2.5 or 10 mglkg
maternal body weight), at concentrations equal to or greater than human dietary
exposure does, not interfere with reproductive development, epididymal sperm
production, or sperm motion parameters, but in vitro sperm fertilizing ability
increased in male offspring (100). Similarly, exposure to GEN (250 and 1000
mglkg diet) from postnatal day 21 to 35 did not alter rat testicular development
(101). Collectively, further study is needed to investigate potential adverse

effects of in utero and postnatal exposure to GEN.

3.4.2 ZEARALENONE

(Zearalenone (ZEA) is a mycoestrogen known as resorcyclic acid lactone
(RAL) and F-2 toxin. It is a family of phenolic compounds produced by several
species of Fusarium (F. graminearum, F. culmorum, F. croolwellense, F.
sambucinum and F. equisetr) that frequently infest many important crops (102).
The concentrations of ZEA and its derivatives (e.g. a-zearalenol, B-zearalenol, a-
zearalanol (zeranol), B-zearalanol (taleranol)) in cereal grains have been
reported by Bottalico (103) at concentrations ranging from 175 to 758 pg/kg food.
Additionally, concentrations of ZEA were measured in the range of 4 to 584 ug/kg
and 55 to 1400 ug/kg in maize for human consumption and animal feed,
respectively, in the UK (104, 105). ZEA was also detected in sewage influents

and effluents of an Italian city at levels below 30 ng/I (106). a-Zearalenol is used

93

as an anabolic growth promoter in cattle in the US (trade names Ralgo®). The
acceptable daily intake (ADI) of zeranol for humans is 0.05 mglkg bw/d and the
limit of zeranol residue in edible meat tissue of cattle implanted with Ralgo® after
70 days is 0.23 mglkg (107, 108). ZEA was detected at a level of 8.7 uglkg in
fresh meat and at a level of 6.9 ug/kg in milk samples (109). A Canadian study
reported that the range of estimated daily exposure to ZEA from food
consumption for young children is 0.05 to 0.10 ug/kg bw/d (110).

The chemical structures of ZEA and its metabolites are similar to the
structure of E2, and thus they have estrogenic and anabolic activities as
documented in in vitro and in vivo tests (111). Consequently, ZEA and its
metabolites have been associated with hyperestrogenism and other reproductive
disorders by competing with E2 in binding to intracellular ERs present in the
uterus, mammary gland, hypothalamus and pituitary gland in various species
(rodent, pigs and monkeys) (24, 112-114). ZEA is metabolized rapidly and its
half-life in the rat intestine is approximately 5 min (115). Compared to E2, ZEA
has an approximately 20-fold lower binding affinity to ERs. a-Zearalenol was as
potent as EE2 and DES in the human endometrial lshikawa cell line (116). The
relative binding affinity of ZEA and its metabolites for ERs in the pig, rat and
chicken have shown that a-zearalenol exhibiting greater affinity than ZEA and
while B-zearalenol had the lowest binding affinity in all species tested.
Furthermore, the relative binding affinity of a-zearalenol was greater in swine
than in the rat and significantly greater than in the chicken because of differences

in ZEA metabolites formed (113).

94

It has been well documented that ZEA disrupts early pregnancy in swine.
Diekman and Green reviewed the adverse effects of ZEA on swine (117). The
age of onset of puberty at 1.8 ppm, ZEA (naturally contaminated milo) fed
prepubertal gilts was decreased, while it was increased in gilts fed 10 ppm
purified ZEA. This result may indicate that ZEA can act as an estrogenic agonist
at a low dose and as an antagonist at a high dose, although it may be due to
difference in purity of ZEA and the strain of test animals. In cycling gilts or sows,
an extended interestrus interval was observed in all animals, regardless of the
purity of ZEA and the treatment period. For the pubertal pig, the no observable
adverse effect level (NOAEL) of ZEA is 0.06 mglkg/day (110). In mated gilts or
sows, postmating day 7 to 10 is a Critical period of gestation for ZEA to exert its
detrimental actions on early embryonic development. Effects of ZEA on
reproduction in boars are decreased libido, testes weights, epididymis weight,
serum testosterone, spennatogenesis, and sperm motility.

On the other hand, the common effects of exposure to ZEA in cattle are
abortion, decreased fertility, abnormal estrus cycles (e.g., prolonged, skipped, or
irregular heats), swollen vulvas, vaginitis, reduced milk production, and
mammary gland enlargement of virgin heifers (118, 119). Weaver et‘al. (120)
observed a reduced conception rate in virgin heifers that consumed ZEA at
greater concentrations than 25 ppm administrated by gelatin capsule (62% vs.
87%) without changes of serum of progesterone concentrations. Collectively, the
potential health risk of ZEA from food will need to be further investigation,

specially for young children.

95

3.5 PHARMACEUTICALS
3.5.1 DIETHYLSTILBESTROL (DES)

Diethylstilbestrol (DES) is a non-steroidal synthetic estrogen that was first
manufactured in London in 1938 (121). In 1947, the US Food and Drug
Administration (FDA) approved DES for preventing habitual abortion. Therefore,
between the late 19405 and 1971, DES was administered to pregnant women to
prevent miscarriage and other pregnancy complications in the US (1.4 to 17.9 9
total dose during pregnancy). As a result, an estimated 4 million pregnant
women have been exposed to DES in utero (81, 88). Furthermore, DES has
been used to restrain lactation, to manage menopausal symptoms, and to treat
breast and prostate cancer (122). In 1953, a study demonstrated that DES did
not prevent miscarriages or premature births (123). However, DES continued to
be prescribed until 1971 in the US and until the late 19705 in European countries
(124). The first adverse reproductive effects of DES, eight cases of vaginal
cancer in Boston area between 1966 and 1969, were reported in 1971. A study
concluded that prenatal exposure to DES causes vaginal and cervical clear cell
adenocarcinoma (CCA) in women younger than 50 (125). When women who
were exposed to DES in utero reached puberty, an increase in cases of genital
cancer (126) and morphological and functional abnormalities in their ovaries,
uterus, and/or oviduct had been reported (127, 128). For example, in utero
exposure to DES caused a 40 times higher incidence of CCA of the vagina and

cervix in female offspring than woman not exposed to DES (126, 129).

96

Studies suggest that estrogenic compounds can alter Miillerian duct
development that occurs between the sixth and eleventh weeks of pregnancy in
humans and laboratory animals. Data from epidemiologic studies demonstrate
that there is astrong correlation between DES exposures in utero and urogenital
and reproductive abnormalities, such as increased cryptorchidism, urogenital
abnormalities epidermal cysts, and hypotrophic testes (130, 131).
Correspondingly, laboratory animal studies demonstrated that significant
increased cryptorchidism (91%) and testicular cancer (8%) occurred in mice that
were exposed to 100 pg DES/kg bw/d from gestational day 9 to 16 (132). The
exact mechanisms by which DES alter development of the reproductive tract are
unknown, but a possible explanation is that DES alters the expression of certain
genes (e.g. insulin-like factor 3, homeobox A10 [Hoxa 10], and Wingless-type
MMTV integration site family member 7a [Wnt-7a]) that are involved in the
development of the reproductive tract during a critical period of development.
For example, studies demonstrated that insulin-like factor 3 has an important role
in the transabdominal phase of testis descent, and in utero exposure to DES
(100 pg/kg maternal body weight) from GD 9 through 17 decreased lnsl3 mRNA
expression in the mouse fetal testis (133).

Environmental estrogenic compounds at high levels of exposure decrease
body weight of laboratory animals but at lower levels of exposure, they may
increase body weight (134). Recently, Newbold et al. (135) demonstrated that in
utero exposure to an extremely low doses of DES (1 part per billion) significantly

increased body weight of mice at 4 months of age. It indicates that there may be

97

an association between in utero exposure to low doses of DES and disruption of
the body's natural weight-control mechanisms resulting in interference of the
normal developmental and homeostatic controls over adipogenesis and energy

balance.

3.5.2 TAMOXIFEN

Tamoxifen is a selective estrogen receptor modulator, which was first
used to treat early and advanced breast cancer in women via regulating growth
of breast cancer cells. It was invented by AstraZeneca Pharmaceutical Company
and it has been commercially available since the mid 1970’s. In the 1950’s, its
potential use as an oral contraceptive was investigated because of its estrogenic
antagonistic activity. It was approved by the US FDA in 1998 for women at risk
developing for breast cancer. Tamoxifen has also been used to treat infertility in
women with anovulatory disorders (10-40 mg/day in days 3 to 7 of the menstrual
cycle).

Tamoxifen and its metabolite (4-hydroxytamoxifen, endoxifen) have both
estrogenic and antiestrogenic activities (136, 137). Compared to the estrogenic
potency of E2 in an recombinant yeast cell assay, tamoxifen is approximately 106

times less potent than E2. Tamoxifen and 4-hydroxytamoxifen have similar
estrogenic effects in an uterotropic assay but the estrogenic activity of tamoxifen
is increased by hydroxylation in a receptor binding assay (138). Endoxifen has
100-fold greater affinity for the ER and in 30 to 100-fold more potent than

tamoxifen in suppressing estrogen-dependent cell proliferation (139).

98

An epidemiologic study reported that endometrial thickness and
abnormalities such as endometrial polyps increased with the duration of
tamoxifen treatment that was longer than 5 years in postmenOpausal breast
cancer patients (140). Suggested mechanisms of action of tamoxifen are to
compete with endogenous estrogen in the body for binding to ERs in breast
tissue. Therefore, transcription of estrogen-stimulated genes are inhibited.
Additionally, the well-known antiestrogen tamoxifen inhibits protein kinase C
activity (141), and thus tamoxifen has the growth-inhibitory and cytotoxic effects
in cultured mouse fibroblast cells at concentrations as low as 5 pM (69).

Effects of tamoxifen on reproduction in laboratory animals have been
reported. A dose-dependent decrease in epididymal sperm production and
testicular tubular atrophy were observed in adult male rats orally exposed to 400
mg tamoxifeng bw/d for 30 days (142). Additionally, pregnenolone biosynthesis
via down regulation of P450$cc gene expression in the testis was decreased
beginning at day 60 of treatment (143). Chronic oral exposure to 0.4 mglkg/day
reduced (74%) the fertility of the male rats with reduced circulating testosterone
and LH (144). However, tamoxifen and endoxifen did not change sperm
production. An increase in the number of abnormal eggs at the 2 to 4 cell stage
was observed in rats exposed to tamoxifen at 0.4 mglkg bw/d from GD 0 to GD 4

(145).

99

3.5.3 17a-ETHYNYL ESTRADIOL (EE2)

Ethynyl estradiol is the major estrogenic component of oral contraceptives
and in a carcinostatic agent for prostate and breast carcinomas.” Its synthesis
was first reported in 1938 by lnhoffen and Hohlweg (146). Doses of EE2 in oral
contraceptives are from 0.4 toZ pg/kg bw/d (147), which doses of 4 to 1400 pg/kg
bw/d (0.3 to 100 mg/day) are used for arresting prostate carcinomas in man
(148). The lowest teratogenic dose of EE2 in mice, 20 pg/kg bwld, was about 10
to 50 times higher than the does of 'EE2 in an oral contraceptive (147, 149). The
maximum tolerated dose (MTD) resulting in reduced body weight gain was 200
pg/kg bwld in male rats treated by gavaging for 28 to 32 days beginning at 7
weeks of age (150). Additionally, the oral lethal doses to 50% of the
experimental animals (LDso) in rats and mice are greater than 5,000 and, 2,500
mglkg bw/d, respectively.

Concern for the adverse effects of EE2 on reproductive health of humans
and animals is increasing, even though there is lack of epidemiological evidence
linking oral contraceptive use and malforrnatlons at birth. Each year,
approximately 2 million women in the United States (US) and the United
Kingdom (UK) are exposed to 0.4 to 2.0 pg EE2/kg bw/d during undetected early
pregnancy (147). Slikker et al. (151) demonstrated placental transfer of EE2 by
daily intravenous administration to pregnant rhesus monkeys on gestational days
108 through 121, whereas endogenous estrogen E2 in the placenta is
metabolized to estrone (E1). In the fetal circulation, 1E1 and EE2 were observed,

whereas E1, E2 and EE2 were observed in the maternal circulation. Therefore,

100

fetuses can be exposed to EE2 in utero during critical stages of development of
the reproductive and central nervous systems (152-154).

Furthermore, there have been human and ecological health concerns
about pharmaceuticals and their metabolites present in drinking water supplies
because of the inability of sewage treatment facilities to remove them from the
waste stream. The major endogenous estrogen metabolite in the urine is 3-
methoxy-2-hydroxy-estrone glucuronide. However, sulfated EE2 is almost
completely absorbed from the gastrointestinal tract and binds to serum albumin.
It appears slowly in the urine, 40% after 5 days, as glucuronides and sulfates,
with a minor amount unconjugated (155). Correspondingly, about 90% of a dose
of titrated EE2 was recovered in both urine and feces in women and the ratio of
eliminated EE2 in urine and feces was 4:6 (156). The half-life'of EE2 in human
and primates is variable (approximately 8 to 27 h) due to different conjugation
profiles produced by tested subjects and different methods of administration
(156-158). Approximately 17 to 29% of sulfate or glucuronide metabolites of
ingested EE2 being used as an oral contraceptive is excreted in the urine (159,
160). In March, 2008, an Associated Press investigation reported that
pharmaceuticals, including EE2, have been detected in the US drinking water
supplies of 24 major metropolitan areas supplying 41 million Americans (161).
The concentrations of EE2 in sewage treatment plant effluents were in the range
of 0.4 to 15 ng/l and 300 to 1800 ng/l in the UK and US, respectively (162-164).

Concentrations of EE2 were measured at 0.48 pg/l in Las Vegas Wash and at

0.52 pg/I in Las Vegas Bay (165). Based on the US Environmental Protection

101

Agency’s (EPA) drinking water exposure estimates, women of childbearing age
(15 to 44 years of age), could be exposed to 6.3x10’5 to 2.5x10‘2 pg EE2/kg bwld
through water consumption, and children younger than one year of age could be
exposed to 2.1x10'5 to 8.3x10’2 pg EE2/kg bwld (166, 167). Therefore, potential
adverse effects of exposure of the fetus and neonate to EE2 through
consumption of drinking water need to be investigated.

Because of different assay methods and different testing cell lines used, it
is difficult to determine estrogenic potency of EE2, compared to E2 or DES.
Studies demonstrated that EE2 and DES may be similar or more potent than E2
based on in vitro E-screen assays (168). In postmenopausal women who
received hormone replacement therapy, E2 and EE2 similarly induced vaginal
epithelium (169). Correspondingly, the potencies of DES and EE2 in in vivo
assays are similar in terms of increasing uterine luminal epithelium hypertrophy
in the immature rat (170). In addition, estrogenic potencies of EE2 in fish have
been shown to be 25 times more potent in in vivo assays (e.g., the vitellogenin
assay and the uterophic assay) compared to in vitro E-screen results (171).

Studies demonstrated that adverse effects of EE2 on the reproductive
health of laboratory animals. Yasuda et al. (149) observed ovotestis and intra-
abdominal testis with persistent Mt’rllerian and Wolffian ducts in male fetuses
exposed in utero to EE2 (0.02, 0.2, or 2.0 mg/kg bw/d) from day 11 through day
17 of gestation, which is prior to gonadal differentiation. Prenatal exposure to
EE2 has long-term effects on the testes such as hyperplasia of Leydig cells and

atrophy of seminiferous tubules in aged mice (172). Leydig cell atrophy and

102

degeneration of germinal epithelium were observed in male rats treated by
gavage with the MT D of EE2 (200 pg/kg bwld) for 28 to 32 days beginning at 7
weeks of age. There was an increase in abnormal spermatozoa and a decrease
in epididymal spermatozoa count in the 10 pg/kg treatment group compared to
controls but these differences were not statistically significant. The male
mammary gland was sensitively affected and feminized by the low dose of EE2
(10 pg/kg) (150). After receiving 10 mg EE2/kg bw/d by daily oral gavage,
epididymal sperm production was significantly decreased in 11- to 12-week-old
rats, whereas the sperm production was slightly changed in the testis (173).
Therefore, high doses of EE2 could decrease sperm production before it affects
sperrnatogenesis in the testis. Additionally, sperm motion parameters such as
percent motile sperm, velocity, linearity, and amplitude of lateral head

displacement (ALH) were significantly decreased (173).

3.6 PESTICIDES
3.6.1 METHOXYCHLOR

Methoxychlor (MXC) is a non-steroidal chlorinated hydrocarbon pesticide.
It has been used to control insects on crops and livestock. In addition, it is used
in gardens and on pets. Its half-life in the water is 20 to 200 days (174). The US
EPA reference dose for MXC is 0.005 mg/kg bwld. Estrogenic activity of MXC
was discovered by Bulger et al in 1978 (175). Furthermore, many studies have
demonstrated its estrogenic effects in different in vivo and in vitro systems (138,

176). Methoxychlor is metabolized by cytochrome P450, and its active estrogenic

103

form is 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) (138, 177). This
metabolite has higher affinity for binding to ERs than MXC, and it possesses ER
agonist and ER antagonist activities (178, 179). Compared to the estrogen
potency of E2, MXC is 1,000 times less potent in the vitellogenin induction assay
(180).

Since studies demonstrated that MXC suppressed pre-implantation
embryo development and increased the rate of embryo abnormalities in female
mice, effects of MXC on the female have been extensively studied and well-
documented (181, 182). Maternal exposure to MXC (33.0 mglkg bw/d) during
the early post-implantation period (days 5—7) significantly decreases sexual
arousal, with a concomitant decrease in testosterone concentrations in adult
males (183). This suggests that MXC may alter the function of the hypothalamic-
pituitary-testicular axis and thus compromise sexual behavior in male offspring.
2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane causes a direct inhibition of
testosterone biosynthesis by Leydig cells in rats , which may result in decreased

male fertility (184).

3.6.2 ATRAZINE

Atrazine (2-chIoro-4-ethylamino-6-isopropylamine-1,2,5-triazine) is the
most commonly used herbicide in the US with an annual application of about
30,000 tone. It also has been used in more than 80 countries (185, 186). It is
produced by Syngenta. Because of concerns about atrazine’s potential ability of

disrupt sex hormones, and the presence of residues in drinking water, it has

104

been banned in Germany, France, Italy, Sweden, Norway and Switzeriand.
Atrazine is routinely found at concentrations as high as 21 ppb in groundwater,
42 ppb in surface water, 102 ppb in river basins in agricultural areas, and up 40
ppb in rainfall in the US midwestem agricultural areas (185, 187, 188). Drinking
water in the US is allowed to have up to 3 ppb atrazine (189).

There were controversial effects of atrazine in laboratory animal studies.
Atrazine has no observed effect levels (NOEL) for reproductive and
developmental toxicity of 5 mglkg bw/d in New Zealand White rabbits, 25 mglkg
bwld in Sprague-Dawley rats, and 5 mglkg/day in a 2-generation feeding study in
SD rats (190, 191). However, serum and intratesticular concentrations of
testosterone were reduced approximately 50% in juvenile rat males orally
exposed to atrazine (50 mglkg) either acutely- or chronically (192). Trentacoste
et al. also observed that 100 mglkg of atrazine for a similar time period (PND 22
to PND 48) significantly decreased serum and intratesticular concentrations of
testosterone in juvenile male rats (193). Furthermore, these in vivo results were
confirmed in a in vitro study. Isolated Leydig cells from rats on PND 49 were
cultured with 232 pM atrazine which resulted in a 35% decrease in testosterone
production (192). This suggests that atrazine may act directly on Leydig cells
and decrease testosterone production. Stoker et al. also demonstrated that
doses as low as 12.5 mglkg bw/d of atrazine administrated from PND 21 until
PND 53 delayed the onset of puberty in the male rat (194). In the female rodent,
atrazine (300 mg/kg bw/d) induced mammary tumors and accelerated

reproductive aging by inhibiting LH release from the pituitary via inhibition of

105

GnRH signaling in the adult rat hypothalamus (195, 196). However, a low dose
of atrazine (25 mglkg bwld) can delay the onset of puberty and alter estrous
cyclicity in the female rat (191). Vaginal opening was significantly delayed In
peripubertal female rats exposed to 30 or 100 mg atrazine/kg bwld, but uterine
weights did not change (197). Further studies are needed to confirm

reproductive toxic effects of atrazine to estimate potential human risk.

3.7 INDUSTRIAL CHEMICALS
3.7.1 BISPHENOL A (BPA)

Bisphenol A (BPA), 2,2-bis(4—hydroxyphenyl)propane, is made by
combining acetone and phenol. It was synthesized by a Russian chemist, A. P.
Dianin, in 1914 (198). Bisphenol A has been used to produce polycarbonate
plastic and epoxy resins which are high-perfonnance plastics that are lightweight
and have high heat resistance. They have a variety of uses including protective
coatings, metal cans, PVC pipe, dental material, automobile parts, adhesives,
plastic bottles, and electrical equipment. In 2003, more than 6.4 billion lb of BPA
was produced globally (199). Heating and washing of polycarbonate products
have been shown to result in an increase in the rate of leaching of BPA (200-
202). As a result, BPA contamination is wide spread in the environment through
municipal waste water. The range of BPA concentrations in the environment has
been reported to be 0.5 to 410 ng/l in rivers and 0.01 to 0.19 mglkg in sediment

(106, 203).

106

Due to an increased production of BPA, exposure of humans to BPA
through intake from food and drinking water has increased. The half-life of BPA
in the human is less than 6 hours and an administrated oral dose of BPA was
were completely recovered in the urine as BPA glucuronide (204). Bisphenol A
has been found in human urine samples at concentrations of 0.4 to 8.0 pg/l and
0.1 to 10.0 pg/l in blood samples (205, 206).

The reference dose of BPA established by the US EPA is 50 pg/kg bwld
(207) and the tolerable daily intake (TDI) established by the European
Commission’s Scientific Committee on Food (ECSCF) is 10 pg/kg bwld, which Is
five times lower than the reference dose established by the US EPA (208).
Furthermore, the daily intake of BPA for infants is about 1.6 pg/kg bwld and for
children 4 to 6 years old, it is about 1.2 pg/kg bwld (209, 210). The migration
limit of BPA in food from BPA containing food containers is 0.6 mglkg in the
European Union (EU) (211).

The reproductive toxicity of BPA at high doses has been reported in
rodents. A continuous breeding toxicity study conducted by the US National
Toxicology Program demonstrated that oral exposure to BPA at doses of 875 or
1750 mglkg bw/d decreased the number of live pups per liter and litters per pair
in CD—1 mice in the first generation (212). Furthermore, intraperitoneal injection
of BPA at 125 mg/kg from gestational day 1 to 15 decreased the number of live
fetuses per litter in rat (213). On the contrary, no significant developmental
toxicity resulted from oral exposure to BPA at doses of 640 mglkg in rats and at

1000 mglkg bwld in mice administrated from GD 6 though GD 15 (214).

107

Even though a positive uterotrophic effect of BPA was demonstrated in
ovariectomized female rats in the 19305 (215, 216), health concerns related to
BPA as an EEM was not an issue until its estrogenic activity was accidentally
discovered by Krishnan’s group when they observed an increased rate of
proliferation of MCF-7 breast cancer cells by BPA that had leached from
autoclaved polycarbonate flasks (217). A study demonstrated that the in vitro
binding affinity of BPA was approximately 20,000-fold lower than that of DES for
both ERor and ERB (90). Additionally, estrogenic activity of BPA is approximately
15,000-fold less than E2, using a yeast-based gene transcription assay (218).

There has been a debate whether there are adverse effects of BPA at low
doses. A study demonstrated that BPA at 100 pM disrupted maturation of mouse
oocytes isolated from the ovary via altering Ca2+ oscillations (219). At maternal
doses as low as 2 and 20 ng BPA/kg bw/d from gestation day 11 to 17
decreased of daily sperm production by approximately 20% and fertility in male
offspring (220). Stimulated development of the mammary gland in mice exposed
in utero to low doses of BPA (25 and 250 pg/kg bwld) was also observed (221 ).
However, other researchers reported no low-dose effects of BPA at the similar
time of pregnancy and in the same strain of mice (222, 223). In order to confirm
potential adverse effects of BPA at low doses, further studies are needed.

4 In addition to reproductive toxicity of induced by BPA, studies
demonstrated that there has a strong association between exposure to BPA and
increased risk of obesity. Bisphenol A in combination with insulin significantly

increased the differentiation of fibroblasts to adipocytes, which means that BPA

108

 

could increase the risk of obesity (224). A study demonstrated that a 2.5-fold
increase in plasma insulin and a 20% decrease in blood glucose levels 30 min
after oral administration of BPA in mice (225). Additionally, BPA rapidly
decreases serum glucose and increases insulin in mice injected twice daily or
orally administrated 10 or 100 pg BPA/kg bw over 4 days (225). BPA treatment
in vitro caused increased glucose uptake in mouse 3T3-F442A adipocytes (226).
Further cohort studies are needed to investigate the possible mechanism of

increased adipocytes by BPA exposure.

3.7.2 PHTHALATES
Phthalate esters are the most widely used industrial chemicals. They are
used principally as plasticizers to improve flexibility and durability in soft vinyl
plastic toys. They are also used in shampoos, soaps, nail polish, and vinyl
flooring. More than 60 different phthalates are also found in products such as
paints, adhesives, inks, dentures, and cosmetics (227). Among the 60 different
phthalates, DEHP in PCV, butyl benzyl phthalate (BBP) in vinyl floor tiles,
dibutyl phthalate (DBP) in paints, and diisononyl phthalate (DINP) in children’s
sucking toys have been investigated for their developmental and reproductive
toxicity because they are able to leach from many phthalate products and thus
contaminate food or drinking water (228-235). Environmental concentration of
DEHP ranged from 0.33 to 97.8 pg/I in surface water, 1.74 to 182 pg/l in
sewage effluents, 27.9 to 154 mglkg in sewage sludge, and 0.21 to 8.44 mglkg

in sediment (203).

109

Based on the NHANES 1999-2000, more than 75% of human urine
samples had detectable concentrations of four phthalate metabolites; mEP,
mono-2-ethylhexyl phthalate (mEHP), mono-n-butyl phthalate (mnBP), and
mBzP (236). Four phthalate monoesters were detected in all urine specimens
from African-American women in the Washington DC. Concentrations were 31
ng/ml for monobenzyl phthalate (mBzP), 53 ng/mL for monobutyl phthalate
(mBP), 211 ng/ml for monoethyl phthalate (mEP), and 7.3 ng/ml for mono-
ethylhexyl phthalate (mEHP) (237). The half-life of phthalate monoester and its
metabolites is approximately 12 hr in humans (237).

The main route of exposure of humans to phthalates is oral. Examples
include ingestion of food, or the chewing of toys by small children (238). Studies
reported that the estimated daily intake of DBP is approximately 0.48 pg/kg bwld,
DEHP is 18 pg/kg bwld, and BBP is 0.11 to 0.29 pg/kg bw/d (239). Other
potential exposure routes may include skin contact from constantly handling
plastic products.

The first documented estrogenicity of a phthalate in vivo was published by
Sharpe et al. (240). In this study, gestational and lactational exposure to BBP
decreases testis weight and daily sperm production in rats. Even though Sharpe
et 3! clearly stated that there was no evidence of a specific estrogenic
mechanism, the endocrine disrupting effects of phthalate and its metabolites in
laboratory animals have been investigated and demonstrated. These effects
include reduced AGD, nipple and areola retention, cleft phallus, hypospadias,

and undescended testes via reducing testosterone synthesis by the fetal testis

110

 

(231, 241, 242). A decreased in testosterone concentrations can be reversed
with clearance of DBP and its metabolites from the testis (243). There was a
dose-response relationship between urine levels of phthalate metabolites such
as mBP and mBzP and lower sperm motility and concentrations in humans (244).
Additionally, there was a significant inverse association between human AGD
and concentrations of phthalate metabolites (245). Dibutyl phthalate and DEHP
treatment decreased of fetal testosterone concentrations by 60 to 85% during the
critical period of development resulting in hypospadlas and cryptorchidism (246).

In contrast to the apparent adverse effects of phthalate on male
reproductive health, females have been thought to be much less sensitive to
phthalate-induced disruption. However, there is evidence that at doses (500 or
1000 mg DBP/kg bw/d from weaning to pregnancy) similar to those that affect
testis function in male rats increased midpregnancy abortions and decreased the
number of delivered live pups (247).

The most recent hypothesis on the mechanisms behind the reproductive
effects of phthalates is that they may have an inhibitory effect on androgen
activity rather than having estrogenic activity. Sohoni and Sumpter demonstrated
that BBP has antagonistic activity in a recombinant yeast-based androgen screen
(248). However, Zacharewski et al. observed that DBP, BBP, and DHP have
weak ER-mediated activity in ER competitive ligand-binding and mammalian-
and yeast-based gene expression assays (249). The fetal testis within one hours
of in utero exposure to 500 mg DBP/kg bwld from GD 12 to GD19, there was

down regulation in the transcription of genes (i.e. SR—B1, StAR, P450scc, and

111

38-HSD) in the tests that are involved in cholesterol transport and the
biosynthesis of testosterone (243, 250). Consequently, testosterone
concentration in fetuses was decreased by 87% as early as GD 17. Taken
together, confirmation of whether current human exposure levels of phthalate
have any consequences for human reproduction as well as whether phthalate
and its metabolites have estrogenic and/or antiandrogenic effects on

reproduction is still needed.

3.8 ENVIRONMENTAL POLLUTANTS
3.8.1 POLYCHLORINATED BIPHENYLS (PCBs)

Polychlorinated biphenyls are a family of 209 congeners, which consist of
two linked phenyl rings and variable chlorination. PCBs were first introduced and
commercially produced in the late 1920s in the US (251). Due to their non-
flammability and chemical stability, PCBs were used widely for various industrial
and commercial purpose such as electrical, heat transfer system, hydraulic
equipment, plastic resins, pigments, dyes, and carbonless copy paper.
Approximately 1.5 billion pounds of PCBs were manufactured in the US until they
was banned in 1977 (252). However, approximately 70% of the manufactured
PCBs are still in use (253, 254).

Persistence of PCBs in the environment and in humans has been well
documented (255). The half-lives for most PCB congeners in the human body
ranged from a few years to 20 years (256). A recent study reported relatively

high serum concentration of PCBs in humans residing near a former

112

manufacturing site in eastern Slovakia that ceased PCB production 20 years
before (3105 ng/g vs. 871 ng/g of lipid in the reference population) (254).

Laboratory animal studies demonstrated that PCBs alter endocrine,
immune, and nervous system functions leading to reproductive tract, alterations,
decreased embryonic growth, delayed implantation, and increased abortion rates
in animals (252, 257, 258). An influence on the sexual differentiation of the brain
by maternal PCB-exposure (40 mglkg) resulted in reduced aromatase activity,
decreased testis weights, and increased uterotrophic effects in rats (259).
Studies indicated that PCB-induced abnormal development of the female
reproductive tract in mice resulted from down-regulation of estrogen-mediated
Wnt genes (Wingless-type MMTV integration site family), which is a known factor
in the reproductive deficits found in mice exposed to DES (252). In vitro studies
using the E-SCREEN assay identified weak estrogenic potency of PCB
congeners (i.e. PCBs 17, 18, 30, 44, 49, 66, 74, 82, 103, 110, 128, and 179) at
environmentally relevant concentrations from a superfund site in Massachusetts
(260).

Laboratory animal studies related to PCBs as EEMs have been clearly
demonstrated and reviewed (261 ). However, even though eidemiological studies
have demonstrated that there was strong association between
neurodevelopmental delay and prenatal or early postnatal PCB-exposure at
environmental concentrations, it is difficult to prove a causative role of PCBs in

producing neurodevelopmental deficits in these cohort studies alone (262, 263).

113

Further epidemiological studies are needed to confirm potential adverse effects

of PCBs as EEMs on reproductive health in humans.

3.8.2 BENZO[A]PYRENE

Benzo[a]pyrene (B[a]P) is a member of the class of lipophilic polycyclic
aromatic hydrocarbons (PAHs), which occur naturally in gas. It is also
continually released into the environment through various combustion processes,
including automobile exhausts, manufacturing of petroleum, aluminum products,
and expulsion of fumes from industrial plants. The major route of human
exposure to B[a]P is ingestion of meat and dairy foods and the minor route of
human exposure is inhalation (264). The estimated average daily intake of B[a]P
in the US population is 1.1 to 2.2 pg per day (264). Benzo[a]pyrene is
biotransformed to polar metabolites by the cytochrome P450 enzymes,
predominantly CYP1A1, 1A2, 2A1, 3A4, and 1B1 (265-269). Main metabolites of
B[a]P are 1-,3-,7-, and 9-OH-B[a]P, 4,5-, 7.8-, and 9,10-diOH-B[a]P, and 1,6-,
3,6-, and 6,12-B[a]P-dione (270-272).

Most B[a]P studies have been focused on its carcinogenic effects.
Laboratory animal studies indicate metabolites of B[a]P, +7,8-oxide (7,8-O), (-)—
dihydrodio (DHD), and (+)-diol-epoxide-2 (DE2), which are capable of covalent
binding to cellular macromolecules such as DNA, RNA, or protein, induce tumor
formation in estrogen-response tissues such as ovary, uterus, and mammary

gland (273-275).

114

In addition to the carcinogenic effects of B[a]P, there have been conflicting
results related to the estrogenic and/or antiestrogenic effects of B[a]P because
B[a]P itself or specific metabolites may be either ER agonists or antagonists.
Benzo[a]pyrene decreases ovarian follicle growth and ovulation, and corpora
lutea formation may due to its antiestrogenic effects through ER-mediated
pathways (276, 277). In vitro studies indicated that B[a]P inhibits estradiol-
dependent reporter activity in hER and decreases E2-induced MCF-7 cell
proliferation (278, 279). Furthermore, one study demonstrated that B[a]P has
estrogenic effects such as uterotrophic effects in the rodent uterus (280) but
another study could not demonstrate such effects (281). Because of these
inconsistent results, it has been hypothesized that the antiestrogenic effects of
B[a]P may be mediated through the AhR. Studies demonstrated that B[a]P
possesses high binding affinity for the AhR (282, 283) and the AhR-ligand
complex alters the transcription rate of E2-inducible genes (284).

Additionally, monohydroxylated metabolites of B[a]P have weak
estrogenic activity or antiestrogenic activity via binding to ER. In a receptor
competition assay using MCF-cells, only monohydroxylated B[a]P metabolites
showed affinity for ERs: 1-, 3-, 7-, and 9-OH-B[a]P caused approximately 20 to
60% displacement of E2 binding to hERor and the IC50 values were 3.3 pM, 90
nM, 10 pM, and 2.0 pM, respectively (IC50 values of E2 were approximately 5.5
nM for ERor and ERB) (281). Overall, 3-OH-B[a]P has the strongest estrogenic

affinity for binding ERs.

115

3.9 BIBLIOGRAPHY

1.

Iguchi T, Sumi M, Tanabe S 2002 Endocrine disruptor issues in Japan.
Congenit Anom (Kyoto) 42:106-1 19

Elias EA, Kincaid RL 1984 Fertility of female mice fed coumestrol and
diethylstilbestrol. J Environ Sci Health B 192441 -451

Kincl FA 1966 Influence of steroid treatment during neonatal life on
sexual maturation. Proc R Soc Med 592817-818

Kavlock RJ, Daston GP, DeRosa C, Fenner—Crisp P, Gray LE, Kaattari
S, Lucier G, Luster M, Mac MJ, Maczka C, Miller R, Moore J, Rolland
R, Scott G, Sheehan DM, Sinks T, Tilson HA 1996 Research needs for
the risk assessment of health and environmental effects of endocrine
disruptors: a report of the US. EPA-sponsored workshop. Environ Health
Perspect 104:715—740

EPA US 1997 Special report on Environmental Endocrine Disruption: An
Effects Assessment and Analysis. In. Washington DC: Office of
Research and Development; EPA/630/R-696/012

European Commission 1997 European Workshop on the Impact of
Endocrine Disrupters on Human Health and the Environment. In. DG XII:
Environment and Climate Research Programme

Dickerson RL, Brouwer A, Earl Gray L, Grothe DR, Peterson RE,
Sheehan DM, M Alfred Wiedow CSM 1998 Dose-response relationships.
In: KENdall R, Dickerson R, Glesy J, Suk W eds. Principles and processes
for evaluating endocrine disruption in wildlife. Danvers, MA: SETAC Press;
69-96

Colbom T 2004 Neurodevelopment and endocrine disruption. Environ
Health Perspect 1122944-949

Brevinl TA, Cillo F, Antonini S, Gandolfi F 2005 Effects of endocrine
disrupters on the oocytes and embryos of farm animals. Reprod Domest
Anim 402291 -299

116

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

Abbassy MS, Ibrahim HZ, el-Amayem MM 1999 Occurrence of
pesticides and polychlorinated biphenyls in water of the Nile river at the
estuaries of Rosetta and Damiatta branches, north of Delta, Egypt. J
Environ Sci Health B 342255-267

Nakada N, Nyunoya H, Nakamura M, Hara A, Iguchi T, Takada H 2004
Identification of estrogenic compounds in wastewater effluent. Environ
Toxicol Chem 23:2807-2815

Peck M, Gibson RW, Kortenkamp A, Hill EM 2004 Sediments are major
sinks of steroidal estrogens in two United Kingdom rivers. Environ Toxicol
Chem 232945-952

Wild SR, Jones KC 1992 Organic chemicals entering agricultural soils in

sewage sludges: screening for their potential to transfer to crop plants and

livestock. Sci Total Environ 1 19285-1 19

Harrison EZ, Oakes SR, Hysell M, Hay A 2006 Organic chemicals in
sewage sludges. Sci Total Environ 367:481-497

Melnick R, Lucier G, Wolfe M, Hall R, Stancel G, Prins G, Gallo M,
Reuhl K, Ho SM, Brown T, Moore J, Leakey J, Haseman J, Kohn M
2002 Summary of the National Toxicology Program's report of the
endocrine disruptors low-dose peer review. Environ Health Perspect

1 102427-431

Almstrup K, Fernandez MF, Petersen JH, Olea N, Skakkebaek NE,
Leffers H 2002 Dual effects of phytoestrogens result in u-shaped dose-
response curves. Environ Health Perspect 110:743—748

Jefferson WN, Newbold RR 2000 Potential endocrine-modulating effects
of various phytoestrogens in the diet. Nutrition 162658-662

North K, Golding J 2000 A maternal vegetarian diet in pregnancy is
associated with hypospadias. The ALSPAC Study Team. Avon
Longitudinal Study of Pregnancy and Childhood. BJU Int 85:107-113

Setchell KD 2001 Soy isoflavones—benefits and risks from nature's
selective estrogen receptor modulators (SERMs). J Am Coll Nutr 2013548-
362$

117

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

Newbold RR, Banks EP, Bullock B, Jefferson WM 2001 Uterine
adenocarcinoma in mice treated neonatally with genistein. Cancer Res
61:4325-4328

Liggins J, Bluck LJ, Runswick S, Atkinson C, Coward WA, Bingham
SA 2000 Daidzein and genistein content of fruits and nuts. J Nutr Biochem
1 1 :326-331

Reinli K, Block G 1996 Phytoestrogen content of foods—a compendium
of literature values. Nutr Cancer 262123-148

Fukutake M, Takahashi M, lshida K, Kawamura H, Sugimura T,
Wakabayashi K 1996 Quantification of genistein and genistin in soybeans
and soybean products. Food Chem Toxicol 342457-461

Etienne M, Jemmali M 1982 Effects of zearalenone (F2) on estrous
activity and reproduction in gilts. J Anim Sci 5521-10

Davis BJ, Weaver R, Gaines LJ, Heindel JJ 1994 Mono-(2-ethylhexyl)
phthalate suppresses estradiol production independent of FSH-CAMP
stimulation in rat granulosa cells. Toxicol Appl Pharmacol 128:224-228

Lovekamp TN, Davis BJ 2001 Mono-(2-ethylhexyl) phthalate suppresses
aromatase transcript levels and estradiol production in cultured rat
granulosa cells. Toxicol Appl Pharmacol 172:217-224

Andrade AJ, Grande SW, Talsness CE, Grote K, Chahoud l 2006 A
dose-response study following in utero and lactational exposure to di-(2-
ethylhexyl)-phthalate (DEHP): non-monotonic dose-response and low
dose effects on rat brain aromatase activity. Toxicology 2272185-192

Sharpe RM 2001 Hormones and testis development and the possible
adverse effects of environmental chemicals. Toxicol Lett 1202221-232

Sharpe RM 2006 Pathways of endocrine disruption during male sexual
differentiation and masculinization. Best Pract Res Clin Endocrinol Metab
20291-1 10

118

30.

31.

32.

33.

34.

35.

36.

37.

38.

Lottrup G, Andersson AM, Leffers H, Mortensen GK, Toppari J,
Skakkebaek NE, Main KM 2006 Possible impact of phthalates on infant
reproductive health. Int J Androl 292172-180

Labrie F, Simard J, Luu-The .V, Pelletier G, Belanger A, Lachance Y,
Zhao HF, Labrie C, Breton N, de Launoit Y 1992 Structure and tissue-
specific expression of 3 beta-hydroxysteroid dehydrogenase/S-ene-4-ene
isomerase genes in human and rat classical and peripheral steroidogenic
tissues. J Steroid Biochem Mol Biol 412421-435

Bellemare V, Faucher F, Breton R, Luu-The V 2005 Characterization of
17alpha-hydroxysteroid dehydrogenase activity (17alpha-HSD) and its
involvement in the biosynthesis of epitestosterone. BMC Biochem 6:12

Whitehead SA, Rice S 2006 Endocrine-disrupting chemicals as
modulators of sex steroid synthesis. Best Pract Res Clin Endocrinol Metab
20245-61

Ohno S, Shinoda S, Toyoshima S, Nakazawa H, Makino T, Nakajin S
2002 Effects of flavonoid phytochemicals on cortisol production and on
activities of steroidogenic enzymes in human adrenocortical H295R cells.
J Steroid Biochem Mol Biol 802355-363

Le Bail JC, Champavier Y, Chulia AJ, Habrioux G 2000 Effects of
phytoestrogens on aromatase, 3beta and 17beta-hydroxysteroid
dehydrogenase activities and human breast cancer cells. Life Sci 6621281-
1291

Wright AS, Thomas LN, Douglas RC, Lazier CB, Rittmaster RS 1996
Relative potency of testosterone and dihydrotestosterone in preventing
atrophy and apoptosis in the prostate of the castrated rat. J Clin Invest
98:2558—2563

Grino PB, Griffin JE, Wilson JD 1990 Testosterone at high
concentrations interacts with the human androgen receptor similarly to
dihydrotestosterone. Endocrinology 126:1 165-1 172

Yamamoto M, Narita A, Kagohata M, Shirai M, Akahori F, Arishima K
2005 Effects of maternal exposure to 3,3',4,4',5-pentachlorobiphenyl
(PCB126) or 3,3',4,4',5,5'-hexachlorobiphenyl (PCB169) on testicular

119

39.

40.

41.

42.

43.

45.

46.

47.

steroidogenesis and sperrnatogenesis in male offspring rats. J Androl
262205-214

Bretveld RW, Thomas CM, Scheepers PT, Zielhuis GA, Roeleveld N
2006 Pesticide exposure: the hormonal function of the female reproductive
system disrupted? Reprod Biol Endocrinol 4230

Stoker TE, Goldman JM, Cooper RL 2001 Delayed ovulation and
pregnancy outcome: effect of environmental toxicants on the
neuroendocrine control of the ovary. Environ Toxicol Pharmacol 92117-129

Costa LG, Olibet G, Murphy SD 1988 Alpha 2-adrenoceptors as a target
for fonnamidine pesticides: in vitro and in vivo studies in mice. Toxicol
Appl Phannacol 932319-328

Young FM, Menadue MF, Lavranos TC 2005 Effects of the insecticide
amitraz, an alpha2-adrenergic receptor agonist, on human luteinized
granulosa cells. Hum Reprod 20:3018-3025

Jongbloet PH 2004 Over-ripeness ovopathy: a challenging hypothesis for
sex ratio modulation. Hum Reprod 192769-774

Dunn JF, Nisula BC, Rodbard D 1981 Transport of steroid hormones:
binding of 21 endogenous steroids to both testosterone-binding globulin
and corticosteroid-binding globulin in human plasma. J Clin Endocrinol
Metab 53:58-68

Kragh-Hansen U 1981 Molecular aspects of ligand binding to serum
albumin. Pharmacol Rev 33:17-53

Sodergard R, Backstrom T, Shanbhag V, Carstensen H 1982
Calculation of free and bound fractions of testosterone and estradiol-17
beta to human plasma proteins at body temperature. J Steroid Biochem
16:801-810

Baker ME 2002 Albumin, steroid hormones and the origin of vertebrates.
J Endocrinol 175:121-127

120

48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

Kahn SM, Hryb DJ, Nakhla AM, Romas NA, Rosner W 2002 Sex
hormone-binding globulin is synthesized in target cells. J Endocrinol
17521 1 3-1 20

Crisp TM, Clegg ED, Cooper RL, Wood WP, Anderson DG, Baetcke
KP, Hoffmann JL, Morrow MS, Rodier DJ, Schaeffer JE, Touart LW,
Zeeman MG, Patel YM 1998 Environmental endocrine disruption: an
effects assessment andanalysis. Environ Health Perspect Suppl 1:11-56

Jones EJ, DeChemey AH 2003 The female reproductive system. In:
Boron WF, Boulpaep EL eds. Medical physiology. Philadelphia: Saunders;
1141-1208

Grow DR 2002 Metabolism of endogenous and exogenous reproductive
hormones. Obstet Gynecol Clin North Am 292425-436

Garreau B, Vallette G, Adlercreutz H, Wahala K, Makela T,
Benassayag C, Nunez EA 1991 Phytoestrogens: new ligands for rat and
human alpha-fetoprotein. Biochim Biophys Acta 1094:339-345

Bergstrand DG, Czar B 1957 Paper electrophoretic study of human fetal
serum proteins with demonstration of a new protein fraction. Scand J Clin
Lab Invest 9:277-286

Mizejewski GJ 2003 Levels of alpha-fetoprotein during pregnancy and
early infancy in normal and disease states. Obstet Gynecol Surv 58:804-
826

Zhu J, Barratt CL, Lippes J, Pacey AA, Lenton EA, Cooke ID 1994
Human oviductal fluid prolongs sperm survival. Fertil Steril 61:360-366

Zhu J, Barratt CL, Lippes J, Pacey AA, Cooke ID 1994 The sequential
effects of human cervical mucus, oviductal fluid, and follicular fluid on
sperm function. Fertil Steril 6121129-1135

Mizejewski GJ 1995 The phylogeny of alpha-fetoprotein in vertebrates:
survey of biochemical and physiological data. Crit Rev Eukaryot Gene
Expr 52281-316

121

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

Mizejewski GJ 2004 Biological roles of alpha-fetoprotein during
pregnancy and perinatal development. Exp Biol Med (Maywood) 2292439-
463

Hodgert JH, Zacharewski TR, Hammond GL 2000 Interactions between
human plasma sex hormone-binding globulin and xenobiotic ligands. J
Steroid Biochem Mol Biol 752167-176

Danzo BJ 1997 Environmental xenobiotics may disrupt normal endocrine
function by interfering with the binding of physiological ligands to steroid
receptors and binding proteins. Environ Health Perspect 105:294-301

Kelce WR, Stone CR, Laws SC, Gray LE, Kemppainen JA, Wilson EM
1995 Persistent DDT metabolite p,p'-DDE is a potent androgen receptor
antagonist. Nature 375:581-585

Kelce WR, Stone CR, Laws SC, Gray LE, Kemppainen JA, Jr., Wilson
EM 1995 Persistent DDT metabolite p,p'-DDE is a potent androgen
receptor antagonist. Nature 3752581

Nardulli AM, Katzenellenbogen BS 1986 Dynamics of estrogen receptor
turnover in uterine cells in vitro and in uteri in vivo. Endocrinology
1 19:2038-2046

Pakdel F, Le Goff P, Katzenellenbogen BS 1993 An assessment of the
role of domain F and PEST sequences in estrogen receptor half-life and
bioactivity. J Steroid Biochem Mol Biol 462663-672

Alarid ET, Bakopoulos N, Solodin N 1999 Proteasome-mediated
proteolysis of estrogen receptor: a novel component in autologous down-
regulation. Mol Endocrinol 13:1522-1534

Lonard DM, Nawaz Z, Smith CL, O'Malley BW 2000 The 265
proteasome is required for estrogen receptor-alpha and coactivator
turnover and for efficient estrogen receptor-alpha transactivation. Mol Cell
52939-948

Alarid ET, Preisler-Mashek MT, Solodin NM 2003 Thyroid hormone is
an inhibitor of estrogen-induced degradation of estrogen receptor-alpha

122

68.

69.

70.

71.

72.

73.

74.

75.

76.

protein: estrogen-dependent proteolysis is not essential for receptor
transactivation function in the pituitary. Endocrinology 144:3469-3476

Fan M, Bigsby RM, Nephew KP 2003 The NEDD8 pathway is required
for proteasome-mediated degradation of human estrogen receptor (ER)-
alpha and essential for the antiproliferative activity of ICI 182,780 in
ERalpha-positive breast cancer cells. Mol Endocrinol 172356-365

O'Brian CA, Liskamp RM, Solomon DH, Weinstein IB 1985 Inhibition of
protein kinase C by tamoxifen. Cancer Res 45:2462-2465

Safe SH 1995 Environmental and dietary estrogens and human health: is
there a problem? Environ Health Perspect 1032346-351

Safe S, Astroff B, Harris M, Zacharewski T, Dickerson R, Romkes M,
Biegel L 1991 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related
compounds as antioestrogens: characterization and mechanism of action.
Pharmacol Toxicol 692400-409

Strichman-Almashanu LZ, Lee RS, Onyango PO, Perlman E, Flam F,
Frieman MB, Feinberg AP 2002 A genome-wide screen for normally
methylated human CpG islands that can identify novel imprinted genes.
Genome Res 122543-554

Reik W, Dean W, Walter J 2001 Epigenetic reprogramming in
mammalian development. Science :0: 293:1089-1093

Jiang YH, Bressler J, Beaudet AL 2004 Epigenetics and human
disease. Annu Rev Genomics Hum Genet 52479-510

Allegrucci C, Thurston A, Lucas E, Young L 2005 Epigenetics and the
gerrnline. Reproduction 129:137-149

Newbold RR, Padilla-Banks E, Jefferson WN 2006 Adverse effects of
the model environmental estrogen diethylstilbestrol are transmitted to
subsequent generations. Endocrinology 147:Si 1-17

123

77.

78.

79.

80.

81.

82.

83.

84.

85.

 

Anway MD, Memon MA, Uzumcu M, Skinner MK 2006
Transgenerational effect of the endocrine disruptor vinclozolin on male
spennatogenesis. J Androl Epub ahead of print

Lee J, Inoue K, Ono R, Ogonuki N, Kohda T, Kaneko-lshino T, Ogura
A, lshino F 2002 Erasing genomic imprinting memory in mouse clone
embryos produced from day 11.5 primordial germ cells. Development
12921807-1817

Li S, Hursting SD, Davis BJ, McLachIan JA, Barrett JC 2003
Environmental exposure, DNA methylation, and gene regulation: lessons
from diethylstilbesterol-lnduced cancers. Ann N Y Acad Sci 9832161-169

Skinner MK, Anway MD 2005 Seminiferous cord formation and germ-cell
programming: epigenetic transgenerational actions of endocrine
disruptors. Ann N Y Acad Sci 1061218-32

Veurink M, Koster M, Berg LT 2005 The history of DES, lessons to be
Ieamed. Phann Worid Sci 272139-143

Newbold RR, Hanson RB, Jefferson WN, Bullock BC, Haseman J,
McLachIan JA 1998 Increased tumors but uncompromised fertility in the
female descendants of mice exposed developmentally to diethylstilbestrol.
Carcinogenesis 19: 1 655-1 663

Li S, Washbum KA, Moore R, Uno T, Teng C, Newbold RR,
McLachIan JA, Negishi M 1997 Developmental exposure to
diethylstilbestrol elicits demethylation of estrogen-responsive lactoferrin
gene in mouse uterus. Cancer Res 57:4356-4359

Li S, Hansman R, Newbold R, Davis B, McLachIan JA, Barrett JC
2003 Neonatal diethylstilbestrol exposure induces persistent elevation of
c-fos expression and hypomethylation in its exon-4 in mouse uterus. Mol
Carcinog 38:78-84

Anway MD, Cupp AS, Uzumcu M, Skinner MK 2005 Epigenetic
transgenerational actions of endocrine disruptors and male fertility.
Science 308:1466-1469

124

86.

87.

88.

89.

90.

91.

92.

93.

94.

95.

Mebrahtu T, Mohamed A, Wang CY, Andebrhan T 2004 Analysis of
isoflavone contents in vegetable soybeans. Plant Foods Hum Nutr 59:55-
61

Mazur W 1998 Phytoestrogen content in foods. Baillieres Clin Endocrinol
Metab 122729-742

Shapiro S, Slone D 1979 The effects of exogenous female hormones on
the fetus. Epidemiol Rev 1 :1 10-123

Mussman HC 1975 Drug and chemical residues in domestic animals. Fed
Proc 342197-201

Kuiper GG, Lemmen JG, Carlsson B, Corton JC, Safe SH, van der
Saag PT, van der Burg B, Gustafsson JA 1998 Interaction of estrogenic
chemicals and phytoestrogens with estrogen receptor beta. Endocrinology
1 39:4252-4263

Belcher SM, Zsamovszky A 2001 Estrogenic actions in the brain:
estrogen, phytoestrogens, and rapid intracellular signaling mechanisms. J
Pharmacol Exp Ther 299:408-414

Murrill WB, Brown NM, Zhang JX, Manzolillo PA, Barnes S,
Lamartiniere CA 1996 Prepubertal genistein exposure suppresses
mammary cancer and enhances gland differentiation in rats.
Carcinogenesis 1 721451-1457

Shen F, Xue X, Weber G 1999 Tamoxifen and genistein synergistically
down-regulate signal transduction and proliferation in estrogen receptor-
negative human breast carcinoma MDA-MB-435 cells. Anticancer Res
19:1657-1662

Setchell KD, Zimmer-Nechemias L, Cai J, Heubi JE 1998 lsoflavone
content of infant formulas and the metabolic fate of these phytoestrogens
in early life. Am J Clin Nutr 68:14538-14618

Setchell KD, Zimmer-Nechemias L, Cai J, Heubi JE 1997 Exposure of
infants to phyto-oestrogens from soy-based infant formula. Lancet 350223-
27

125

96.

97.

98.

99.

100.

101.

102.

103.

104.

 

Flynn KM, Ferguson SA, Delclos KB, Newbold RR 2000 Effects of
genistein exposure on sexually dimorphic behaviors in rats. Toxicol Sci
55:31 1-319.

Jefferson WN, Couse JF, Padilla-Banks E, Korach KS, Newbold RR
2002 Neonatal exposure to genistein induces estrogen receptor (ER)alpha
expression and multioocyte follicles in the maturing mouse ovary:
evidence for ERbeta-mediated and nonestrogenic actions. Biol Reprod
67:1285—1296

Jefferson WN, Padilla-Banks E, Newbold RR 2006 Studies of the
effects of neonatal exposure to genistein on the developing female
reproductive system. J AOAC Int 89:1189-1 196

Hughes CL, Liu G, Beall S, Foster WG, Davis V 2004 Effects of
genistein or soy milk during late gestation and lactation on adult uterine
organization in the rat. Exp Biol Med (Maywood) 2292108-1 17

Fielden MR, Samy SM, Chou KC, Zacharewski TR 2003 Effect of
human dietary exposure levels of genistein during gestation and lactation
on long-term reproductive development and sperm quality in mice. Food
Chem Toxicol 41:447-454.

Fritz WA, Cotroneo MS, Wang J, Eltoum IE, Lamartiniere CA 2003
Dietary diethylstilbestrol but not genistein adversely affects rat testicular
development. J Nutr 133:2287-2293.

Minervini F, Dell'Aquila ME, Maritato F, Minoia P, Visconti A 2001
Toxic effects of the mycotoxin zearalenone and its derivatives on in vitro
maturation of bovine oocytes and 17 beta-estradiol levels in mural
granulosa cell cultures. Toxicol In Vitro 152489-495

Bottalico A 1998 Fusarium diseses of creals: species complex and
related mycotoxin profiles, in Europe. Journal of Plant Pathology 80285-103

Scudamore KA, Nawaz S, Hetmanski MT 1998 Mycotoxins in
ingredients of animal feeding stuffs: ll. Determination of mycotoxins in
maize and maize products. Food Addit Contam 15:30-55

126

105.

106.

107.

108.

109.

110.

111.

112.

113.

114.

Scudamore KA, Patel S 2000 Survey for aflatoxins, ochratoxin A,
zearalenone and fumonisins in maize imported into the United Kingdom.
Food Addit Contam 172407-416

Lagana A, Bacaloni A, De Leva I, Faberi A, Fago G, Marino A 2004
Analytical methodologies for determining the occurrence of endocrine
disrupting chemicals in sewage treatment plants and natural waters.
Analytica Chemica Acta 501 :79-88

Doyle E 2000 Human safety of hormone implants used to promote growth
in cattle. In:

Galbraith H 2002 Hormones in international meat production: biological,
sociological and consumer issues. Nutrition Research Reviews 15:293-
314

EI-Hoshy SM 1999 Occurrence of zearalenone in milk, meat and their
products with emphasis on influence of heat treatments on its level. Archiv
fur Lebensmittelhygiene 502140-143

Kuiper-Goodman T, Scott PM, Watanabe H 1987 Risk assessment of
the mycotoxin zearalenone. Regul Toxicol Pharmacol 72253-306

Etienne M, Doumiad JY 1994 Effects of zearalenone or glucosinolates in
the diet on reproduction in sows: a review. Livestock Porudction Science
40299-113

Boyd PA, Wittliff JL 1978 Mechanism of Fusarium mycotoxin action in
mammary gland. J Toxicol Environ Health 421-8

Fitzpatrick DW, Picken CA, Murphy LC, Buhr MM 1989 Measurement
of the relative binding affinity of zearalenone, alpha-zearalenol and beta-
zearalenol for uterine and oviduct estrogen receptors in swine, rats and
chickens: an indicator of estrogenic potencies. Comp Biochem Physiol C
942691-694

Kiang DT, Kennedy BJ, Pathre SV, Mirocha CJ 1978 Binding
characteristics of zearalenone analogs to estrogen receptors. Cancer Res
38:3611-3615

127

115.

116.

117.

118.

119.

120.

121.

122.

123.

124.

125.

Ramosa AJ, Hemandezb E, Pla-Delfinac JM, Merino M 1996 Intestinal
absorption of zearalenone and in vitro study of non-nutritive sorbent
materials. Int J Pharrn 128:129-137

Le Guevel R, Pakdel F 2001 Assessment of oestrogenic potency of
chemicals used as growth promoter by in—vitro methods. Hum Reprod
1621030—1036

Diekman MA, Green ML 1992 Mycotoxins and reproduction in domestic
livestock. J Anim Sci 70:1615—1627

Kallela K, Ettala E 1984 The oestrogenic Fusarium toxin (zearalenone) in
hay as a cause of early abortions in the cow. Nord Vet Med 362305-309

Mirocha CJ, Schauerhamer B, Pathre SV 1974 Isolation, detection, and
quantitation of zearalenone in maize and bariey. J Assoc Off Anal Chem
5721104-1 1 10

Weaver GA, Kurtz HJ, Behrens JC, Robison TS, Seguin BE, Bates FY,
Mirocha CJ 1986 Effect of zearalenone on the fertility of virgin dairy
heifers. Am J Vet Res 47:1395—1397

Dodds EC, Goldberg L, Larson W, Robinson R 1938 Estrogenic activity
of certain synthetic compounds. Nature 1412247

Noller KL, Fish CR 1974 Diethylstilbestrol usage: Its interesting past,
important present, and questionable future. Med Clin North Am 58:793-
810

Dieckmann WJ, Davis ME, Rynkiewicz LM, Pottinger RE 1953 Does
the administration of diethylstilbestrol during pregnancy have therapeutic
value? Am J Obstet Gynecol 66:1062-1081

Giusti RM, lwamoto K, Hatch EE 1995 Diethylstilbestrol revisited: a
review of the long-term health effects. Ann Intern Med 122:778-788

Herbst AL, Ulfelder H, Poskanzer DC 1971 Adenocarcinoma of the
vagina. Association of maternal stilbestrol therapy with tumor appearance
in young women. N Engl J Med 284:878-881

128

126.

127.

128.

129.

130.

131.

132.

133.

134.

135.

Herbst AL, Ulfelder H, Poskanzer DC 1971 Adenocarcinoma of the
vagina. N Engl J Med 284:878-881

DeChemey AH, Cholst I, Naftolin F 1981 Structure and function of the
fallopian tubes following exposure to diethylstilbestrol (DES) during
gestation. Fertil Steril 1981 Dec;36(6):741-5 362741 -745

Greco TL, Duello TM, Gorski J 1993 Estrogen receptors, estradiol, and
diethylstilbestrol in early development: the mouse as a model for the study
of estrogen receptors and estrogen sensitivity in embryonic development
of male and female reproductive tracts. Endocrine reviews 14259-71

Noller KL 1977 The ”moming-after pill". Fertil Steril 282211

Gill WB, Schumacher GF, Bibbo M, Straus FHn, Schoenberg HW 1979
Association of diethylstilbestrol exposure in utero with cryptorchidism,
testicular hypoplasia and semen abnormalities. J Urol 122236-39

Storgaard L, Bonde JP, Olsen J 2006 Male reproductive disorders in
humans and prenatal indicators of estrogen exposure. A review of
published epidemiological studies. Reprod Toxicol 2124-15

Newbold RR, Bullock BC, McLachIan JA 1987 Testicular tumors in mice
exposed in utero to diethylstilbestrol. J Urol 13821446-1450

Emmen JM, McLuskey A, Adham IM, Engel W, Verhoef Post M,
Themmen AP, Grootegoed JA, Brinkmann A0 2000 Involvement of
insulin-like factor 3 (lnsl3) in diethylstilbestrol-induced cryptorchidism.
Endocrinology 141 2846-849

Baillie-Hamilton PF 2002 Chemical toxins: a hypothesis to explain the
global obesity epidemic. J Altem Complement Med 8:185—192

Newbold RR, Padilla-Banks E, Snyder RJ, Jefferson WN 2005
Developmental exposure to estrogenic compounds and obesity. Birth
Defects Res A Clin Mol Teratol 732478-480

129

136.

137.

138.

139.

140.

141.

142.

143.

144.

145.

Jordan VC 1995 Tamoxifen: toxicities and drug resistance during the
treatment and prevention of breast cancer. Annu Rev Pharmacol Toxicol
35

Gill-Sharma MK, Gopalkrishnan K, Balasinor N, Parte P, Jayaraman
S, Juneja HS 1993 Effects of tamoxifen on the fertility of male rats. J
Reprod Fertil 992395-402

Shelby MD, Newbold RR, Tully DB, Chae K, Davis VL 1996 Assessing
environmental chemicals for estrogenicity using a combination of in vitro
and in vivo assays. Environ Health Perspect10421296-1300

Coezy E, Borgna JL, Rochefort H 1982 Tamoxifen and metabolites in
MCF7 cells: correlation between binding to estrogen receptor and
inhibition of cell growth. Cancer Res 422317-323

Hann LE, Giess CS, Bach AM, Tao Y, Baum HJ, Barakat RR 1997
Endometrial thickness in tamoxifen-treated patients: correlation with
clinical and pathologic findings. AJR Am J Roentgenol 168:657-661

Sabenlval GS, Gill-Sharma MK, Balasinor N, Choudhary J, Padwal V,
Juneja HS 2003 Tamoxifen, protein kinase C and rat sperm mitochondria.
Cell Biol Int 272761-768

D'Souza UJ 2004 Effect of tamoxifen on spennatogenesis and tubular
morphology in rats. Asian J Androl 62223-226

Parte P, Balasinor N, Glll-Sharma MK, Maitra A, Juneja HS 2002
Temporal effect of tamoxifen on cytochrome P450 side chain cleavage
gene expression and steroid concentration in adult male rats. J Steroid
Biochem Mol Biol 822349-358

Gill-Shanna MK, Balasinor N, Parte P, Aleem M, Juneja HS 2001
Effects of tamoxifen metabolites on fertility of male rat. Contraception
632103-1 09

Balasinor N, Parte P, Gill-Sharma MK, Juneja HS 2001 Effect of
tamoxifen on sperm fertilising ability and preimplantation embryo
development. Molecular and Cellular Endocrinology 178:199-206.

130

146.

147.

148.

149.

150.

151.

152.

153.

‘1 54.

Hyder SM, Chiappetta C, Stancel GM 1999 Synthetic estrogen 17alpha-
ethinyl estradiol induces pattern of uterine gene expression similar to
endogenous estrogen 17beta-estradiol. J Phannacol Exp Ther 2902740-
747

Thayer KA, Ruhlen RL, Howdeshell KL, Buchanan DL, Cooke PS,
Preziosi D, Welshons WV, Haseman J, Vom Saal FS 2001 Altered
prostate growth and daily sperm production in male mice exposed
prenatally to subclinical doses of 17alpha-ethinyl oestradiol. Hum Reprod
162988-996.

Miyamoto Y, Ueda K, Oshida K, Ohmori E 2000 Collaborative work to
evaluate toxicity on male reproductive organs by repeated dose studies in
rats 2). Testicular toxicity in rats treated orally with ethinylestradiol for 2
weeks. J Toxicol Sci 25:33-42.

Yasuda Y, Kihara T, Tanimura T, Nishimura H 1985 Gonadal
dysgenesis induced by prenatal exposure to ethinyl estradiol in mice.
Teratology 32:219-227.

Andrews P, Freyberger A, Hartrnann E, Eiben R, Loof I, Schmidt U,
Temerowski M, Folkerts A, Stahl B, Kayser M 2002 Sensitive detection
of the endocrine effects of the estrogen analogue ethinylestradiol using a
modified enhanced subacute rat study protocol (OECD Test Guideline no.
407). Arch Toxicol 762194-202

Slikker W, Jr., Bailey JR, Newport D, Lipe GW, Hill DE 1982 Placental
transfer and metabolism of 17 alpha-ethynylestradiol-17 beta and
estradiol-17 beta in the rhesus monkey. J Pharmacol Exp Ther 2232483-
489

Kallen B, Mastroiacovo P, Lancaster PA, Mutchinick O, Kringelbach
M, Martinez-Frias ML, Robert E, Castilla EE 1991 Oral contraceptives in
the etiology of isolated hypospadias. Contraception 442173-182

Li DK, Daling JR, Mueller BA, Hickok DE, Fantel AG, Weiss NS 1995
Oral contraceptive use after conception in relation to the risk of congenital
urinary tract anomalies. Teratology 51:30-36

DeSesso JM 1997 Comparative embryology. Handbook of
developmental toxicology. In. New York:: CRC Press; 111-174

131

155.

156.

157.

158.

159.

160.

161.

162.

163.

164.

Longcope C, Gorbach S, Goldin B, Woods M, Dwyer J, Warram J
1985 The metabolism of estradiol; oral compared to intravenous
administration. J Steroid Biochem 23:1065—1070

Speck U, Wendt H, Schulze PE, Jentsch D 1976 Bio-availability and
pharrnacokinetics of cyproterone acetate-14C and ethinyloestradioI-3H
after oral administration as a coated tablet (SH B 209 AB). Contraception
142151-163

Goldzieher JW, Mileikowsky G, Newburger J, Dorantes A,
Stavchansky SA 1988 Human pharrnacokinetics of ethynyl estradiol 3-
sulfate and 17-sulfate. Steroids 51 :63-79

Pasqualini JR, Castellet R, Portols MC, Hill JL, Kincl FA 1977 Plasma
concentrations of ethynyl oestradiol and norethindrone after oral
administration to women. J Reprod Fertil 492189-193

Maggs JL, Grimmer SF,,Orme ML, Breckenridge AM, Park BK,
Gilmore IT 1983 The biliary and urinary metabolites of [3H]17 alpha-
ethynylestradiol in women. Xenobiotica 132421-431

Williams MC, Goldzieher JW 1980 Chromatographic patterns of urinary
ethynyl estrogen metabolites in various populations. Steroids 362255-282

Donn J, Mendoza M, Pritchard J 2008 AP Probe Finds Drugs in Drinking
Water. In: The Associated Press. New York

Aheme GW, Briggs R 1989 The relevance of the presence of certain
synthetic steroids in the aquatic environment. Pharrn Pharmacol 41:735-
736.

Tabak HH, Bloomhuff RN, Bunch RL 1981 Steroid hormones as water
pollutants, ll. Studies on the persistence and stability of natural urinary
and synthetic ovulation-inhibiting hormones in untreated and treated
wastewaters. Dev Ind Microbiol 222497-519

Williams RJ, Johnson AC, Smith JJ, Kanda R 2003 Steroid estrogens
profiles along river stretches arising from sewage treatment works
discharges. Environ Sci Technol 3721744-1750

132

165.
166.

167.

168.

169.

170.

171.

172.

173.

174.

Roofer P, Snyder S, Zegers RE, Rexing DJ, Fronk JL 2000 Endocrine-
disrupting chemicals in a so'urce water. J AWWA 92:52-58

US EPA 2000 Estimated per capita water ingestion in the United States.
In. Washington DC: US EPA, Office of Water 1301; 40

US EPA 2001 National primary drinking water regulations; arsenic and
clarifications to compliance and new source contaminants monitoring; final
rule. In. Washington DC: US EPA

Gutendorf B, Westendorf J 2001 Comparison of an array of in vitro
assays for the assessment of the estrogenic potential of natural and

synthetic estrogens, phytoestrogens and xenoestrogens. Toxicology
1 66:79-89.

Von Schoultz B 1988 Potency of different oestrogen preparations. In:
Studd JWW, Whitehead Ml eds. The menopause. Oxford: Blackwell
Scientific Publications; 130-137

Branham WS, Zehr DR, Sheehan DM 1993 Differential sensitivity of rat
uterine growth and epithelium hypertrophy to estrogens and antiestrogens.
Proc Soc Exp Biol Med 203:297-303

Van den Belt K, Berckmans P, Vangenechten C, Verheyen R, Witters
H 2004 Comparative study on the in vitro/in vivo estrogenic potencies of
17beta-estradiol, estrone, 17alpha-ethynylestradiol and nonylphenol.
Aquat Toxicol 662183-195

Yasuda Y, Ohara I, Konishi H, Tanimura T 1988 Long-term effects on
male reproductive organs of prenatal exposure to ethinyl estradiol. Am J
Obstet Gynecol 1 59:1246-1250

Kaneto M, Kanamori S, Hishikawa A, Kishi K 1999 Epididymal sperm
motion as a parameter of male reproductive toxicity: sperm motion,
fertility, and histopathology in ethinylestradiol-treated rats. Reprod Toxicol
13(4): 2279-289.

Lafuente A, Marquez N, Pousada Y, Pazo D, Esquifino Al 2000
Possible estrogenic and/or antiandrogenic effects of methoxychlor on
prolactin release in male rats. Arch Toxicol 742270-275

133

175.

176.

177.

178.

179.

180.

181.

182.

183.

Bulger WH, Muccitelli RM, Kupfer D 1978 Interactions of chlorinated
hydrocarbon pesticides with the BS estrogen-binding protein in rat testes.
Steroids 322165-177

Suzuki M, Lee HC, Chiba S, Yonezawa T, Nishihara M 2004 Effects of
methoxychlor exposure during perinatal period on reproductive function
after maturation in rats. J Reprod Dev 502455-461

Laws SC, Carey SA, Ferrell JM, Bodman GJ, Cooper RL 2000
Estrogenic activity of octylphenol, nonylphenol, bisphenol A and
methoxychlor in rats. Toxicol Sci 5421 54-167

Gaido KW, Leonard LS, Maness SC, Hall JM, McDonnell DP, Saville
B, Safe S 1999 Differential interaction of the methoxychlor metabolite 2,2-
bis-(p-hydroxyphenyI)-1,1,1-trichloroethane with estrogen receptors alpha
and beta. Endocrinology 140:5746-5753

Gaido KW, Maness SC, McDonnell DP, Dehal SS, Kupfer D, Safe S
2000 Interaction of methoxychlor and related compounds with estrogen
receptor alpha and beta, and androgen receptor: structure-activity studies.
Mol Phannacol 582852-858

Smeets JM, van Holsteijn I, Giesy JP, Seinen W, van den Berg M 1999
Estrogenic potencies of several environmental pollutants, as determined
by vitellogenin induction in a carp hepatocyte assay. Toxicol Sci 50:206-
21 3

Cummings AM 1997 Methoxychlor as a model for environmental
estrogens. Crit Rev Toxicol 272367-379

Tiemann U 2008 In vivo and in vitro effects of the organochlorine
pesticides DDT, TCPM, methoxychlor, and lindane on the female
reproductive tract of mammals: a review. Reproductive toxicology
(Elmsford, NY 252316-326

Amstislavsky SY, Amstislavskaya TG, Eroschenko VP 1999
Methoxychlor given in the periimplantation period blocks sexual arousal in
male mice. Reprod Toxicol 132405-411

134

184.

185.

186.

187.

188.

189.

190.

191.

192.

Akingbemi BT, Ge RS, Klinefelter GR, Gunsalus GL, Hardy MP 2000 A
metabolite of methoxychlor, 2,2-bis(p-hydroxyphenyI)-1,1, 1-
trichloroethane, reduces testosterone biosynthesis in rat leydig cells
through suppression of steady-state messenger ribonucleic acid levels of
the cholesterol side-chain cleavage enzyme. Biol Reprod 622571-578

Dalton R 2002 Frogs put in the gender blender by America's favourite
herbicide. Nature 4162665-666

Steeger T 2003 White paper on potential developmental effects of
atrazine on amphibians. In. Washington, DC: Office of Prevention,
Pesticides and Toxic Substances, Office of Pesticide Programs,
Environmental Fate and Effects Division; 1-95

Gammon DW, Aldous CN, Carr WCJ, Sanbom JR, Pfeifer KF 2005 A
risk assessment of atrazine use in California: human health and ecological
aspects. Pest Manag Sci 612331-355

Solomon KR, Baker DB, Richards RP, Dixon KR, Klaine WJ, LaPoint
TW, Kendall RJ, Weisskopf CP, Giddings JM, Giesy JP, Hall LWJ,
Williams WM 1996 Ecological risk assessment of atrazine in North
American surface waters. Environ Toxicol Chem 15:31-76

Du Preez LH, Jansenvan Rensburg PJ, Jooste AM, Carr JA, Giesy JP,
Gross TS, Kendall RJ, Smith EE, Van Der Kraak G, Solomon KR 2005
Seasonal exposures to triazine and other pesticides in surface waters in
the western Highveld com-production region in South Africa. Environ
Pollut 135:131-141

Hauswirth JW, Wetzel LT 1998 Toxicity characteristics of the 2-
chlorotriazines atrazine and simazine. In: Ballantine LG, McFarland JE,
Hackett DS eds. Triazine herbicides: Risk assessmen. Washington, DC:
Oxford University Press; 370-383

Laws SC, Ferrell JM, Stoker TE, Schmid J, Cooper RL 2000 The
effects of atrazine on female wistar rats: an evaluation of the protocol for

assessing pubertal development and thyroid function. Toxicol Sci ;()
582366-376

Friedmann AS 2002 Atrazine inhibition of testosterone production in rat
males following peripubertal exposure. Reprod Toxicol 162275-279

135

193.

194.

195.

196.

197.

198.

199.

200.

201.

202.

Trentacoste SV, Friedmann AS, Youker RT, Breckenridge CB, Zirkin
BR 2001 Atrazine effects on testosterone levels and androgen-dependent
reproductive organs in peripubertal male rats. J Androl 222142-148

Stoker TE, Laws SC, Guidici DL, Cooper RL 2000 The effect of atrazine
on puberty in male wistar rats: an evaluation in the protocol for the
assessment of pubertal development and thyroid function. Toxicol Sci
58:50-59

Cooper RL, Stoker TE, Tyrey L, Goldman JM, McElroy WK 2000
Atrazine disrupts the hypothalamic control of pituitary-ovarian function.
Toxicol Sci 532297-307

Eldridge JC, Wetzel LT, Stevens JT, Simpkins JW 1999 The mammary
tumor response in triazine-treated female rats: a threshold-mediated
interaction with strain and species-specific reproductive senescence.
Steroids 642672-678

Ashby J, Tinwell H, Stevens J, Pastoor T, Breckenridge CB 2002 The
effects of atrazine on the sexual maturation of female rats. Regul Toxicol
Pharmacol 352468-473

Dianin AP 1914 Molecular structure in relation to oestrogenic activity.
Compounds without a phenanthrene nucleus. Russ Phys Chem Soc
3621310

Burridge E 2003 Bisphenol A: product profile. Eur Chem News 14-20217

Kang JH, Kito K, Kondo F 2003 Factors influencing the migration of
bisphenol A from cans. J Food Prot 6621444-1447

Olea N, Pulgar R, Perez P, Olea-Serrano F, Rivas A, NovilIo-Fertrell A,
Pedraza V, Soto AM, Sonnenschein C 1996 Estrogenicity of resin-based
composites and sealants used in dentistry. Environ Health Perspect
1042298-305

Kang JH, Kondo F 2002 Determination of bisphenol A in canned pet
foods. Res Vet Sci 732177-182

136

203.

204.

205.

206.

207.

208.

209.

210.

211.

212.

Fromme H, Kuchler T, Otto T, Pilz K, Muller J, Wenzel A 2002
Occurrence of phthalates and bisphenol A and F in the environment.
Water Res 3621429-1438

Volkel W, Colnot T, Csanady GA, Filser JG, Dekant W 2002
Metabolism and kinetics of bisphenol a in humans at low doses following
oral administration. Chem Res Toxicol 15:1281-1287

Calafat AM, Kuklenyik Z, Reidy JA, S.P. C, Ekong J, Needham LL
2005 Urinary concentrations of bisphenol A and 4-nonylphenol in a human
reference population. Environ Health Perspect 113:391-395

Ikezuki Y, Tsutsumi O, Takai Y, Kamei Y, Taketani Y 2002
Determination of bisphenol A concentrations in human biological fluids
reveals significant early prenatal exposure. Hum Reprod 17:2839—2841

vom Saal FS, Hughes C 2005 An extensive new literature concerning
low-dose effects of bisphenol A shows the need for a new risk
assessment. Environ Health Perspect1132926-933

ECSCF 2002 Opinion of the Scientific Committee on Food on Bisphenol
A. SCF/CS/PM/3938 Final. In: BrusselszEuropean Commission Scientific
Committee on Food

Degen GH, Bolt HM 2000 Endocrine disruptors: update on
xenoestrogens. Int Arch Occup Environ Health 732433-441

Bolt HM, Janning P, Michna H, Degen GH 2001 Comparative
assessment of endocrine modulators with oestrogenic activity: I. Definition
of a hygiene-based margin of safety (HBMOS) for xeno-oestrogens
against the background of European developments. Arch Toxicol 74:649-
662

Kang JH, Kondo F, Katayama Y 2006 Human exposure to bisphenol A.
Toxicology Epub ahead of print

Morrissey RE, Lamb JCt, Morris RW, Chapin RE, Gulati DK, Heindel
JJ 1989 Results and evaluations of 48 continuous breeding reproduction
studies conducted in mice. Fundam Appl Toxicol 132747-777

137

213.

214.

215.

216.

217.

218.

219.

220.

221.

Hardin 80, Bond GP, Sikov MR, Andrew FD, Beliles RP, Niemeier RW
1981 Testing of selected workplace chemicals for teratogenic potential.
Scand J Work Environ Health 7:66-75

Morrissey RE, George JD, Price CJ, Tyl RW, Marr MC, Kimmel CA
1987 The developmental toxicity of bisphenol A in rats and mice. Fundam
Appl Toxicol 82571-582

Dodds EC, Lawson W 1936 Synthetic estrogenic agents without the
phenanthrene nucleus. Nature 1372996

Dodds EC, Lawson W 1938 Molecular stmcture in relation to oestrogenic
activity. Compounds without a phenanthrene nucleus. Proceedings of the
Royal Society London B 1252222-232

Krishnan AV, Stathis P, Pennuth SF, Tokes L, Feldman D 1993
Bisphenol-A2 an estrogenic substance is released from polycarbonate
flasks during autoclaving. Endocrinology 132:2279-2286

Gaido KW, Leonard LS, Lovell S, Gould JC, Babai D, Portier CJ,
McDonnell DP 1997 Evaluation of chemicals with endocrine modulating
activity in a yeast-based steroid hormone receptor gene transcription
assay. Toxicol Appl Pharmacol 1432205—212

Mohri T, Yoshida S 2005 Estrogen and bisphenol A disrupt spontaneous
[Ca(2+)](i) oscillations in mouse oocytes. Biochem Biophys Res Commun
326:166-173

vom Saal FS, Cooke PS, Buchanan DL, Palanza P, Thayer KA, Nagel
SC, Parrnigiani S, Welshons WV 1998 A physiologically based approach
to the study of bisphenol A and other estrogenic chemicals on the size of
reproductive organs, daily sperm production, and behavior. Toxicol Ind
Health 142239-260

Markey CM, Luque EH, Munoz De Toro M, Sonnenschein C, Soto AM
2001 In utero exposure to bisphenol A alters the development and tissue
organization of the mouse mammary gland. Biol Reprod 65:1215-1223

138

222.

223.

224.

225.

226.

227.

228.

229.

230.

231.

Ashby J, Tinwell H, Haseman J 1999 Lack of effects for low dose levels
of bisphenol A and diethylstilbestrol on the prostate gland of CF1 mice
exposed in utero. Regul Toxicol Phannacol 3021 56-166

Cagen SZ, Waechter JMJ, Dimond SS, Breslin WJ, Butala JH, Jekat
FW, Joiner RL, Shiotsuka RN, Veenstra GE, Harris LR 1999 Normal
reproductive organ development in CF-1 mice following prenatal exposure
to bisphenol A. Toxicol Sci 50236-44

Masuno H, Kidani T, Sekiya K, Sakayama K, Shiosaka T, Yamamoto
H, Honda K 2002 Bisphenol A in combination with insulin can accelerate
the conversion of 3T3-L1 fibroblasts to adipocytes. J Lipid Res 432676-684

Alonso-Magdalena P, Morimoto S, Ripoll C, Fuentes E, Nadal A 2006
The estrogenic effect of bisphenol A disrupts pancreatic beta-cell function
in vivo and induces insulin resistance. Environ Health Perspect 1142106-
1 12

Sakurai K, Kawazuma M, Adachi T, Harigaya T, Saito Y, Hashimoto N,
Mori C 2004 Bisphenol A affects glucose transport in mouse 3T3-F442A
adipocytes. Br J Pharmacol 141:209-214

Thomas JA, Thomas MJ 1984 Biological effects of di-(2-ethylhexyl)
phthalate and other phthalic acid esters. Crit Rev Toxicol 132283-317

Stroheker T, Cabaton N, Nourdin G, Regnier JF, Lhuguenot JC,
Chagnon MC 2005 Evaluation of anti-androgenic activity of dl-(2-
ethylhexyl)phthalate. Toxicology 20821 15-121

Mahood IK, Hallmark N, McKinnell C, Walker M, Fisher JS, Sharpe RM
2005 Abnormal Leydig cell aggregation in the fetal testis of rats exposed
to di (n-butyl) phthalate and its possible role in testicular dysgenesis.
Endocrinology 146261 3-623

Fennell TR, Krol WL, Sumner SC, Snyder RW 2004 Phannacokinetics
of dibutylphthalate in pregnant rats. Toxicol Sci 822407-418

Hotchkiss AK, Parks-Saldutti LG, Ostby JS, Lambright C, Furr J,
Vandenbergh JG, Gray LEJ 2004 A mixture of the "antiandrogens"

139

232.

233.

234.

235.

236.

237.

238.

239.

240.

Iinuron and butyl benzyl phthalate alters sexual differentiation of the male
rat in a cumulative fashion. Biol Reprod 71:1852-1861

Salazar V, Castillo C, Ariznavarreta C, Campon R, Tresguerres JA
2004 Effect of oral intake of dibutyl phthalate on reproductive parameters
of Long Evans rats and pre-pubertal development of their offspring.
Toxicology 2052131-137

Masutomi N, Shibutani M, Takagi H, Uneyama C, Takahashi N, Hirose
M 2003 Impact of dietary exposure to methoxychlor, genistein, or
diisononyl phthalate during the perinatal period on the development of the
rat endocrine/reproductive systems in later life. Toxicology 1922149—170

Sohoni P, Sumpter JP 1998 Several environmental oestrogens are also
anti-androgens. J Endocrinol 1582327-339

Jobling S, Reynolds T, White R, Parker MG, Sumpter JP 1995 A
variety of environmentally persistent chemicals, including some phthalate
plasticizers, are weakly estrogenic. Environ Health Perspect 1032582-587

Silva MJ, Barr DB, Reidy JA, Malek NA, Hodge CC, Caudill SP, Brock
JW, Needham LL, Calafat AM 2004 Urinary levels of seven phthalate
metabolites in the US. population from the National Health and Nutrition
Examination Survey (NHANES) 1999-2000. Environ Health Perspect

1 122331 -338

Hoppin JA, Brock JW, Davis BJ, Baird DD 2002 Reproducibility of
urinary phthalate metabolites in first morning urine samples. Environ
Health Perspect 110:515-518

Schettler T 2006 Human exposure to phthalates via consumer products.
Int J Androl 292134-139

Meek M, Chan PJ 1994 Bis(2-ethylhexyl)phthalate: evaluation of risks to
health from environmental exposure in Canada. J Environ Sci Health Part
C2179-194

Sharpe RM, Fisher JS, Millar MM, Jobling S, Sumpter JP 1995
Gestational and lactational exposure of rats to xenoestrogens results in

140

241 .

242.

243.

244.

245.

246.

247.

248.

249.

reduced testicular size and sperm production. Environ Health Perspect
10321136-1143

Ema M, Miyawaki E, Kawashima K 2000 Critical period for adverse
effects on development of reproductive system in male offspring of rats
given di-n-butyl phthalate during late pregnancy. Toxicol Lett 1112271-278

Moore RW, Rudy TA, Lin TM, Ko K, Peterson RE 2001 Abnormalities of
sexual development in male rats with in utero and lactational exposure to
the antiandrogenic plasticizer Di(2-ethylhexyl) phthalate. Environ Health
Perspect 1092229-237

Thompson CJ, Ross SM, Gaido KW 2004 Di(n-butyl) phthalate impairs
cholesterol transport and steroidogenesis in the fetal rat testis through a
rapid and reversible mechanism. Endocrinology 14521227—1237

Duty SM, Silva MJ, Barr DB, Brock JW, Ryan L, Chen Z, Herrick RF,
Christiani DC, Hauser R 2003 Phthalate exposure and human semen
parameters. Epidemiology 142269-277

Marsee K, Woodruff TJ, Axelrad DA, Calafat AM, Swan SH 2006
Estimated daily phthalate exposures in a population of mothers of male
infants exhibiting reduced anogenital distance. Environ Health Perspect
114:805-809

Fisher JS 2004 Environmental anti-androgens and male reproductive
health: focus on phthalates and testicular dysgenesis syndrome.
Reproduction 127:305-315

Gray LEJ, Laskey J, Ostby J 2006 Chronic di-n-butyl phthalate exposure
in rats reduces fertility and alters ovarian function during pregnancy in
female Long Evans hooded rats. Toxicol Sci 932189-195

Sohoni P, Sumpter JP 1998 Several environmental oestrogens are also
anti-androgens. J Endocrinol 1582327-339

Zacharewski TR, Meek MD, Clemons JH, Wu ZF, Fielden MR,
Matthews JB 1998 Examination of the in vitro and in vivo estrogenic
activities of eight commercial phthalate esters. Toxicol Sci 462282-293

141

250.

251.

252.

253.

254.

255.

256.

257.

258.

259.

Thompson CJ, Ross SM, Hensley J, Liu K, Heinze SC, Young SS,
Gaido KW 2005 Differential steroidogenic gene expression in the fetal
adrenal gland versus the testis and rapid and dynamic response of the
fetal testis to di(n-butyl) phthalate. Biol Reprod 732908-917

Battershill JM 1994 Review of the safety assessment of polychlorinated
biphenyls (PCBs) with particular reference to reproductive toxicity. Hum
Exp Toxicol 132581 -597

Ma R, Sassoon DA 2006 PCBs exert an estrogenic effect through
repression of the Wnt7a signaling pathway in the female reproductive
tract. Environ Health Perspect 114:898-904

Davidson NE 1998 Environmental estrogens and breast cancer risk. Curr
Opin Oncol 102475-478

Petrik J, Drobna B, Pavuk M, Jursa S, Wimmerova S, Chovancova J
2006 Serum PCBs and organochlorine pesticides in Slovakia: age,
gender, and residence as determinants of organochlorine concentrations.
Chemosphere 652410-418

Kimbrough RD, Krouskas CA 2003 Human exposure to polychlorinated
biphenyls and health effects: a critical synopsis. Toxicol Rev 222217-233

Ogura 12004 Half-life of each dioxin and PCB congener in the human
body. Organohalogen compounds 66:3376-3384

Colbom T, Smolen MJ 1996 Epidemiological analysis of persistent
organochlorine contaminants in cetaceans. Rev Environ Contam Toxicol
146291 -1 72

Kholkute SD, Rodriguez J, Dukelow WR 1994 Reproductive toxicity of
Aroclor-1254: effects on oocyte, spermatozoa, in vitro fertilization, and
embryo development in the mouse. Reprod Toxicol 82487-493

Hany J, Lilienthal H, Sarasin A, Roth-Harer A, Fastabend A,
Dunemann L, Lichtensteiger W, Winneke G 1999 Developmental
exposure of rats to a reconstituted PCB mixture or aroclor1254: effects on
organ weights, aromatase activity, sex hormone levels, and sweet
preference behavior. Toxicol Appl Pharmacol 1 582231 -243

142

260.

261.

262.

263.

264.

265.

266.

267.

268.

269.

DeCastro BR, Korrick SA, Spengler JD, Soto AM 2006 Estrogenic
activity of polychlorinated biphenyls present in human tissue and the
environment. Environ Sci Technol 4022819-2825

Winneke G, Walkowiak J, Lilienthal H 2002 PCB-induced
neurodevelopmental toxicity in human infants and its potential mediation
by endocrine dysfunction. Toxicology 181—182:161-165

Jacobson JL, Jacobson SW 1996 Intellectual impairment in children
exposed to polychlorinated biphenyls in utero. N Engl J Med 335:783—789

Newman J, Aucompaugh AG, Schell LM, Denham M, DeCaprio AP,
Gallo MV, Ravenscroft J, Kao CC, Hanover MR, David D, Jacobs AM,
Tarbell AM, Worswick P, Environment. ATFot 2006 PCBs and cognitive
functioning of Mohawk adolescents. Neurotoxicol Teratol 282439-445

Hattemer-Frey HA, Travis CC 1991 Benzo-a-pyrene: environmental
partitioning and human exposure. Toxicol Ind Health 72141-157

Hall M, Forrester LM, Parker DK, Grover PL, Wolf CR 1989 Relative
contribution of various forms of cytochrome P450 to the metabolism of
benzo[a]pyrene by human liver microsomes. Carcinogenesis 1021815-
1 821

Shimada T, Gillam EM, Oda Y, Tsumura F, Sutter TR, Guengerich FP,
Inoue K 1999 Metabolism of benzo[a]pyrene to trans-7,8-dihydroxy-7, 8-
dihydrobenzo[a]pyrene by recombinant human cytochrome P450 1B1 and
purified liver epoxide hydrolase. Chem Res Toxicol 122623-629

Shou M, Korzekwa KR, Crespi CL, Gonzalez FJ, Gelboin HV 1994 The
role of 12 cDNA-expressed human, rodent, and rabbit cytochromes P450
in the metabolism of benzo[a]pyrene and benzo[a]pyrene trans-7,8-
dihydrodiol. Mol Carcinog 102159-168

Kim JH, Stansbury KH, Walker NJ, Trush MA, Strickland PT, Sutter
TR 1998 Metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-diol by
human cytochrome P450 1B1. Carcinogenesis 1921847-1853

Keshava C, Divi RL, Whipkey DL, Frye BL, McCanlies E, Kuo M,
Poirier MC, Weston A 2005 Induction of CYP1A1 and CYP1 B1 and

143

270.

271.

272.

273.

274.

275.

276.

277.

278.

279.

formation of carcinogen-DNA adducts in normal human mammary
epithelial cells treated with benzo[a]pyrene. Cancer Lett 221 :21 3-224

Estabrook RW, Saeki Y, Chacos N, Capdevila J, Prough RA 1980
Polycyclic hydrocarbon metabolism: a plethora of phenomena. Adv
Enzyme Regul 1923-17

Gelboin HV 1980 Benzo[alphalpyrene metabolism, activation and
carcinogenesis: role and regulation of mixed-function oxidases and related
enzymes. Physiol Rev 6021 107-1166

Pelkonen O, Nebert DW 1982 Metabolism of polycyclic aromatic
hydrocarbons: etiologic role in carcinogenesis. Phannacol Rev 342189-222

Huggins CB, Pataki J, Harvey RG 1967 Geometry of carcinogenic
polycyclic aromatic hydrocarbons. Proc Natl Acad Sci U S A 58:2253-2260

Yang NC, Castro AJ, Lewis M, Wong TW 1961 Polynuclear aromatic
hydrocarbons, steroids and carcinogenesis. Science 1342386-387

Grover PL, MacNicoll AD, Sims P, Easty GC, Neville AM 1980
Polycyclic hydrocarbon activation and metabolism in epithelial cell
aggregates prepared from human mammary tissue. Int J Cancer 262467-
475

Mattison DR, Singh H, Takizawa K, Thomford PJ 1989 Ovarian toxicity
of benzo(a)pyrene and metabolites in mice. Reprod Toxicol 32115-125

Swartz WJ, Mattison DR 1985 Benzo(a)pyrene inhibits ovulation in
057BL/6N mice. Anat Rec 212:268-276

Tran DQ, lde CF, McLachIan JA, Arnold SF 1996 The anti-estrogenic
activity of selected polynuclear aromatic hydrocarbons in yeast expressing
human estrogen receptor. Biochem Biophys Res Commun 229:101-108

Arcaro KF, O'Keefe PW, Yang Y, Clayton W, Gierthy JF 1999
Antiestrogenicity of environmental polycyclic aromatic hydrocarbons in
human breast cancer cells. Toxicology 13321 15-127

144

 

280.

281.

282.

283.

284.

Cook JH, Dodds EC 1933 Sex hormones and cancer-producing
compounds. Nature 131:205-206

Fertuck KC, Matthews JB, Zacharewski TR 2001 Hydroxylated
benzo[a]pyrene metabolites are responsible for in vitro estrogen receptor-
mediated gene expression induced by benzo[a]pyrene, but do not elicit
uterotrophic effects in vivo. Toxicol Sci 592231-240

Bigelow SW, Nebert DW 1982 The Ah regulatory gene product. Survey
of nineteen polycyclic aromatic compounds' and fifteen benzo[a]pyrene
metabolites' capacity to bind to the cytosolic receptor. Toxicol Lett 102109-
1 18

Okey AB, Dube AW, Vella LM 1984 Binding of benzo(a)pyrene and
dibenz(a,h)anthracene to the Ah receptor in mouse and rat hepatic
cytosols. Cancer Res 4421426-1432

Duan R, Porter W, Samudio I, Vyhlidal C, Kladde M, Safe S 1999
Transcriptional activation of c-fos protooncogene by 17beta-estradiol:
mechanism of aryl hydrocarbon receptor-mediated inhibition. Mol
Endocrinol 1321511-1521

145

CHAPTER4
Effects of In Utero and Lactational Exposure of Female Mice to

Diethylstilbestrol, 17a-Ethynyl Estradiol, and Genistein on Ovulation and In
Vitro Fertilizing Ability of Eggs.

4.1 ABSTRACT

In this study, the effects of in utero and lactational exposure of mice to
diethylstilbestrol (DES), 17a-ethynyl estradiol (EE2), and genistein (GEN) on
ovulation and in vitro fertilizing ability of eggs collected from female offspring (F-
1) were examined. Pregnant C57BL/6 mice (F-0), bred with DBA/2 males, were
gavaged with DES (0, 0.1, 1.0, or 10 pg/kg of body weight per day (bw/d)), EE2
(0, 0.1, 1.0, or 10 pg/kg bw/d), or GEN (0, 0.1, 0.5, 2.5, or 10 mglkg bw/d) from
gestational day (GD) 12 to postnatal day (PND) 20. The female offspring were
examined at two different stages of reproductive development: PND 21 to 42
(peripuberty) and PND 56 to 105 (postpuberty). There was no significant
change in the number of eggs ovulated by peripubertal female offspring exposed
to DES, EE2, or GEN. However, the number of eggs ovulated by female
offspring on PND 56 to 105 significantly increased (46%) in the 0.1 pg DES/kg
bw/d treatment group, while the number of eggs ovulated decreased (31 %) in the
10 pg EE2/kg bw/d group, compared to the control group. Fertilizing ability of
eggs at peripuberty was significantly decreased in the 10 pg EE2/kg bw/d
treatment group (P < 0.01). Maternal exposure to GEN did not affect ovulation
rate nor the fertilizing ability of eggs ovulated by F-1 mice. Our results suggest
that in utero and lactational exposure to DES and EE2 altered ovulation and

fertilization, whereas GEN had no observable effects on the number of ovulated

146

eggs and fertilizing ability of eggs from female offspring.

4.2 INTRODUCTION

Estrogen plays a key role in regulating ovarian development and function
during growth of the fetus. The primary function of the ovaries is to produce
eggs, which originate from a permanent cell population established before birth.
The ovaries of the fetus are constantly exposed to hormonal stimuli during the
critical periods of fetal and perinatal development to acquire competence for
ovulation and for fertilizing ability of eggs. Therefore, in utero and perinatal
exposure to exogenous chemicals, which mimic endogenous hormones, may
disrupt fertility in adulthood.

Fetal and perinatal exposure to exogenous chemicals known as
estrogenic endocrine modulators (EEMs) has been .linked to a variety of adverse
effects on reproductive health in both humans and wildlife (1-4). Estrogenic
endocrine modulators are naturally occurring or synthetic chemicals that may
cause transient alteration of endocrine function within the physiological or
homeostatic range. In this study, we examined the effects of three: EEMs:
diethylstilbestrol (DES), 17a-ethynyl estradiol (EE2), and genistein (4’,5,7-
trihydrozylsoflavone or GEN) on female mice after in utero and lactational
exposure.

Diethylstilbestrol is a synthetic estrogen, which was administered to
pregnant women to prevent miscarriages in the late 19403 and 19503. Reduced

fertility and greater reproductive tract abnormalities and genital cancer have been

147

documented as consequences of fetal exposure to DES In humans (5-7). Total
doses of DES prescribed to women ranged from 1.4 to 17.9 9 during pregnancy
(8, 9). DES was banned in 1971 because of carcinogenic effects in offspring
attributed to in utero exposure. Because of the adverse reproductive effects,
many epidemiological and toxicological studies have been conducted assessing
DES to examine and compare toxicities in various tissues (10-14).

Interest in the effects of the synthetic estrogen EE2 on the reproductive
health of humans and animals is increasing. The chemical is used as the major
component of oral contraceptives (15, 16). Each year, there are approximately
2 million women in the United States (US) and the United Kingdom (UK) exposed
to 0.4 to 2.0 pg EE2/kg bw/d during undetected early pregnancy (15), thus
resulting in exposure of the fetus to this synthetic estrogen. In animal studies,
matemally administrated EE2 can cross the placenta and be transferred to the
fetus resulting in decreased fetal body weight, increased number of fetal deaths,
hyperplasia of follicular cells and degeneration of primordial follicles in a dose-
dependent manner (17-20).

Furthermore, there have been human and ecological health concerns
about pharmaceuticals and their metabolites present in drinking water supplies
because of the inability of sewage treatment facilities to remove them from the
waste stream. In March, 2008, an Associated Press investigation reported that
pharmaceuticals, including estrogenic compounds such as EE2, have been
detected in the US drinking water supplies of 24 major metropolitan areas

supplying 41 million Americans (21). Approximately 17 to 29% of sulfate or

148

glucuronide metabolites of ingested EE2 being used as an oral contraceptive are
excreted in the urine (22, 23). The concentrations of EE2 in sewage treatment
plant effluents were in the range of 0.4 to 15 ng/l and 300 to 1800 ng/l in the UK
and US, respectively (24-26). Concentrations of EE2 were measured at 0.48
pg/l in Las Vegas Wash and at 0.52 pg/l in Las Vegas Bay (27). Based on the
US Environmental Protection Agency’s (EPA) drinking water exposure estimates,
women of childbearing age (15 to 44 years of age), could be exposed to 6.3x10'6
to 2.5x10'2 pg EE2/kg bw/d through water consumption, and children younger
than one year of age could be exposed to 2.1x10'5 to 8.3x10’2 pg EE2/kg bwld
(28, 29). Therefore, potential adverse effects of exposure of the fetus and
neonate to EE2 through consumption of drinking water need to be investigated.
There are also concern and controversy regarding the potential adverse
effects of fetal and neonatal exposure to GEN. Genistein is a phytoestrogen
isoflavonoid found in the seeds of leguminous plants. Some soy-based infant
formulas contain as much as 40 mg/I of soy lsoflavones of which approximately
65% is GEN and its glycosidic conjugates (30). The estimated consumption of
total isoflavones by infants fed a soy-based formula is 4.5 to 8.0 mglkg bwld.
Mouse studies have shown that in utero exposure to GEN decreased body
weight at birth (31) and increased the incidence of uterine adenocarcinomas at
adulthood in mice (32). Furthermore, neonatal exposure to GEN increased
multioocyte follicles (33), and prolonged the estrous cycle in mice (34) in a dose-
dependent fashion. Female mice exposed to GEN as neonates have reduced

fertility and litter sizes as adults. Based on animal studies, further study is

149

 

needed to examine potential adverse effects of GEN exposure at human dietary
concentrations.

Ovarian response to EEMs may differ at different stages of biological
maturation (e.g. prepubertal, pubertal, and postpubertal) (35). In the present
study, we tested the hypothesis that in utero and lactational exposure to DES,
EE2 and GEN causes long-term alterations in ovarian function and affects
ovulation and fertility of female offspring (F-1). We examined the hormonal
response of ovaries to superovulation at two different ages in female offspring
(PND 21 to 42 [peripuberty; PEP] and PND 56 to 105 [postpuberty; POP). Egg
fertilizing ability was assessed using an in vitro fertilization (IVF) assay to avoid
confounding treatment effects on sexual behavior. Doses of DES, EE2 and

GEN were similar to clinical and recommended dietary doses used by humans.

4.3 MATERIALS AND METHODS
Animals

EIeven-week-old virgin C57BL/6 female (F-O) and seven- to eight-week-
old DBA/2 male (F-0) were obtained from Charles River Laboratories (Portage,
MI, USA) for each of three trials assessing the effects of DES, EE2, and GEN
independent of one another (each test compound has its own control group for
comparison). Previous studies from our laboratory have shown that B602 F-1,
offspring from the C57BL/6 x DBA/2 cross mating, superovulate a large number
of eggs and have a consistently high rate of fertilization (36-38). All mice were

housed in polycarbonate cages with cellulose fiber chips (Aspen Chip Laboratory

150

Bedding, Northeastern Products, Warrensberg, NY, USA) as bedding. The
animal room was maintained at 23 °C with 30-40% humidity. The lights were on
a 12-hour cycle.

All animals had unlimited access to deionized water provided in glass
bottles with rubber stoppers and AlN-76A rodent feed (Research Diets, New
Brunswick, NJ, USA). AlN-76A rodent feed is a casein-based, open-forrnula
purified diet that contains non-detectable concentrations of the estrogenic
isoflavones genistein, diadzein and glycitein (39). Offspring were provided with

standard 2019 rodent feed (Harlen Teklad 22/5, Madison, WI, USA)..

Maternal treatment
When animals arrived, CS7BL/6 female and DBA/2 male mice (F-O) were
separated by sex, housed two per cage and acclimatized for three to seven
days. At the beginning of the study, two females were housed with a male for
mating. Following evidence of pregnancy, dams were separated from the
males, housed individually, and randomly assigned to one of the three
estrogenic endocrine modulators treatment groups. Ten to twelve pregnant
females per each treatment were used.
Diethylstilbestrol, 17a-ethynylestradiol, and corn oil were obtained from
Sigma Chemical Co. (St. Louis, MO, USA). Genistein (>98% pure) was
obtained from lndofine Chemical Company (Somerville, NJ, USA). Pregnant
C57BL/6 F-0 mice were treated by daily gavage with diethylstilbestrol (0, 0.1, 1.0,

or 10 pg/kg bw), 17a-ethynylestradiol (0, 0.1, 1.0, 10 pg/kg bw), or genistein (0,

151

0.1, 0.5, 2.5, or 10 mglkg bw) in 0.1 ml corn oil from GD 12 to postnatal day
(PND) 20 (Figure 4-1). Dams were not treated on the day of parturition (PND O)
to avoid cannibalism of pups by the dams. Body weights of dams were
measured daily between 09:00 AM and 12:00 AM to administer treatment.
Offspring were separated by sex on PND 21 (weaning) and fed AlN-76A until

necropsy. All F-O mice were euthanized on PND 21.

 

Paiing female (c5731.) and male pert) Birth I302) We in
Mating Gestation Lactation
F.0 llllllllllllllllll llllllllllllllllll

Offspring

Maternal treatment period
GesMionaldaylZ PostnataldayZO

. Dams were treated by gavage with DES (0, 0.1, 1.0,
10 pglrg maternal bwlday, EE2 (D, 0.1, 1.0, 10119119
maternal bwlday), GEN (0, 0.1, 0.5, 2.5, 10 mglkg mat
emal bwlday) from gestational day 12 to postnatal day

 

 

We try 20
Fflllllllllllll
F1 Offspring
- _
Age Dayo Day? Day 14 Day21 Day211039 Day56t0105
Litter size Anogenital distance Body weight Body weight
(AGD) Organ weight Organ weight
Body weights Ovulalion Ovulation

Egg fertilizing ability Egg fertilizing abil'ly

Figure 4-1. Schedule of maternal treatments and parameters measured in their
female offspring (F-1).

152

Offspring (F- 1) development and necropsy

B602 F-1 body weight and anogenital distance (AGD; the length of the
perineum from the base of the genital tubercle to the center of the anus when the
skin was naturally extended without stretching) were measured on PND 7, PND
14, and PND 21. A dissecting microscope (Nikon, Melville, NY, USA) with an
ocular micrometer was used for measurement of AGD. The anogenital area
was extended slightly without stretching and measured to the nearest 0.1 mm.
At the times of the in vitro fertilization assay at PEP or POP, female F-1 body

weights were recorded.

Superovulatr'on

To examine the responsiveness of female offspring exposed to DES, EE2
or GEN in utero and during lactation to ovulation induction stimuli, female
offspring at PEP or POP were injected intraperitoneally (i.p.) with 10 international
units (IU) of pregnant mare’s serum gonadotropin (Sigma Chemical Co., St. Louis,
MO, USA) in 0.1 ml saline followed 48 h later with 10 IU human Chorionic
gonadotropin (Sigma Chemical Co., St. Louis, MO, USA) in 0.1 ml saline. The
superovulated eggs from each female were collected from the ampulla 14 to 16 h
later and incubated in a 1-ml organ culture dish (60 mm diameter x 15 mm high)
(Becton Dickinson Falcon, Franklin Lakes, NJ, USA) containing 0.9 ml Brinster’s
BMOC-3 medium supplemented with penicillin and streptomycin (Gibco/BRL,

Grand Island, NY, USA).

153

In vitro fertilization assay

Sperm used for the in vitro fertilization assay were collected by puncturing
the caudal epididymides of reproductively mature non-treated male mice with a
25-gauge needle and placed in a 1-ml organ culture dish containing 1.0 ml
BMOC—3. The sperm suspension was incubated at 37 °C under a humidified
5% 002 air environment for 60 min before insemination. Sperm was diluted in
BMOC-3 medium with the concentration being measured using a CellSoft
computer (CASA; CRYO Resources Inc, New York, NY, USA). Diluted sperm
(0.1 ml) was added to the 0.9 ml medium containing the eggs in an organ culture
dish to achieve a final sperm concentration of 3 x 104 sperm per 1-ml dish. After
insemination, eggs were incubated for 24 h, and 50 pl of 35 pM bisBenzimide
(Sigma Chemical Co., St. Luis, MO, USA) was added to the dish. After 20 min
incubation with the stain, the eggs were examined using a Nikon Optophot
fluorescent microscope (Melville, NY, USA) equipped with a 100-watt mercury
bulb, a 365/10-nm excitation filter, a 400-nm dichromic mirror, and 400-nm
barrier filter. Eggs were counted and scored as fertilized if they were at the two-
cell stage or at the one-cell stage containing spindle, two pronuclei, and a second
polar body. Additionally, eggs were evaluated for fragmentation and other signs

of degeneration.
Statistical analysis

SAS version 8.2 (SAS Inc, Cary, NC, USA) was used for all data analyses.

All data were tested for normality by using the Shapiro-Wilk test and residual plot.

154

Litter was considered to be the experimental unit. Comparisons of offspring
body weight, AGD, number of eggs ovulated, percent of egg fertilizing ability and
percent of egg degeneration between control and treatment groups were
analyzed with ANOVA using the MIXED procedure of SAS, where treatment was
a fixed effect and dam was a random effect within treatment (a nested design).
Dunnett’s test was used for comparisons between control and treatment groups
for each compound. No within dose comparisons were made, nor were EEMs
treatments compared to one another. When parameters were analyzed, litter
size on PND 0, body weight, and age at necropsy were included as a covariate, if
significant correlations were confirmed (40). In vitro fertilization data were
analyzed in two age groups (PEP and POP) and were adjusted by age of animal.
Eggs were not included in data analysis, if it was not possible to determine the
stage of development. Statements of significance are based on a P value less
than 0.05.
4.4 RESULTS
Body weights of F-1 female mice

Body weights on PND 7 and 21 were analyzed with and without
adjustment for litter size on PND 0, because, as expected, body weight was
negatively correlated with litter size (P < 0.05) (Table 4-1). With litter size
adjustment, body weight in the 10 pg DES treatment group was significantly
decreased on PND 21. Conversely, the 1.0 pg DES treatment increased body
weight at POP (P < 0.05). Maternal EE2 treatment significantly decreased F-1

female body weight in the 1.0 and 10 pg treatment groups at PND 7 and this

155

effect was persistent only in the 10 pg treatment groups until PND 21. After litter
size adjustment, there was no significant difference in body weights observed,
compared to the control group. Unlike the DES and EE2 treatments, there were
no significant body weight changes in the GEN treatment groups with or without

litter size adjustment.

Anogenital distance
DES, EE2, or GEN treatment did not affect AGD regardless of AGD

adjustment by body weight (Table 4-2).

Ovulation

The number of ovulated eggs and in vitro fertilizing ability was determined
at two different ages (PEP and POP). There was no significant change in the
number of ovulated eggs from DES-, GEN-, and EE2-exposed female offspring
at PEP (Table 4-3). The number of ovulated eggs was greater than controls in
the 0.1 pg DESI kg bwld treatment group (P < 0.05). There was no significant '
change in the number of ovulated eggs in the EE2 or GEN maternal treatment
groups. The number of ovulated eggs was less than controls in the 10 pg

EE2/kg bwld treatment group at POP but the P value was 0.09.

Fertilizing ability of eggs

156

The percent of fertilized eggs in the DES treatment groups was not
different from controls at PEP and POP (Table 4-4). Fertilizing ability of eggs
was less than controls in the 10 pg EE2 female offspring PEP (P < 0.05). GEN
treatment did not affect fertilizing ability of eggs. The relative percentage of

fertilizing ability of eggs is presented in Table 4-4a (when the control = 100%).

Percentage of degenerated eggs

DES, EE2, or GEN did not affect degeneration of eggs regardless of age

(Table 4-5).

157

 

35.9.05: 5o 5E: «an an E25253

 

 

5555 55.5 n 55.5 5555 55.5 n 55.5 5555 5.55 H 55.5 5555 55.5 n 55.5 5555 55.5 n 55.5 a 525
5555 55.5 n 55.5 5555 55.5 n 55.5. 5555 55.5 n 5:. 5555 55.5 n «5.5 5555 55.5 n 55.5 5 525
5 oze :5 3.5 .25. 5. 5525555!
5555 55.5 n 3.5 5555 :55 n 55.5 5555 5.5 n 5.5 5555 55.5 H 5.5 5555 55.5 n 55.5 a 525
5:555 55 n «I. 5555 55.5 a 55.5 5555 55.5 n 53V 5555 55.5 n 55.5 5555 R5 5 55.5 5 525
55255555 555555
5. 25.5; 5555.
55 5.5 5.5 5.5 5
$335 55.qu5 535.com he soon 352.55. sz
55: 55.5 n «5.55 5:55 555 a 55.55 3555 55.5 n E55 3555 55.5 n «5.55 55559555
55: 55.5 e 55.55 3:55 555 e 55.55 35: 55.5 n 55.3 A :5: 55.5 5 R55 5.5555555

5555 35.5 n F:
5:55 555 n 5.5

5:555 555 n 55.5
3555 3.5 n 3.5

35$ .55 n 3.5
35$ 95 n {.5

>32er 5o 5E: am one an EoEuwaF<
525$ 55.5 ... 55.5 a 525
35$ 35 n 55.5 5 525
5 52.. 55 on.» .35. 5 52555.3

 

 

.5555 55.5 n 55.5 5:555 55.5 n 55.5 35$ 55.5 n 55.5 35$ 55 n 55.5 a 525
.5355 55.5 n 55.5 .3555 25 n 55.5 35$ 55.5 5 55.5 35$ 55.5 n 53 5 525
EoEamafia 5.85:5
5. 25.5; 5.8
55 5.5 ..5 5
$5555 9:9: .2525: $550.82. .0 one: 3535:. «mm
35: 55.5 e 55.55 .555 55.5 e 55. a 355 55.5 H 55.55 55: 55.5 n 3.55 55559555
55: 55.5 n 5.55 :5: 55.5 n 55. 3 £555 :55 n 55.: 5:55 555 n 55.: 5.855555

255$ 55.5 a 55.5

$58 :20 H mod

3555 55.5 n 55.5
3555 55.5 n 55.5

55$ 55.5 e 3.5
55$ 35 n 55.5

>323: ..o 5E: as one 3 E25325
358 55.5 n 55.5 a 525
3555 S5 5 55.5 5 525
5 oze :5 en.» .3... 5. 52555.3

 

.5598. 55595 55 55 55555855555 zmo 555 .mm .35 .5? 52555 5555 55555555 5.552 5 £555; 585 .3 5.555

55$ :5 n 5.5 5:555 55.5 a 55.5 55$ :5 n 55.5 3555 55.5 n 55.5 a 525
5555 55.5 n 55.5 3555 55.5 n 55.5. 55$ :5 H 5.5 3555 3.5 n 55.5 5 025
E25253 505:5
5. 2.5.5; 558
55 5.. 5.5 5
mmo

.5235 9.53 .2585553525 2.5 855 .5535:

158

.55 v 1: ”mod v n? ”5595 65:8 :55 5:25.56 25:85:55 .
$55 .3558: ”02.5
.3555: ,6 :onEac \ ......mEEm 50 558:5 50 mm H 5:55.: 95:55 5555. mm 52:55.95 Em 505.55 __<

 

55: 55.5 H 55.55
55: 55.. H 55.55

:5: 55.5 H 55.55
35 55.5 H 55.5.

55: 55.5 5 E55 55: 55.5 a 2.55 5:: t5 5 3.53 5555985
555 5: a 55.3 55$ 5: H 55.5. 555 5: H 55.3 55555555
5.58 5-5 5.55:

159

.28 .9958 ”02a
.Emz: Ho 59:5: H mEEEm .0 55:55 .6 mm H game 993 .32 nwucmmma 2m mm:_m> __<

53 «to H H3 889 2.0 H one 82.2 2.0 H 9} 853 :d H Be 859 02. H H? 3 02¢

8:3 Hod H o: 332 Hod H a: 3:3 Hod H :3 8:3 Hod H :2 858 Hod H 9: H 02¢
:55 £90; 2.3 3 “5.53.2

33$ 36 H 9} 888 «3 H 36 53v :6 H m: 853 «to H H3 683 N3 H one a 32.”.

8.53 mod H HHH 858 mod H x: 833 23 H 84 $9: 8.0 H x: 658 Hod H m3 H 02¢
:55 “5.52.?“ 522.5

ow<
3 3 m... to c
335 9:95 ESmEam Ho anon 353a: zmo
8:99 2d H m3 533 Ed H 3m 35$ are H de 8:2: H; H Xe to. 02¢
8:5 «to H 3N 6:3 2.0 H H? 35$ :6 H 2: 53$ :6 H 84 H oza
:55 £925 .62. 3 .5563?
6:3 8.0 H «me $83 Ed H and 358 mad H 8n 3:03 5.0 H 31‘ E oza
6:3 «:0 H vow 3:va o; H H3 858 :.o H Hg 8:93 Ed H 23 H 02¢
:55 “cassava 285:5
ao<
2 3 to o
.23.. 9.53 352.8 355ng .o 8% .233: «mm
8:333 H N? 3:8 2.0 H N? 33$ 2.0 H Re 359 mod H N; 5 02¢
832 8.0 H :3 3:08 mod H H? am: «no H 23 2:89 vod H 23 H ozn.
.55. £225 :68. 3 “552.3
8S3 HE H HIV GEE m; H m: 33$ H; H H3 558 Ed H m: 3 02¢
659 mod H 8H 3:3 mod H 8H 8:9; 8d H SH 389 mod H H3 H oza
i=5 “5.58%.“ .855:
oo<
3 3 to o
.23 9.53 ....zoeznaszu Ho 38 352a: mmo
8:38. 5:25 9 H3 .2253.“ so: 2.5 Ba .Nmm .mmo 5E 38: 2:3 Ho gumbo 23m. Ho 80$ 8.5% 2%,: .3 2an

160

.86 v or 539m .928 E0: EmHmtfi >_Emoc_cm_m .
A925 Ho .0983: : 2955 Ho .8825 Ho mm H .888 993 $8. “825me 9m .82? __<

 

 

88: 8.0 H 3.2 EN: 8; H cm. 5 88: 88 H 8. 5 88: 8.8 H 2.3 8:: 8.”. H 3.8 82368
88: 8.8 H 8.8 85 SH H H88. 85 8.8 H 3.9 83 H; H 8.3. 8:8 88 H 8.8 883:8
on? 082:3 Ho .3532
2 m a m a H a o 23

:55 9:95 533:3 Ho awou 35322

 

833 mm.» H 8.: 8S8 22 H 8.8 38$ 88 H mHHN
88: «NH H 88m 85% 88 H 8.3, :5: 88 H 8H,?

8:88 8.8 H Hv.H~ b.8368

8:0: 5.8 H 8.8 83:98
mama Boa—2:3 Ho .3532

 

3 3 2.
8:3 9:83 682.8 3553: Ho 3% .2522

9 «mm

 

Emu SH H 8.2 8:3 8.8 H 3.8 . 8:3 38 H 8.8
88: 8+ H 3.3 85: «3 H 5.9. 85% a? H 9.3

88.: 88 H 8.8 833988
8:3 one H NH. 2. 823:8
uuuo $2326 “—0 53:52

 

3 3 3
8:2. 9.31. .23352268 .0 83 .2332

o mwo

 

.cozflofl H3395 Nwflmc .9833me Eat sz ucm .wa .wmo 5:5 umfimb mEmo Ho macamto 29:3 E9. $3 8535 Ho .3832 .mé mfim...

1
6
1

.80 v Q. ”0:05 6.2.00 E0... 0.0.00.0 2.0005005 ..
8.00.. .0 505:... . 208.00 00 50:55 .0 mm H 2.00.: 90:8 #80. 08:80.0 0.0 829 __<

 

 

 

 

 

 

 

6.9. 8.8 H 88 EN: 8.... H 8.8 .92. 8... H 8.8 8.2. 88 H 2.5 SE 8.8 H 8.8 383.83
.8: 8..” H 5.8 it 88 H 3.8 $8. 8... H 8.8 33. 8... H 8.8 68. 3+ H 8.8 5.838..
0......“ 8.3.5“. .x.
2 3 ...... H... ..
.035 9:95 520.000 .0 0000 .0530: zme
$8: 8.8 H 8.8 823. 8.8 H 83 32%. 8.8 H 8.8 8:8. «8 H 8.8 383.83
.88: 8.. H 8.8 5.3. 8.. H 8.8 :8: 8.. H 8.8 . ES. 8... H 8.8 38383
0......“ 83:5“. ...
3 3 H... a
.23: 9:8. 3.02.8 35.53: .0 80: 353...: «mm
:8: 9: H 8.8 $8. 8.2 H 8.8 :8. 8... H 8...... 8.9. E H 8.8 2.83.83
68. 8.8 H 8.2. .8 H. Bx H 8.8 E8. .38 H 8.8 8.5. 8.8 H 8.8 38383
8.8 83.03“. .x.
S 3 H... o
.23: 9:8. 383.838.: .0 one: 3522... mm:
.388.

83.... 9 ..8 83.588 59. 2me 8m .88 .80 5.; 8.8.. 28... ..o 8.380 298. 52.. 88 u.0 ......8 88.8. 9..: s ..1 2an

162

P1

.86 v Q. EH65 63:8 E0: Embtfi 228535 .
.me: ho .mnEsc \ 295cm ho .3835 Ho mm H 259: Ems—um “mam. 3:533 05 829 __<

 

 

 

 

 

 

Ana: «.0 H N8 EN: no H 0.3 as: to H 0:: 68: Yo H v.02 Er: 3.2 E2338
:5: 00 H 3.2 $5 no H $2 $8 to H 08. S3 to H «.8 35V 3.2 3.83%;
3......“ 9.3.3:“. .x.
3 ....N m... 2. o
.23.. 9:95 503.com .0 32. 3:3qu zww
3mm 3 H 3”: 858 No H Q8 22.0.3 90 H 32 8:3 32 5.3338
.83: E H mam 5:8 No H H. 2: EB: E H «.8 EB: 92: €835“.
3:3.“ 9.3.33". .x.
3 3 3 o
.23.. 9:9: 33.32. 3:38.52 3 Son .253: «mm
:5 3 0o H ”.8 3516 H 52 43$ 3 H 32 8a: 92: émaamon.
65: mm H 6% En: «N H «.8 £53 to H 0.8? 8:3 92: 3.33:8
3......“ 9.3.3.9. .\.
3 3 E. o
.23.. 9:9: .3893286 .0 82. .333: mmo

 

.5388. 298.5 N? awn .mco_§wmm

82. zmo Em NH. .80 55, Emma memo Ho actuate 22.2 52. m8» 8 5% 35:5 E: s 3 $3528 9%? HI. 2an

163

 

Awe... .o .383. \ «.mEEm .o .3835 .0 mm H 9.8:. 933 “mam. 8.535 2m 93.? __<

 

 

 

 

 

 

8a: SN H 8..” Eu: 3.3 H 3d 8.333 H 8.0 a.” a? H 8.» .3333 H cm. 3 3.8398,.
.33. 8a H 9.... 3.5 mom H Be .3. an H 3: 53 So H B... as. 3.m H 23 2.83.5.
mono 33.2895 ox.
3 3 m... 3 o
5:5 9:95 583:3 .0 300 3532.. zmo
.23. mod H 8.0 .35. 6a H .3 saw. m5 H 3... 6:8. 8N H 9... 2.83.8.
8.3. 3.3 H 3..” .333 8... H mm... fa: 8.0 H on... 2:3. No.3 H 8.3 2.835..
ammo wanna—890 .x.
3 3 3... a u m
.23.. 9.3... 352.8 3:332 .0 38 3532... m
in: mud H :3 33. 3.... H 38 $3. 8... H 3.3 8.9. an H 8.3 3.2398.
.23. 3.3 H no... .5: 8.. H and E8. 3... H SN 65. X! H no... 3.83:8
ago ocuflacouco .x.
3 o 3 3 o a mug

.23 9.3... .2.§..=u..§o.u .0 88 352“:

 

.8598. case... 3 E. .mcoasmmmrefi zmo 2m .mm .80 5.; 8.8.. 25.. 39.5%.. 23.... EB. mum» 8.99.qu .o 28.9. .3 mam»

a

1

4.5 DISCUSSION

The present study demonstrated that in utero and lactational exposure to
the high dose (10 ug/kg bwld) of DES decreased body weight on PND 21, while
the low-dose (1.0 of ug/kg bwld) of DES increased postpubertal body weight in
female offspring (Table 4—1). High dose (10 pg/kg bw/d) of EE2 decreased body
weights in female offspring until weaning. These results suggest that in utero
and lactational exposure to EEMs may alter body weights of infants and wildlife
at an early stage of development and during the postpubertal period.

Maternal exposure to estrogen reduces fetal body weight due to reduced
placental blood flow during late pregnancy (41, 42). Yasuda et al. (18)
demonstrated that daily oral gavage of mice with EE2 (0.02, 0.2, and 2.0 mglkg
bw) from GD 11 to GD 17 decreased average fetal body weight. Even though
our doses used in the present study were 2- to ZOO-fold less than in the study by
Yasuda et al. (18), we also observed decreased body weight at the high doses of
DES and EE2 until weaning. On the other hand, based on dramatically
increased obesity after the 1980’s and an important role of estrogen in the natural
weight-control mechanisms, a hypothesis has been formulated that EEMs at low
concentration alter the body's natural weight-control mechanisms resulting in
interference of the normal developmental and homeostatic controls over
adipogenesis and energy balance (43-45). In this study, the increase in body
weight of female offspring maternally exposed to 1.0 of pg DES/kg bw/d at POP
is in agreement with the results reported by Newbold et al. (43). In their study,

in utero exposure to DES (1 part per billion or ppb) and GEN (50 parts per million

165

or ppm) in drinking water significantly increased body weight at 4 months of age
(PND 112). Furthermore, ERa knockout mice have increased body fat content
with a 10-fold increase in E2 (46, 47). A proposed mechanism is that low doses
of EEMs increase signaling of ERB or block signaling of ERa and contribute to
obesity by altering the body's natural weight-control mechanisms. Taken
together, further research is needed to investigate whether in utero and
lactational exposures to low doses of EEMs are a potential cause of obesity.
Anogenital distance is considered a sensitive indicator of
demasculinization of male genitalia. However, it may not be a sensitive
biomarker to examine estrogenicity of EEMs in female offspring. We observed
that in utero and lactational exposure to DES, EE2, and GEN treatment did not
significantly affect AGD of F-1 females regardless of AGD adjustment for body
weight (Table 4-2). In contrast to our results, AGD was significantly increased in
female rats exposed in utero to 0.2 pg DES/kg bw on GD 11 through GD 17 by
daily subcutaneous injection (48). Clark et al. (49) confirmed that the growth
rate of the AGD was controlled by dihydroxytestosterone rather than by
testosterone. Consequently, EEMs that interfere with the synthesis of
dihydroxytestosterone or decrease its bioactivity can decrease the growth rate of
AGD. Evan et al. (50) reported that GEN inhibits the activity of 5a-reductase,
which converts testosterone to dihydroxytestosterone in genital skin fibroblasts.
A GEN-induced decrease of AGD in female offspring on PND 7 was observed in
female rats exposed in utero to 5,000 pg GEN/kg bw on GD 16 through GD 20 by

daily subcutaneous injection (51). The difference in AGD results observed in

166

this study compared to results from other studies may be due to species
differences, differences in doses, and the effectiveness of different routes of
administration to pregnant dams (gavage vs. subcutaneous injection).

Results from the present study suggest that in utero and lactational
exposure to synthetic estrogens and phytoestrogens may have different effects
on ovarian responses of postpubertal female offspring to pregnant mare’s serum
gonadotropin injection followed by human chorionic gonadotropin injection, which
induces ovulation. We observed that in utero and lactational exposure to DES
(0.1 pg DES/kg bwld) altered the number of ovulated eggs in female offspring at
POP, while EE2 and GEN has no effects on ovulation in female offspn'ng at the
same stage of development. The different ovarian responses to superovulation
at later stages of life may be due to differences in estrogen potency between
DES and the phytoestrogen. Compared to the potency of endogenous estrogen,
the relative potencies of DES were similar or greater compared to 17B-estradiol
(E2) (if E2 is assigned an arbitrary relative potency value of 1, then the relative
potency of DES and EE2 is 1.25 to 2.5) (52). The relative potency of GEN was
10'4 to 10'5 less than that of E2.

Furthermore, our studies indicated that exposure to low doses of DES
and EE2 in utero and during lactation enhanced ovulatory capacity when
compared with control animals, whereas animals exposed to higher doses
ovulated fewer eggs. Oocytes are a permanent cell population generated
before birth. Therefore, the total number of oocytes present in the mature ovary

originates from a definite number of primordial germ cells, which migrate into the

167

developing fetal ovary (53). Studies demonstrated that in utero or neonatal
exposure to an EEM induces multioocyte follicles in adult female offspring (34, 54,
55). Iguchi et al. (56) observed significantly decreased ovulation and increased
incidences of multioocyte follicles (up to nine oocytes per follicle) in 7-week-old
female mice subcutaneously injected daily with 1.0 pg DES/kg bw from PND 1 to
PND 5, compared to age-matched control mice. The incidence of multioocyte
follicles was significantly increased when neonatal DES treatment was begun on
PND 0 to PND 3, but was decreased when started after PND 1O (54). These
results indicate that in utero and lactational exposure to EEMs may decrease the
total population of germ cells through direct action on the fetal ovary via induction
of multioocyte follicles and/or indirectly through negative feedback on the fetal
hypothalamic-pituitary axis (57, 58). Further study is necessary to more fully
elucidate the mechanism of induction of multioocyte follicles. In addition to the
exposure concentration, the timing of exposure is an important factor in terms of
the increased incidence of multioocyte follicles in adult females through alteration
of the genetic program of the fetal and neonatal ovary. However, similar to
results in the present study, Wordinger et al. (59) observed increased ovulation in
6- to 8-week-old female mouse offspring exposed in utero to DES (10 pg/kg
maternal bw) on GD 15 or GD 16 by daily subcutaneous injection. The
underlying mechanisms for the biphasic effects of DES and EE2 reported here
need to be investigated further.

In contrast to our results for the GEN-exposed animals, Jefferson et al.

(33) reported that neonatal exposure to GEN (1.0, 10, or 100 pg/pup/d,

168

equivalent to 667, 6,667 or 66,667 jig/kg bwld) by subcutaneous injection for 5
days increased the number of ovulated oocytes in the 1.0 pg GEN treated mice,
while the number of ovulated eggs decreased in the 10 and 100 pg GEN-treated
mice on PND 19. They observed a dose-related increase in multioocyte follicles
(up to four oocytes per follicle). They also demonstrated that ERB plays a role
in the occurrence of multioocytes in that ERB knockout mice did not have
multioocytes in the ovaries. In addition, epidemiological studies indicated that
the consumption of isoflavone-rich soy foods extends the length of the follicular
phase and decreases serum follicle-stimulating hormone and luteinizing hormone
concentrations (60, 61). This suggests that GEN may alter oocyte maturation
via a delay in follicular development. Taken together, this study demonstrated
that in utero and lactational exposure to GEN at relevant human exposure
concentrations did not have adverse effects on ovulation.

Our in vitro fertilizing results from in utero and lactational exposure to
DES are in agreement with those from epidemiological studies, which indicated
that DES-exposed women are less likely to become pregnant than unexposed
controls (62). In the present study, there was a decreasing trend in in vitro
fertilizing ability of eggs collected from female offspring exposed to high doses of
DES. In addition, the high dose of EE2 significantly decreased in vitro fertilizing
ability during the peripubertal period. Studies demonstrated that multioocyte
follicles from female mice injected daily with 1.0 pg DES for 5 days from birth,
had lower embryo cell division (8-cell stage embryos: 77% in the control animals

vs. 47% in the DES-exposed mice, respectively) (56, 63). These results

169

indicate that neonatal exposure to DES may significantly increase multioocytic
follicles leading to decreased embryo cell division and thus fertilization.
Conversely, Wordinger et al. (59) observed that there was no significant
difference in the in vitro development of 2-cell embryos to the blastocyst stage
between control and female offspring of females subcutaneously injected with 10
pg DES /kg bw on GD 15 or GD16. On the other hand, there were no significant
effects of in utero and lactational exposure to GEN on fertilizing ability of eggs.
Further study needs to be done to explain the effects of maternal DES and EE2
exposure on fertilizing ability of eggs of female offspring.

The present study demonstrated that in utero and lactational exposure to
DES, EE2, and GEN did not affect the number of degenerated eggs. Our
results are different from those of other researchers who have shown that the
numbers of degenerated eggs increased in female offspring exposed to EEMs
during the in utero and/or neonatal period (19, 63). These researchers suggest
that EEMs delay cell cycles by degrading centrosomal proteins and perturbing

the spindle microtubule organization and chromosome segregation (64, 65).

Acknowledgements

This work was supported by a grant from US EPA (R827-402-01-0) to KC, PMS,

and T2.

170

4.6 BIBLIOGRAPHY

1.

10.

11.

Norgil Damgaard I, Main KM, Toppari J, Skakkebaek NE 2002 Impact
of exposure to endocrine disrupters in utero and in childhood on adult
reproduction. Best Pract Res Clin Endocrinol Metab 16:289-309

Hutchinson TH 2002 Reproductive and developmental effects of
endocrine disrupters in invertebrates: in vitro and in vivo approaches.
Toxicol Lett 131:75—81

Brevini TA, Cillo F, Antonini S, Gandolfi F 2005 Effects of endocrine
disrupters on the oocytes and embryos of farm animals. Reprod Domest
Anim 40:291-299

Kaiser J 2005 Developmental biology. Endocrine dianpters trigger fertility
problems in multiple generations. Science 308:1391-1392

Herbst AL, Ulfelder H, Poskanzer DC 1971 Adenocarcinoma of the
vagina. N Engl J Med 284:878-881

DeChemey AH, Cholst l, Naftolin F 1981 Structure and function of the
fallopian tubes following exposure to diethylstilbestrol (DES) during
gestation. Fertil Steril 1981 Dec;36(6):741-5 36:741-745

Greco TL, Duello TM, Gorski J 1993 Estrogen receptors, estradiol, and
diethylstilbestrol in early development: the mouse as a model for the study
of estrogen receptors and estrogen sensitivity in embryonic development
of male and female reproductive tracts. Endocrine reviews 14:59-71

Smith OW, Smith GVS 1949 Usa of diethylstilbestrol to prevent fetal loss
from complications of late pregnancy. N Engl J Med 241 :562-568

Smith CW 1948 Diethylstilbestrol in the prevention and treatment of
complications of pregnancy. Am J Obstet Gynecol 56:821-834

McLachIan JA 1977 Prenatal exposure to diethylstilbestrol in mice:
toxicological studies. J Toxicol Environ Health 2:527—537

McLachIan JA, Newbold RR, Shah HC, Hogan MD, Dixon RL 1982

171

 

12.

13.

14.

15.

16.

17.

18.

19._

20.

Reduced fertility in female mice exposed transplacentally to
diethylstilbestrol (DES). Fertil Steril 382364-371

Newbold RR, McLachIan JA 1982 Vaginal adenosis and
adenocarcinoma in mice exposed prenatally or neonatally to
diethylstilbestrol. Cancer Res 42:2003-2011

Haney AF, Newbold RR, McLachIan JA 1984 Prenatal diethylstilbestrol
exposure in the mouse: effects on ovarian histology and steroidogenesis
in vitro. Biol Reprod 30:471-478

Yasuda Y, Ohara I, Konishi H, Tanimura T 1988 Long-term effects on
male reproductive organs of prenatal exposure to ethinyl estradiol. Am J
Obstet Gynecol 1 59: 1 246-1 250

Thayer KA, Ruhlen RL, Howdeshell KL, Buchanan DL, Cooke PS,
Preziosi D, Welshons WV, Haseman J, Vom Saal FS 2001 Altered
prostate growth and daily sperm production in male mice exposed
prenatally to subclinical doses of 17alpha-ethinyl oestradiol. Hum Reprod
16:988—996

Hyder SM, Chiappetta C, Stancel GM 1999 Synthetic estrogen 17alpha-
ethinyl estradiol induces pattern of uterine gene expression similar to
endogenous estrogen 17beta-estradiol. J Pharmacol Exp Ther 290:740-
747

Slikker W, Jr., Bailey JR, Newport D, Lipe GW, Hill DE 1982 Placental
transfer and metabolism of 17 alpha-ethynylestradioI-17 beta and
estradiol-17 beta in the rhesus monkey. J Pharmacol Exp Ther 2232483-
489

Yasuda Y, Kihara T, Nishimura H 1981 Effect of ethinyl estradiol on
development of mouse fetuses. Teratology 23:233—239

Yasuda Y, Kihara T, Tanimura T, Nishimura H 1985 Gonadal dysgenesis
induced by prenatal exposure to ethinyl estradiol in mice. Teratology
322219-227

Yasuda Y, Konish H, Tanimura T 1987 Ovarian follicular cell hyperplasia
in fetal mice treated transplacentally with ethinyl estradiol. Teratology

172

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

36:35-43

Donn J, Mendoza M, Pritchard J 2008 AP Probe Finds Drugs in Drinking
Water. In: The Associated Press. New York

Maggs JL, Grimmer SF, Onne ML, Breckenridge AM, Park BK,
Gilmore IT 1983 The biliary and urinary metabolites of [3H]17 alpha-
ethynylestradiol in women. Xenobiotica 13:421-431

Williams MC, Goldzieher JW 1980 Chromatographic patterns of urinary
ethynyl estrogen metabolites in various populations. Steroids 36:255-282

Aheme GW, Briggs R 1989 The relevance of the presence of certain
synthetic steroids in the aquatic environment. Pharrn Pharmacol 41:735-
736

Tabak HH, Bloomhuff RN, Bunch RL 1981 Steroid hormones as water
pollutants, II. Studies on the persistence and stability of natural urinary and
synthetic ovulation-inhibiting hormones in untreated and treated
wastewaters. Dev Ind Microbiol 22:497-519

Williams RJ, Johnson AC, Smith JJ, Kanda R 2003 Steroid estrogens
profiles along river stretches arising from sewage treatment works
discharges. Environ Sci Technol 37:1744-1750

Roefer P, Snyder S, Zegers RE, Rexing DJ, Fronk JL 2000 Endocrine-
disrupting chemicals in a source water. J AWWA 92:52-58

US EPA 2000 Estimated per capita water ingestion in the United States. In.

‘ Washington DC: US EPA, Office of Water 1301; 40

US EPA 2001 National primary drinking water regulations; arsenic and
clarifications to compliance and new source contaminants monitoring; final
rule. In. Washington DC: US EPA

Setchell KD, Zimmer-Nechemias L, Cai J, Heubi JE 1998 Isofiavone
content of infant formulas and the metabolic fate of these phytoestrogens
in eariy life. Am J Clin Nutr 68:14538-14618

173

31.

32.

33.

34.

35.

36.

37.

38.

39.

Flynn KM, Ferguson SA, Delclos KB, Newbold RR 2000 Effects of
genistein exposure on sexually dimorphic behaviors in rats. Toxicol Sci
55:311-319

Newbold RR, Banks EP, Bullock B, Jefferson WM 2001 Uterine
adenocarcinoma in mice treated neonatally with genistein. Cancer Res
61:4325-4328

Jefferson WN, Couse JF, Padilla-Banks E, Korach KS, Newbold RR
2002 Neonatal exposure to genistein induces estrogen receptor (ER)alpha
expression and multioocyte follicles in the maturing mouse ovary:
evidence for ERbeta—mediated and nonestrogenic actions. Biol Reprod
67:1285-1296

Jefferson WN, Padilla-Banks E, Newbold RR 2006 Studies of the effects
of neonatal exposure to genistein on the developing female reproductive
system. J AOAC Int 89:1189-1196

Fitzgerald C, Elstein M, Spona J 1999 Effect of age on the response of
the hypothalamo-pituitary—ovarian axis to a combined oral contraceptive.
Fertil Steril 71 :1079-1084

Chou K, Cook RM 1994 Paraoxon inhibits fertilization of mouse gameters
in vitro. Bull Environ Contm Toxicol 53:863-868

Fielden MR, Halgren RG, Fong CJ, Staub C, Johnson L, Chou K,
Zacharewski TR 2002 Gestational and lactational exposure of male mice
to diethylstilbestrol causes long-term effects on the testis, sperm fertilizing
ability in vitro, and testicular gene expression. Endocrinology 143:3044-
3059

Fielden MR, Samy SM, Chou KC, Zacharewski TR 2003 Effect of
human dietary exposure levels of genistein during gestation and lactation
on long-term reproductive development and sperm quality in mice. Food
Chem Toxicol 41 :447-454

Odum J, Tinwell H, Jones K, Van Miller JP, Joiner RL, Tobin G,
Kawasaki H, Deghenghi R, Ashby J 2001 Effect of rodent diets on the
sexual development of the rat. Toxicol Sci 61 :115-127

174

 

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

Gallavan RH, Jr., Holson JF, Stump DG, Knapp JF, Reynolds VL 1999
Interpreting the toxicologic significance of alterations in anogenital
distance: potential for confounding effects of progeny body weights.
Reproductive Toxicology 13:383-390

Beydoun SN, Abdul-Karim RW, Haviland ME 1974 The regulatory effect
of estrogens on fetal growth. III. Placental deoxyribonucleic acid,
ribonucleic acid, and proteins. Am J Obstet Gynecol 120:918-921

Shepherd RW, Stanczyk FZ, Bethea CL, Novy MJ 1992 Fetal and
maternal endocrine responses to reduced uteroplacental blood flow. J Clin
Endocrinol Metab 75:301-307

Newbold RR, Padilla-Banks E, Snyder RJ, Jefferson WM 2005
Developmental exposure to estrogenic compounds and obesity. Birth
Defects Res A Clin Mol Teratol 73:478-480

Grun F, Blumberg B 2006 Environmental obesogens: organotins and
endocrine disruption via nuclear receptor signaling. Endocrinology
147:850-55

Baillie-Hamilton PF 2002 Chemical toxins: a hypothesis to explain the
global obesity epidemic. J Altem Complement Med 8:185-192

Naaz A, Zakroczymski M, Heine P, Taylor J, Saunders P, Lubahn D,
Cooke PS 2002 Effect of ovariectomy on adipose tissue of mice in the
absence of estrogen receptor alpha (ERalpha): a potential role for
estrogen receptor beta (ERbeta). Horm Metab Res 34:758-763

Heine PA, Taylor JA, lwamoto GA, Lubahn DB, Cooke PS 2000
Increased adipose tissue in male and female estrogen receptor-alpha
knockout mice. Proc Natl Acad Sci USA 97:12729-12734

Honma S, Suzuki A, Buchanan DL, Katsu Y, Watanabe H, Iguchi T
2002 Low dose effect of in utero exposure to bisphenol A and

diethylstilbestrol on female mouse reproduction. Reprod Toxicol 16:117-
122

Clark RL, Antonello JM, Grossman SJ, Wise LD, Anderson C, Bagdon
WJ, Prahalada S, MacDonald JS, Robertson RT 1990 External genitalia

175

 

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

abnormalities in male rats exposed in utero to finasteride, a 5 alpha-
reductase inhibitor. Teratology 42:91 -1 00

Evans BA, Griffiths K, Morton MS 1995 Inhibition of 5 alpha-reductase
in genital skin fibroblasts and prostate tissue by dietary lignans and
isofiavonoids. J Endocrinol 147:295-302

Levy JR, Faber KA, Ayyash L, Hughes CL, Jr. 1995 The effect of
prenatal exposure to the phytoestrogen genistein on sexual differentiation
in rats. Proc Soc Exp Biol Med 208:60-66

Gutendorf B, Westendorf J 2001 Comparison of an array of in vitro
assays for the assessment of the estrogenic potential of natural and
synthetic estrogens, phytoestrogens and xenoestrogens. Toxicology
166279-89

Byskov AG 1994 Embryology of mammalian gonads and ducts. In: Knobil
E, Neill JD eds. The physiology of reproduction. New York: Raven Press;
487-540

Iguchi T, Takasugi N, Bern HA, Mills KT 1986 Frequent occurrence of
polyovular follicles in ovaries of mice exposed neonatally to
diethylstilbestrol. Teratology 34:29-35

Guillette LJJ, Moore BC 2006 Environmental contaminants, fertility, and
multioocytic follicles: a lesson from wildlife? Semin Reprod Med 24:134-
141

Iguchi T, Fukazawa Y, Uesugi Y, Takasugi N 1990 Polyovular follicles in
mouse ovaries exposed neonatally to diethylstilbestrol in vivo and in vitro.
Biol Reprod 43:478-484

Gharib SD, Wierrnan ME, Shupnik MA, Chin W 1990 Molecular
biology of the pituitary gonadotropins. Endocr Rev 11 :177-199

Shupnik MA 1996 Gonadotropin gene modulation by steroids and
gonadotropin-releasing hormone. Biol Reprod 542279-286

Wordinger RJ, Brown D, Atkins E, Jackson FL 1989 Superovulation

176

 

 

60.

61.

62.

63.

64.

65.

and early embryo development in the adult mouse after prenatal exposure
to diethylstilboestrol. J Reprod Fertil 85:383-388

Cassidy A, Bingham S, Setchell KB 1994 Biological effects of a diet of
soy protein rich in isofiavones on the menstrual cycle of premenopausal
women. Am J Clin Nutr 60:333-340

Cassidy A, Bingham S, Setchell K 1995 Biological effects of isoflavones
in young women: importance of the chemical composition of soyabean
products. Br J Nutr 74:587-601

Bibbo M, Gill WB, Azlzi F, Blough R, Fang VS, Rosenfield RL,
Schumacher GF, Sleeper K, Sonek MG, Wied GL 1977 Follow-up study
of male and female offspring of DES-exposed mothers. Obstet Gynecol
49:1-8

Iguchi T, Kamiya K, Uesugi Y, Sayama K, Takasugi N 1991 In vitro
fertilization of oocytes from polyovular follicles in mouse ovaries exposed
neonatally to diethylstilbestrol. In Vrvo 5:359-363

Can A, Semiz O 2000 Diethylstilbestrol (DES)—induced cell cycle delay
and meiotic spindle disruption in mouse oocytes during in-vitro maturation.
Mol Hum Reprod 6:154-162

Can A, Semiz O, Cinar O 2005 Bisphenol-A induces cell cycle delay and
alters centrosome and spindle microtubular organization in oocytes during
meiosis. Mol Hum Reprod 112389-396

177

CHAPTER 5

Effects of in utero and Lactational Exposure of Male Mice to 17a-Ethynyl
Estradiol on Sperm Production, Quality and In Vitro Fertilizing Ability.

5.1 ABSTRACT

The objective of this study was to examine the effect of in utero and
lactational exposure of male mice to 17a-ethynyl estradiol (EE2), the estrogenic
component of oral contraceptives, on sperm production, quality and fertilizing
ability. Pregnant CS7BU6 mice (F-O), bred with DBA/2 males, were gavaged
with O, 0.1, 1.0, or 10 pg EE2/kg body weight per day (bwld) from gestational day
(GD) 12 to postnatal day (PND) 20. The male offspring (F-1) were examined for
sperm production and quality in young adult (PND 105 to 117) and middle-aged
(PND 315 to 337) mice by using computer-aided sperm analysis and an in vitro
fertilization (IVF) assay. Body weights were decreased in the 1.0 and 10 pg/kg
bwld groups on PND 7. Testis weights were decreased in the 10 pg/kg bwld
group on PND 21. Kidney weights were decreased in the 10 pg/kg bwld group in
middle-aged males. There was no significant treatment effect on anogenital
distance (AGD), regardless of dose and age. Sperm production in the 10 pg/kg
bwld group was significantly decreased in middle-aged males, as compared to
the control group. Even though sperm production was decreased in the 10 pg/kg
bwld group, there was no difference in fertilizing ability of sperm. The results
demonstrated that in utero and lactational exposure to 10 pg EE2/kg bw/d could
decrease sperm production without changing sperm motion patterns and sperm

fertilizing ability.

178

5.2 INTRODUCTION

Estrogens have been considered the predominant female sex steroid
hormones, but many studies have demonstrated that estrogens are also
produced in a variety of tissues in mammalian males such as adipose tissue,
brain, liver, adrenal glands, mammary glands, and testes (1-5). The serum
concentrations of estrogens are about 20 to 40 pg/ml in male humans (6), 35 to
45 pg/ml in male rats (7), and 2 to 180 pg/ml in males of other species (8, 9).
Ten to 20% of the circulating estrogens in males is produced by the testes; the
remaining 85% comes from peripheral aromatization of androgen precursors in
different tissues (10, 11). Similar to human testes, testes of the adult rat
synthesize approximately 10 to 25% of circulating estrogens (11, 12).

Estrogens in the male are derived by irreversible conversion of
testosterone or androstenedione into 176-estradiol (E2) by aromatase coded in
the CYP19 gene (13). To exert their biological effects, estrogens bind to the
intracellular estrogen receptors (ERs) or putative plasma membrane ER (14).
Studies using ER knockout mouse models demonstrated that estrogens are
essential for the development and normal functioning of testes (9), as well as for
the processes of sperrnatogenesis (15) and steroidogenesis (16) in males.

Concerns have been raised over the potential adverse effects of
estrogenic endocrine modulators (EEMs) on the development of the male
reproductive system. Humans and animals can be exposed to many estrogenic
and anti-estrogenic chemicals, including polychlorinated biphenyls (PCBs),

dichloro-diphenyl-trichloroethane (DDT), diethylstilbestrol (DES) and

179

phytoestrogens through environmental media, food and pharmaceutical products.
Our previous DES and genistein (GEN) studies demonstrated that in utero and
lactational exposure to DES (10 pglkg bwld) decreased sperm production, the
number of Sertoli cells, and the in-vitro fertilizing ability of sperm without apparent
abnormalities in testes and sperm motion parameters (17). On the other hand, in
utero and lactational exposure to GEN at doses of 0.1, 0.5, 2.5 or 10 mglkg bw/d
increased in vitro fertilizing ability of sperm from male offspring without changing
sperm production (18).

In the present study, we examined the effects of in utero and lactational
exposure of mice to 17a-ethynylestradiol (EE2) on male reproductive
development. Additionally, we compared results of the present study with results
from our previous DES and GEN studies. EE2, a synthetic estrogen, is widely
used as a component of oral contraceptives and as a carcinostatic agent for
prostate and breast carcinomas. The doses of EE2 used for oral contraceptives
are approximately 0.4 to 2.0 pglkg bw/d (19) and for arresting prostate
carcinomas, doses range from 4.0 to 1400 pglkg bw/d (20). It has been
estimated that approximately two million women per year in the United States
(US) and the United Kingdom (UK) are exposed to EE2 during undetected early
pregnancy (19, 21). Slikker et al. (22) demonstrated placental transfer of EE2 by
daily intravenous administration to pregnant rhesus monkeys on gestational days
108 through 121, whereas endogenous estrogen E2 in the placenta is
metabolized to estrone (E1). In the fetal circulation, E1 and EE2 were observed,

whereas E1, E2 and EE2 were observed in the maternal circulation. These

.180

 

results indicated that EE2 can be transferred to the fetus and affect development
of the fetal reproductive and central nervous systems (23—25).

In addition, there have been human and ecological health concerns over
pharmaceuticals and their metabolites present in drinking water supplies
because of human wastes. Significant proportions (approximately 17 to 29%) of
ingested sulfate or glucuronide metabolites of EE2 excreted in the urine (26, 27)
are present in effluents from wastewater treatment facilities. Concentrations of
EE2 in sewage treatment plant effluent has been measured at concentrations
ranging from less than 0.4 ng/l to 15 ng/l and from 300 to 1800 ng/l in the UK and
US, respectively (28-30). In 1997, concentrations of EE2 were measured at 0.48
pg/I in the Las Vegas Wash and at 0.52 pg/l in Las Vegas Bay (31). In 2006, it
was reported in a study that 4.7 ng EE2/I, 1.2 ng EM and 0.83 ng E2/l were
detected in seawater from the Acushnet River Estuary (Massachusetts, USA) (32,
33). Furthermore, in March, 2008, an Associated Press report indicated that
pharmaceuticals including estrogenic compounds such as EE2 have been
detected in the US drinking water supplies of 24 major metropolitan areas
supplying 41 million Americans (34). Based on the US Environmental Protection
Agency’s (EPA) drinking water exposure estimates, women of childbearing age
(15 to 44 years of age), could be exposed to 6.3x10'6 to 2.5x10‘2 pg EE2/kg bw/d
through water consumption, and children younger than one year old could be
exposed to 2.1x10’5 to 8.3x10‘2 pg EE2/kg bw/d (35, 36). To examine effects of

in utero and lactational exposure to EE2 on the reproductive development of

181

male mouse offspring, we selected doses of 0.1, 1.0 and 10 pglkg bw/d based on

potential environmental exposures.

5.3 MATERIALS AND METHODS
Animals

Eleven-week-old virgin C57BL/6 female and 7- to 8-week-old DBA/2
proven breeder male mice were obtained from Charles River Laboratories
(Portage, MI, USA) and housed in polycarbonate cages with cellulose fiber chips
(Aspen Chip Laboratory Bedding, Northeastern Products, Warrensberg, NY,
USA) as bedding. BGD2 F-1 mice, offspring from the CS7BL/6 x DBA/2 cross
mating, were used for the IVF assay since a large number of oocytes obtained
from superovulated B6D2 F-1 mice had consistent and reproducible fertilization
in control animals (37). The animal room was maintained at 30 to 40% humidity
and 23 °C. Room lighting was on a 12:12 lightzdark cycle. All animals had free
access to deionized water provided in glass bottles with mbber stoppers and
AlN-76A rodent feed (Research Diets, New Brunswick, NJ, USA). AlN-76A
rodent feed is a casein-based, open-fomiula purified diet that contains non-
detectable concentrations of the estrogenic isoflavones genistein, diadzein and
glycitein (38, 39). B6D2 F-1 female mice that were used as oocyte donors were

provided with standard rodent feed (Harlen Teklad 22/5, Madison, WI, USA).

182

 

Treatments

Upon arrival, C57BL/6 females and DBA/2 males were separated by sex,
housed two per cage and acclimatized for three to seven days. At the beginning
of the study, two females were housed with a male for mating. Following
evidence of pregnancy, dams were separated from the males, housed
individually, and randomly assigned to one of four treatment groups (0, 0.1, 1.0,
or 10 pglkg bwld).

Corn oil and 17a-ethynylestradiol (298% pure) were obtained from Sigma
Chemical Co. (St. Louis, MO, USA). Pregnant dams were treated by daily
gavage of 17a-ethynylestradiol (0, 0.1, 1.0, 10 pglkg bw) in 0.1 ml corn oil from
gestational day (GD) 12 to postnatal day (PND) 20 (Figure 5-1). The dose of test
chemical was adjusted each day to the dam’s body weight, which was measured
before dosing between 0900 and 1200 h. Offspring were separated by sex on

PND 21 and fed AlN-76A until necropsy. F-0 mice were euthanized on PND 21.

Measurements:
a. Anogenital distance

The anogenital distance (AGD) is the distance between the anus and the
base of the external genitals. AGD is considered a sensitive indicator of
demasculinization of male genitalia. Therefore, F-1 AGD was measured with
body weight on PND 7, PND 14, and PND 21. Measurements were made with a
dissecting microscope equipped with an ocular micrometer (Nikon, Melville, NY,

USA). Measurements were recorded to the nearest 0.1 mm. To minimize

183

 

operator variation and increase precision, two individuals measured AGD
independently. AGD were averaged when the two independent readings were

different.

Pa'ring female (CSIBL) and male (DBA) Birth (3502) Wearl'ng

Mating Gestation Lactation
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

Offspring

   

 

 

Maternal treatment period
Gestational day12 Postnatal day 20

oDams were heated by gavage with EE2 (o, 0.1, 1.0,
10 pgfrg maternal bwlday) from gestational day 12 to
postnatal dav 20

B'rth We 'ng

Fol-IIIIIIIIII
orrspn'ng
F-1 —
Age Dayll oayr Day14 Day 21 Day 105t0117 Day315to337

Litter size Anogenital distance Bodyweight Body weight
(AGD) Organ weight Organ weight
Body weights Sperm concentration Sperm concentration
Sperm motion parameters Sperm motion parameters
Sperm fertilizing ability Sperm fertilizing ability

 

Figure 5-1. Schedule of maternal treatments and parameters measured in their male
offspring (F-1).

184

b. Testis weight, sperm production and motion analysis

Testis weight of male offspring (F-1) was measured on PND21 and when
animals were sacrificed for the in vitro fertilization assay at young adulthood or
middle age. Male offspring were sacrificed by cervical dislocation. Sperm were
collected by piercing the pair of cauda epididymides with a 25 gauge needle in an
organ culture dish (Becton Dickinson, Franklin Lakes, NJ, USA) that contained 1
ml of BMOC-3 medium (37°C) (Gibco/BRL, Grand Island, NY, USA), which is a
capacitation supporting medium. Sperm suspensions were incubated for 30 min
at 37°C in humidified air (95% Oz, 5% C02) before sperm concentration and
motion parameters were analyzed. Sperm suspensions (20 pl) were placed on a
warmed 20 pm counting chamber and analyzed using a Series 4000, computer-
assisted digital image analysis system (CellSoft, Series 4000, CRYO Resources
Ltd., New York, NY, USA). For motility, number of motile sperm, velocity,
linearity, amplitude of lateral head displacement, and beat/cross frequency, a
minimum of 100 sperm cells was analyzed for each animal. Sperm concentration
is expressed as the number of spermatozoa/ml suspension medium. “Motile
count” refers to the number of sperm that moved faster than 20 pm/s/ml. Velocity
is defined as the average distance (pm) traveled by motile sperm in 1 s. Linearity
represents the ratio of the length of a straight line to the actual distance traveled
(x10) by each sperm cell. Amplitude of lateral head displacement is a
measurement of the lateral movement of the sperm head from the curve mean of

its track. The beat/cross frequency (Hz) is the number of beats (or crosses) of

185

the sperm across its curve mean/s. All measurements for each sperm collection

were performed in duplicate and averaged.

c. Sperm fertilizing ability

Non-treated females (3-week-old B602 F-1) serving as oocyte donors
were injected intraperitoneally with 10 IU of pregnant mare’s serum (PMSG)
(Sigma Chemical Co., St. Louis, MO, USA) followed by 10 IU of human chorionic
gonadotropin (Sigma Chemical Co., St. Louis, MO, USA) 48 h later. Fourteen to
16 h later, the superovulated oocytes from each female were collected from the
proximal oviducts and incubated in a 1 ml organ culture dish (60 mm diameter x
15 mm high) (Becton Dickinson Falcon, Franklin Lakes, NJ, USA) containing 0.9
ml Brinster’s BMOC-3 medium supplemented with penicillin and streptomycin
(Gibco/BRL, Grand Island, NY, USA). Sperm diluted with Brinster’s BMOC—3
medium (0.1 ml) were added to the 0.9 ml medium organ culture dish containing
the oocytes to achieve a final sperm concentration of 3 x 104 sperm/ml/dish. In
order to measure sperm fertilizing ability, the optimal concentration of sperm was
selected in the IVF assays to increase the assay sensitivity for detecting positive
and negative changes in sperm fertilizing ability. After insemination, oocytes
were incubated for 24 h, and then 50 pl of 35 pM bisBenzimide (Sigma Chemical
Co., St. Louis, MO, USA) was added to the dish. The oocytes were incubated
with stain for an additional 20 min before being examined for fertilization, using a
Nikon Optophot fluorescent microscope (Melville, NY, USA) equipped with a 100-

watt mercury bulb, a 365/10-nm excitation filter, a 400-nm dichromic mirror, and

186

400-nm barrier filter. Oocytes were counted and scored as fertilized if the
oocytes were at the two-cell stage or at the one-cell stage containing spindle, two
pronuclei, and a second polar body. Fragmentation and other signs of

degeneration of oocytes were also recorded.

Statistical analysis

All data analyses were performed using SAS (version 8.2, SAS Inc, Cary,
NC, USA). Data were tested for normality by using the Shapiro-Wilk test and a
residual plot. When data were not normally distributed such as velocity and
amplitude of later head displacement of sperm, log transformation was applied to
the data with subsequent retesting. Data were analyzed using litter as the
experimental unit to estimate litter effect as experimental error. The MIXED
procedure of SAS was used for analyzing a nested design, which treatment was
a fixed effect and dam was a random effect within treatment. To examine
correlation between testis weights and kidney weights, Pearson’s correlation test
was used. When analyzing offspring body weight, organ weight, and AGD, litter
size at PND 0 and age at necropsy were included as covariates (40). For
analyzing IVF data, data set were separately analyzed by two age groups (young
adult or middle-aged males) and adjusted by age of each animal. The MIXED
procedure was also used for IVF data. Dunnett’s test was used for comparisons
between control and treatment groups. The level of significance was set at P <

0.05.

187

5.4 RESULTS
Developmental effects on F-1 male mice

Maternal exposure to 1.0 and 10 pg EE2/kg bwld resulted in changes in
male F-1 body weight while changes in organ weights were restricted to the 10
pg EE2/kg bwld group. Body weights in both the 1.0 and 10 pg EE2/kg bwld
groups were significantly decreased at PND 7 (P < 0.05) (Table 5-1). There was
a trend of decreased body weight in young adult males. Kidney weights were
decreased in the 10 pglkg bwld group in middle-aged males. Testis weights of
males in the 10 pg EE2/kg bw/d group were significantly less compared to
controls on PND 21. The correlations between body weights and kidney weights
were P = 0.19 on PND 21 and P < 0.01 at middle age, respectively. The
correlations between body weights and testis weights were P < 0.01 both on
PND 21 and at middle age, respectively. The correlation between testis weights
and kidney weights were P = 0.08 on PND 21 and P = 0.03 at middle age,
respectively. When organ weights were adjusted for body weight, however, there
were no statistically significant EE2 treatment effects in organ weights.

With a single exception, in utero and lactational exposure to EE2 did not
result in gross abnormalities in the reproductive tract of male offspring.

Furthermore, there were no significant differences in AGD (Table 5-2).

188

Sperm production and quality
In utero and lactational exposure to EE2 at doses of 10 pglkg bwld had a
significant effect on epididymal sperm concentration of sperm in middle-aged

mice (Table 5-3). There was no significant change in sperm motion parameters.

Sperm fertilizing ability

Although there was a statistically significant decrease in epididymal sperm
concentrations (Table 5-3), in utero and lactational exposure to EE2 did not affect
in vitro fertilizing ability of sperm from F-1 male offspring (Table 5-4). The
relative percentage of fertilizing ability of sperm was presented in Table 5-4a

(when the control = 100%).

189

 

 

 

 

 

 

 

 

Em: 88 H .8... 8:8: 88 H 8... 88v 88 H 8... 88: 88 H 8... 88-8.85
8.9 88 H 8... Ea 8.8 H 8.8 88. 8.8 H .88 88: 8.8 H R... 9.88 88>
888 88 H 8... c... .v .88 H 8... 888 88 H 8.8 8.8: R. H 8.8 .8 828

o 888 .o .8. 2.8.83 .2... 8.8.2
E8: ... .. H 8.8. 8.8.: 88 H 858 888 B. .. H 858 88: 88. H 8. .8 8888.822
88. 8.8 H 8.8. Se .88. H 8.8. 688 8.8. H 888. 88: .88. H 8.8. 89.88 88>
.85 88. H 8.8 A. ... .v 8... H 88.3. 88. 8.8 H .8. .8 8.8: 8:. H .38 .N 828
3.5 2925 no.3»
.58: 8.8 H 8. .8 8.8.: 8.8 H 88.88 68. 8.8 H 8.88 88: 8.8 H 888 888-2822
58. 8.8 H 8.88 Se 88. H 88.88 88. 8.8 H 8.8.. 68: 88. H 8.8.. 89.88 888>
53 8.8 H 8.8. is 88. H 8.8 E: 88.8 H 88.. 58 8.8 H 888... .N 828
as. .8833 8:28.
£8: .88 H ....8 8.2.: 88 H 8.3 88. 88 H ....E as: 8.8 H 8.... 888.2822
88. 88 H 88.8 E8 88 H 888.. 8.8 8... H 8.8 88: 88 H 8.8.. 88.88 88>
88. #8. H 8.8 A. ... .V 8.8. H 8.8 .88. 88 H 8.8 8.8: 8.8. H 8.8 .N 828
3.5 23.83 8:83...

ca: 8.8 H 8.. 8:8: ...8 H 8.. 88. ..8 H 8.8 88: 8.8 H ...8 8888.825.
88. 888 H 8... E8 88 H 8.. 6.8V 88 H 8.. 68: 88 H 8.. 89.88 888>
88. 88 H 88 :2. .v 88 H 88 8.3 88 H 88 8.8: 8.8 H 8.8 .N 828

a. £8.83 .2...

E8: 8.. H .88 8:8: 8.. H 8.8 6.8 .88 H 8.8 $8: 8.. H 8.8.. 8 888.2822

588 8.. H 8.8 SE 8.. H 88 A85 8.. H 8.8 68: 8.. H :8 8 88.38 888>
8.8. 88 H 8.. 8.83 88 H ...8 888 88 H 8.8 8:89 88 H 8.8 .8 828
8.83 88 H 88 3:3 .88 H 88 A888 88 H 88 8.83 88 H 88 ... 828
.689 8.8 H 88 8:83 ..8 H 88 888 88 H 8.... 8.83 :8 H 8... . 828

.3 £8.83 88
8. 8.. .8 8

6.38 9:83 3.82.8 .8388: .8 Son .8538:

 

.coonflicmaocfi N. >88 .93sz% E9. «mm 53> no.8... 8888 .o matawto o_mE .o 8.599. compo ucm mango; room ..A». 038.

 

190

 

.modva . ”8:05 68:00 E9. €932: 3:85:05 .
.8898: .0 8E: 8.. E995 ..on ..o. 3.83.8.8. o

._o.ooE omx=2 :_ .3998: .o 9:: .m mom :0. 3.3.6.4

n

885 888.2 s 8 828 88 .88. 8 8.8.8 8; £98; .88 m
.92.: .0 :3E:: . m_mE_:m .o .8835 .0 mm H mcmoE 28:88 .82 En mo:_m> __<

 

 

 

E3 88 H 88 48...... 888 H 88 .8388 H 88 .88.. 88 H .88 88.285
.88. 888 H 88 E8. 88 H 88 .88. 88 H «.8 .88.. 88 H ..8 9.88 88>
.88. 88 H .88 .. ... 5 888 H 88 .88. 88 H .88 8.8.. 88 H 88 .N 828

8 8.38 .o .8. £88; 8.8. 8.8.3

:8: 88 H 8. 6...... 88 H .8. 6.8. 88 H 8.. 48.8.. 88 H 8.. 88-2822
.88. 88 H E. E8. .88 H 8.. .88. 88 H E. .88.. 88 H 8.. 9.88 88>
EN. 88 H 8.. E... 88 H 8.. ...: 88.8 H 8.. .88. 88 H 8.. .8 828

8 p88 .8 .8. £8.83 8:8. 8.8.3.

€2.88 H 8.8 48...... .88 H «.8 .88. 88.8 H 8.8 88: 88 H ..8 888822
.88. .88 H 8.8 .8. .88 H :8 6.8. .88 H v.8 .88.. .88 H 8.8 8.38 88>
.88. 88 H R8 .. ... .. 8.8 H 88 .88. 8.8 H 88 8.8: 88 H 88 .8 828

o 8.38 .8 .8. £8.83 8:55 8.8.3
8.88 .-8 28b

 

191

 

.58988888 09. .o 8:... .8 £982, >83 .0 .09 3:0 8:. 80. 8288.68.

8

.88... 88.8.2 5 8.8888 8 88 E88; 888 88 888.8 88; :9. 8

.29.: .0 .3E:: . 8_mE_:m .o 85:83:. .0 mm H 8:92.. 98:88 .888. 2.8 8829 =<

 

 

 

8:88. ...8 H 88.8 8.8... 8.8 H 88.8 8.8. 8.8 H 888 8.88. 8.8 H 8.8 .8 82..

8:88. 8.8 H 8.8 8.83. 8.8 H 88.. A888. 8.8 H 38.. 8:88. 3.8 H 38.8 3. 82:

8:88. 8.8H38. A.:3. ...8H88. A888. 8.8H88. 8:3. ...8H88. 882:
V 8A8..8.EE. 898 8.8:. 8.88.28

8:88. 88.8 H .88 8:8... 88.8 H 88.8 A888. 88.8 H 8.8 8.8... 88.8 H 83.8 .8 82:

8:88. 88 H 38.8 8.83. 88.8 H 838 A888. 88.8 H .88 8:88. 88.8 H 88.8 3. :2:

8:88. 8.8 H 83.8 A. :3. 8.8 H 88 A888. 8.8 H 88.8 8:3. 8.8 H .88 8 oz:
88:... oo< 8.8.2

8:88. 88.8 H .88 8.8.. 88.8 H 88.8 888. 88.8 H 8.8 88:83. 88.8 H 88.8 .8 :2:

8:88. 88 H .88 8.83. 88.8 H 88.8 A888. .88 H 88.8 8:88. 88.8 H 88.8 3. :2:

8:88. 8.8 H 88.8 A. :3. 8.8 H 38 A888. 8.8 H 88.8 8:3. 8.8 H 38.8 8 :2:
Ass. :98 8.8:

 

2.

o._.

F...

0

A28... 9:8... .2882. 38858.88. .8 888 .8528:

 

.5388. :wfiefi 8. >80 .m:o_.m.8mm E9. «mm 5.3 80.80.. 8E8: .o m::amto 28:: .o 08:38:. _m._:ma:< 8.8 8.3.8...

192

.00.0vn_ . ”0:90 .0008 E0: $9006 20:85:05 .
.mw H 0009: 90:00 .000. 20 mm:_m> =<

 

 

 

 

 

 

 

«v.0 H 0.20.. 3.0 H 9.0? 00.0 H 3.8. 0Y0 H V0.0? 0000-202:
0Y0 H 00.0.. 00.0 H 00.0w 0v.0 H 3.0? 00.0 H 0.0.? 02:00 93>
3.: 0.000360% 000.8 “mam
5.0 H 0.0 3.0 H 00.»~ 00.0 H 006 «0.0 H 00.0 300-2025.
2.0 H 3.0 9.0 H :3. 00.0 H 004. 9.0 H 00.0 9.300 93>
3.1355320»... cam: .30. .0 0033.003
30 H 00.0 3.0 H 0:. 00 H 5v 0'0 H and 800-2025.
0P0 H 006 9.0 H 006 0V0 H 004q 2.0 H 00.0‘ $300 93>
5:35..
000 H 00.00? 0.0.0 H 00.0: 00.0 H v0.02. ~00 H 00.03 800.2025.
0N.N H 00.0: 0:. H 00.5? 00.~ H 00.0NF VON H N009 02:00 0::o>
£53 $8.;
.3; H V0.00 00.? H 00.00 00.? H v0.0.0 00.? H v0.0a 0000.062:
N0; H 05.8 3H H v0.00 00.? H 00.0w F0... H 00.00 0:000 0::o>
...-:00 :0 003223000 .5on
#0? Acta 800 400: 803622
3;: 6%: So: $0: 258 98>
A905. 00 50:5: \ 0.9020 00 .00.:an
0... 9.0 0

3
.225 9.33.2

00.33 $550.3... .0 «not 3530!

 

000800. 50:25 me >00 .mcoafimmd E0: 000 55, 09000 0800 ..ometmwto 90E 00 5030 0:0 0000:0808 .5QO 3,5223 .00 2an

193

 

00000 ..0002 05 2 000020 00: 20 “05 0000 “—0 E0200 0

.mm H 0000:. 000000 0000. 0.0 000_0> __<

 

 

 

 

 

 

 

00.0 H 00.0 00.0 H VV.3 0: H V0.0 V0.0 H V0.0 00000.02:

00.0 H 0: 00.0 H 30.0 00.0 H 0: 00.0 H V0.0 0:000 0008»
0000 0300800.: .3063... .x.

V0.0.H 03.0 00.0 H 00.0 0V0 H 0: 00.0 H 03.0 00000.02:

00.0 H 00.3 00.0 H 00.0 V0.0 H 00.0 0V.0 H 00.0 0:000 0:0o>
00000 08009000.: ..00000 ox.

0V3 H 00.0 0V3 H 00... 00.3 H 30.0 00.3 H 0..... 00000.02:

00.3 H 3.0 00.0 H 00.0 00.0 H 30.3 V0.0 H 00... 0:000 0008»
0000 00000500.“. .x.

00.0 H 00.0V 00.0 H 00.0V V0.0 H 00. V 00.0 H 00.0V 00000.02:

00.0 H 00.00 00.x. H 00.00 00.0 H 00.30 0V0 H 00.V0 0:000 0c0o>
003.00”. =00.0:O .x.

30.0 H 00.00 03.0 H 00.00 V0.V H 00.00 0V0 H V0.00 00000.02:

03.0 H 00.00 V0.0 H 00.00 00V H 30.00 V0.0 H 00.00 0:000 0c0o>
35% 9.3.0.5". .x.

Em: SS: @100 000: 0000-000:

50: So: GE :0: 0 0:80 93>
0.00: 00 .00E0: \ 0.0.500 .0 0000.02:

03 0.3 3.0 0

:22. 9:03 .2258 .2053: .o 3% .0520:

 

.8038. 00:25 «2% .mcozsmmefi 000 .0; 02000 0500 03:30.8 0.05 EB. 50% 0:02.28 .0 $5320.00. S: 5 V0 003

194

 

.mm H 0:00E 0.0000 0000. 0.0 0029 ..<

 

 

 

 

0.0 H 0.00 0.0 H 0.00 0.V H 0V0 0.00.. 00000.02:

00 H v.0: 0.3 H 0.3 0.0 H V2 0.02 93%. 28>
03.0.8 __3.0=0 ...

0.0 H 0.3 0.0 H 0.5 0.0 H32 0.02 800.002.).

3 H002 to H 0.02 3 H 0.5 0.02 0:30 88>
. 0:30 050.03". V.

$2. .20.: 8.0. .00: 800-0002

.20. E2. .05 .0: 0 0:80 28>
.000.. “.0 .0200: . 0.0.0.00 .0 000.002:

2 3 2. o

.23.. 9:0... .2058 3550.5: .0 080 .0530:

 

8.000 50:25 2

:00 00005000 80.... 0mm 5.? 000000 00.00 00 0000000 0.08 E0... 0:000 .0E>0_0_00 00 3:50 00.0.0.0. 00.3 E “.0 0000.00.00 02.0.00 .0V.0 0.00..

195

5.5 DISCUSSION

The present study demonstrated that in utero and lactational exposure to
EE2 at high doses (1.0 and 10 pglkg bw/d) decreased early postnatal body
weights in male offspring. Commercially available oral contraceptives provide
daily doses ranging from 0.4 to 2.0 pg EE2/kg bw (19). Therefore, early
developmental delays in an infant may result from the mother taking oral
contraceptives during an undetected early pregnancy. We expected that in utero
and lactational exposure to EE2 have a similar effect on body weights of male
offspring as we observed in our previous DES study because studies indicated
that the effective doses of DES and EE2 in cell proliferation and ER binding
assays were similar or greater compared to E2 (41-45). However, the decrease
in early postnatal body weights reported here is not in agreement with our
previous results from the DES study in that there were no significant treatment
effects on body weights of male offspring (17). The reasons for these conflicting
results may be that EE2 has a different estrogenic mode of action in the in vivo
assay system compared to DES. Therefore, it is difficult to predict the in vivo
estrogenic effects of EEMs.

Epidemiological and laboratory animal studies demonstrated that in utero
exposure to EEMs at low doses increases body weights in adulthood via
interference of the normal developmental and homeostatic controls over
adipogenesis and energy balance (46-49). For example, Newbold et al. (50)
demonstrated that in utero exposure to DES (1 part per billion or ppb) and GEN

(50 parts per million or ppm) in drinking water significantly increased mouse body

196

 

 

 

weight at 4 months of age. Furthermore, ERa knockout mice have increased
body fat content with a 10-fold increase in E2 (51, 52). A proposed mechanism
is that low doses of EEMs increase signaling of ERB or block signaling of ER):
and contribute to obesity by altering the body's natural weight-control
mechanisms. However, in the present study, we observed a trend of decreased
body weights of male offspring in both young adult and middle-aged males at 1
and 10 pg EE2/kg bwld. Taken together, further research is needed to
investigate whether in utero and lactational exposure to low doses of EEMs is a
potential cause of obesity.

In addition to decreased early postnatal body weights by in utero and
lactational exposure to EE2, decreases in kidney weights were observed in the
10 pg EE2/kg bw/d male offspring at middle age as well as decreases in testis
weights were observed at PND 21. However, when organ weights were adjusted
for body weight, there were no statistically significant EE2 treatment effects in
organ weights. These results suggest that low doses of in utero and lactational
exposure to EE2 may not affect organ weight, which is in agreement with the
results of a 28-day repeatedoral toxicity study in the rat (53).

The development of reproductive organs is a complex process that occurs
during early gestation and is related to hormonal mechanisms that control the
differentiation of bipotential gonads into the testes or ovaries. In the present
study, we observed one incidence of herrnaphroditic organs in a male in the 0.1
pg EE2/kg bw/d treatment group on PND 105 (1 out of 113 mice maternally

exposed to EE2). This animal’s testis weight was approximately 47% less than

197

 

the mean testes weight of the control group. The cauda epididymis contained no
sperm cells. Similar to our observation, Yasuda et al. (54) demonstrated
ovotestis and intra-abdominal testis with persistent Miillerian and Wolffian ducts
in matemally—exposed male mouse fetuses whose dams were exposed to EE2
(20, 200, or 2000 pglkg bwld) from day 11 through day 17 of gestation, which
was prior to gonadal differentiation. Additionally, studies demonstrated that high
doses of EE2 (200 pglkg bw/d) administered for 2 or 7 days to pregnant mice
caused 17 to 20% cryptorchidism in fetal and postnatal mice (55, 56). In our
previous study (17), there was only one incidence of unilateral cryptorchidism on
PND 315 in a total of 116 male mice offspring maternally exposed to 10 pg
DES/kg bw/d. Since spontaneous herrnaphroditism in the common laboratory
rodent is rare (57-59), our case may be related to the effects of exposure to low
doses of EE2 from GD 12 through lactation. However, further investigation is
needed to determine whether this incidence of herrnaphroditic organs occurred
by chance.

In general, AGD is considered to be a sensitive indicator of
demasculinization of male genitalia. The AGD in males is approximately twice as
long as the AGD in females. This is because dihydroxytestosterone produced by
the gonads increases the length of the perineum separating the anus from the
testicles via stimulation of cell proliferation (60-62). The growth rate of AGD in
male rats is increased around gestational day 15, when testosterone synthesis
begins (63). In the present study, administration of EE2 to pregnant dams was

initiated on GD 12, when the gonads begin to produce dihydroxytestosteone.

198

 

However, AGD in male offspring was not changed. Our finding corresponds to
the results from our previous studies with DES and GEN (17, 18). Furthermore,
few studies demonstrated that EE2 exposure affects AGD in males. (64, 65).
Collectively, in utero and lactational exposure to EE2 at potential environmental
exposure concentration dose not result in demasculinization of male genitalia.

There have been concerns about decreases in sperm concentration due
to in utero exposure to EEMs. In this study, we observed an approximate 20%
decrease in sperm production that was associated with a 10% decrease in testes
weight at middle age in the 10 pglkg bwld group. Similarly, in our previous
studies, a 20% decrease in sperm production was associated with an 11%
decrease in testicular weight due to a DES-induced decrease in the number of
Sertoli cells (25%, 17%, and 32% relative to controls on PND 21, 105, and 315,
respectively) (17). In contrast, a 31% increase in sperm production associated
with an approximate 10% increase in testicular weight was observed in the 10
mg GEN/kg bw/d treatment group (18). However, clinically relevant doses of
EE2 (0.002, 0.02, 0.20, and 2.0 pglkg bw/d) from GD 0 to GD 17, which is
equivalent to gestation week 16 in humans, decreased sperm production without
changing testis weights in 50-day-old mice (19). Collectively, the data indicate
that in utero exposure to EE2 has long-term effects on sperm production and
testicular weight, which may be useful criteria to evaluate male reproductive
toxicity.

In utero exposure to EE2 resulting in decreased testis weight and a

concomitant decrease in sperm production may be at least in part a result of

199

 

 

inhibition of testicular steroidogenesis and local loss of ERa-mediated estrogen
action, which are important in testicular development. Leydig cells and Sertoli
cells are the main synthesis sites of steroid hormones. During PND 10 to 26,
Leydig cells and Sertoli cells in the immature rat are dividing and undergoing
functional maturation (66). Synthesis of steroid hormones in the testis is
dependent on two key groups of steroidogenic enzymes; cytochrome P450
enzymes (e.g. cholesterol side chain cleavage enzyme and aromatase) and
hydroxysteroid dehydrogenases (HSD) (e.g. 3B-HSD and 17B-HSD) (67). An in
vitro study demonstrated that E52 inhibits activities of 3B-HSD and 17a—HSD in
human testicular tissue (68). At 2- or 200-fold higher doses of EE2 compared to
the highest dose (10 pg EE2/kg bw/d) used in the present study, atrophy of
Sertoli cells in fetal mouse testes on GD 18 as well as atrophy of seminiferous
tubules, Leydig cell hyperplasia, and a 67% decrease in testosterone
concentrations in aged (20- to 22-month-old) male mice resulted from in utero
exposure to 20, 300, or 2,000 pg EE2/kg from GD 11 to GD 17 (54, 69, 70). At
the maximum tolerated dose (200 pglkg bw/d) of EE2, Leydig cell atrophy and
degeneration of germinal epithelium were observed in rats (71). After receiving
10,000 pg EE2/kg bw/d, which is 1,000-fold higher dose in the highest dose used
in the present study, by oral gavage for 3 and 5 days, epididymal sperm
production was significantly decreased in 11- and 12-week—old rats (72).
Furthermore, estrogen receptor or knockout (aERKO) mice had normal prenatal
development of the reproductive tract, but at approximately 20 weeks of age,

there was a significant decrease in testis weights (73). Compared with age-

200

# J

 

 

matched, wild-type male mice, there was an approximately 2-fold increase in LH
concentrations, whereas FSH concentrations remained unchanged in aERKO
mice (74). Increased LH secretion resulted in Leydig cell hyperplasia and a 2-
fold increase in testosterone concentrations. These data suggested that in utero
and lactational exposUre to EE2 decrease weights of the testes and reproductive
tract via local loss of ERa-mediated estrogen action. Additionally, the effects of
in utero and lactational exposure to EE2 would be occurring at the level of the
hypothalamus through the negative feedback loop to decrease GnRH pulse
frequency and at the level of the pituitary to decrease responsiveness to GnRH
(9, 75, 76). Therefore, synthesis of LH and FSH, which were stimulated by
GnRH, would result in decreased concentrations of testosterone leading to
altered testicular develOpment, which in turn would alter sperrnatogenesis in the
adult testis. On the other hand, there is a possibility of a direct action of estrogen
on germ cells during various stages of germ cell development. Li et al. (77)
demonstrated that increased differentiating sperrnatogonia type A cells were
stimulated by a 1 pM dose of E2 but not by higher doses.

The present study demonstrated that in utero and lactational exposure to
EE2 at relevant doses for the human did not affect fertilizing ability of sperm
without changing sperm motion parameters. However, this finding is contrary to
results from our previous DES and GEN studies. A dose of 0.1 pg DES/kg bw/d
resulted in increases in sperm fertilizing ability and beat cross frequency on PND
315 (17). On the other hand, GEN (10 mglkg bw) significantly increased in vitro

fertilizing ability of sperm without changes in any sperm motion parameters (18).

201

 

WIT

 

At a 1,000-fold higher dose of EE2, Kaneto et al. (70) reported decreased
percentages of motile sperm, velocity and ALH displacement in rats orally
exposed to 10,000 pg EE2/kg bw/d for 4 weeks as well as reduced male
fertilizing ability in vivo (72). In conclusion, this study demonstrates that in utero
and lactational exposure to relevant doses of EE2 for the human decrease
postnatal body weight and has long-term effects on sperm production and
without changing fertilizing ability of sperm and sperm motion parameters in male

mice.

Acknowledgements

This work was supported by a grant from US EPA (R827-402-01-0) to KC,
PMS, and T2.

202

 

 

5.6 REFERENCES

1.

Schlinger BA, Arnold AP 1991 Brain is the major site of estrogen
synthesis in a male songbird. Proc Natl Acad Sci 88:4191-4194

Roselli CE, Resko JA, Stormshak F 2003 Estrogen synthesis in fetal
sheep brain: effect of maternal treatment with an aromatase inhibitor. Biol
Reprod 682370-374

Patchev AV, Gotz F, Rohde W 2004 Differential role of estrogen receptor
isofonns in sex-specific brain organization. The FASEB Journal Express
Article

Brodie AM 1979 Recent advances in studies on estrogen biosynthesis. J
Endocrinol Invest 2:445-460

Kula K, Walczak-Jedrzejowska R, SIowikowska-Hilczer J, Wranicz JK,
Kula P, Oszukowska E, Marchlewska K 2005 Important functions of
estrogens in men—breakthrough in contemporary medicine. Przegl Lek
62:908-915

Nagata C, Takatsuka N, Shimizu H, Hayashi H, Akamatsu T, Murase K
2001 Effect of soymilk consumption on serum estrogen and androgen
concentrations in Japanese men. Cancer Epidemiol Biomarkers Prev
102179-184

Brewster ME, Anderson WR, Pop E 1997 Effect of sustained estradiol
release in the intact male rat: correlation of estradiol serum levels with
actions on body weight, serum testosterone, and peripheral androgen-
dependent tissues. Physiol Behav 61:225-229

Weinstein RL, Kelch RP, Jenner MR, Kaplan SL, Grumbach MM 1974
Secretion of unconjugated androgens and estrogens by the normal and
abnormal human testis before and after human chorionic gonadotropin. J
Clin Invest 53:1-6

Akingbemi BT 2005 Estrogen regulation of testicular function. Reprod
Biol Endocrinol 3:51

203

 

 

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

Gennari L, Nuti R, Bilezikian JP 2004 Aromatase activity and bone
homeostasis in men. J Clin Endocrinol Metab 89:5898—5907

Levine AC, Kirschenbaum A, Gabrilove JL 1997 The role of sex
steroids in the pathogenesis and maintenance of benign prostatic
hyperplasia. Mt Sinai J Med 64:20-25

Dorrington JH, Fritz IB, Armstrong DT 1978 Control of testicular
estrogen synthesis. Biol Reprod 18:55-64

Robertson KM, O'Donnell L, Jones ME, Meachem SJ, Boon WC,
Fisher CR, Graves KH, McLachIan RI, Simpson ER 1999 lmpairrnent of
sperrnatogenesis in mice lacking a functional aromatase (cyp 19) gene.
Proc Natl Acad Sci U S A 96:7986—7991

Benten WP, Lieberherr M, Giese G, Wunderlich F 1998 Estradiol
binding to cell surface raises cytosolic free calcium in T cells. FEBS Lett
422:349—353

O'Donnell L, Robertson KM, Jones ME, Simpson ER 2001 Estrogen
and sperrnatogenesis. Endocr Rev 222289-318

Simpson E, Rubin G, Clyne C, Robertson K, O'Donnell L, Jones M,
Davis S 2000 The role of local estrogen biosynthesis in males and
females. Trends Endocrinol Metab 1 1 :184—188

Fielden MR, Halgren RG, Fong CJ, Staub C, Johnson L, Chou K,
Zacharewski TR 2002 Gestational and lactational exposure of male mice
to diethylstilbestrol causes long-term effects on the testis, sperm fertilizing
ability in vitro, and testicular gene expression. Endocrinology 143:3044-
3059

Fielden MR, Samy SM, Chou KC, Zacharewski TR 2003 Effect of
human dietary exposure levels of genistein during gestation and lactation
on long-term reproductive development and sperm quality in mice. Food
Chem Toxicol 41 :447-454

Thayer KA, Ruhlen RL, Howdeshell KL, Buchanan DL, Cooke PS,
Preziosi D, Welshons WV, Haseman J, Vom Saal FS 2001 Altered
prostate growth and daily sperm production in male mice exposed

204

 

 

20.

21.

22.

23.

24.

25.

26.

27.

28.

prenatally to subclinical doses of 17alpha-ethinyl oestradiol. Hum Reprod
16:988-996

Miyamoto Y, Ueda K, Oshida K, Ohmori E 2000 Collaborative work to
evaluate toxicity on male reproductive organs by repeated dose studies in
rats 2). Testicular toxicity in rats treated orally with ethinylestradiol for 2
weeks. J Toxicol Sci 25:33—42

Smithells RW 1981 Oral contraceptives and birth defects. Dev Med Child
Neurol 23:369-372

Slikker W, Jr., Bailey JR, Newport D, Lipe GW, Hill DE 1982 Placental
transfer and metabolism of 17 alpha-ethynylestradiol-17 beta and
estradiol-17 beta in the rhesus monkey. J Pharmacol Exp Ther 223:483-
489

Kallen B, Mastroiacovo P, Lancaster PA, Mutchinick O, Kringelbach
M, Martinez-Frias ML, Robert E, Castilla EE 1991 Oral contraceptives in
the etiology of isolated hypospadias. Contraception 44:173-182

Li DK, Daling JR, Mueller BA, Hickok DE, Fantel AG, Weiss NS 1995
Oral contraceptive use after conception in relation to the risk of congenital
urinary tract anomalies. Teratology 51 :30-36

DeSesso JM 1997 Comparative embryology. Handbook of
developmental toxicology. In. New York:: CRC Press; 111-174

Maggs JL, Grimmer SF, Onne ML, Breckenridge AM, Park BK,
Gilmore IT 1983 The biliary and urinary metabolites of [3H]17 alpha-
ethynylestradiol in women. Xenobiotica 13:421-431

Williams MC, Goldzieher JW 1980 Chromatographic patterns of urinary
ethynyl estrogen metabolites in various populations. Steroids 36:255-282

Aheme GW, Briggs R 1989 The relevance of the presence of certain
synthetic steroids in the aquatic environment. Pharrn Pharmacol 41:735-
736

205

 

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

Tabak HH, Bloomhuff RN, Bunch RL 1981 Steroid hormones as water
pollutants, ll. Studies on the persistence and stability of natural urinary
and synthetic ovulation-inhibiting hormones in untreated and treated
wastewaters. Dev Ind Microbiol 222497-519

Williams RJ, Johnson AC, Smith JJ, Kanda R 2003 Steroid estrogens
profiles along river stretches arising from sewage treatment works
discharges. Environ Sci Technol 37:1744-1750

Reefer P, Snyder S, Zegers RE, Rexing DJ, Fronk JL 2000 Endocrine-
disrupting chemicals in a source water. J AWWA 92252-58

Zuo Y, Zhang K, Deng Y 2006 Occurrence and photochemical
degradation of 17alpha-ethinylestradiol in Acushnet River Estuary.
Chemosphere 63:1 583-1 590

Zuo Y ZK, Deng Y. 2006 Occurrence and photochemical degradation of
17alpha-ethinylestradiol in Acushnet River Estuary. Chemosphere ;():
63:1583-1590

Donn J, Mendoza M, Pritchard J 2008 AP Probe Finds Drugs in Drinking
Water. In: The Associated Press. New York

US EPA 2000 Estimated per capita water ingestion in the United States.
In. Washington D.C.: US EPA, Office of Water 1301; 40

US EPA 2001 National primary drinking water regulations; arsenic and
clarifications to compliance and new source contaminants monitoring; final
rule. In. Washington DC: US EPA

Chou K, Cook RM 1994 Paraoxon inhibits fertilization of mouse gameters
in vitro. Bull Environ Contm Toxicol 53:863-868

Reeves PG 1997 Components of the AlN-93 diets as improvements in the
AlN-76A diet. J Nutr 127:8388-8418

Thigpen JE, Setchell KD, Ahlmark KB, Locklear J, Spahr T, Caviness
GF, Goelz MF, Haseman JK, Newbold RR, Forsythe DB 1999

206

40.

41.

42.

43.

45.

46.

47.

48.

Phytoestrogen content of purified, open- and closed-formula laboratory
animal diets. Lab Anim Sci 49:530-536

Gallavan RH, Jr., Holson JF, Stump DG, Knapp JF, Reynolds VL 1999
Interpreting the toxicologic significance of alterations in anogenital
distance: potential for confounding effects of progeny body weights.
Reproductive Toxicology 13:383-390

Gutendorf B, Westendorf J 2001 Comparison of an array of in vitro
assays for the assessment of the estrogenic potential of natural and
synthetic estrogens, phytoestrogens and xenoestrogens. Toxicology
166279-89 A

Branham WS, Zehr DR, Sheehan DM 1993 Differential sensitivity of rat
uterine growth and epithelium hypertrophy to estrogens and antiestrogens.
Proc Soc Exp Biol Med 203:297-303

Van den Belt K, Berckmans P, Vangenechten C, Verheyen R, Witters
H 2004 Comparative study on the in vitro/in vivo estrogenic potencies of
17beta-estradiol, estrone, 17alpha-ethynylestradiol and nonylphenol.
Aquat Toxicol 66:183-195

* Shapiro S, Slone D 1979 The effects of exogenous female hormones on

the fetus. Epidemiol Rev 1 :1 10-123

Mussman HC 1975 Drug and chemical residues in domestic animals. Fed
Proc 34:197-201

Schildkraut JM, Demark-Wahnefried W, DeVoto E, Hughes C, Laseter
JL, Newman B 1999 Environmental contaminants and body fat
distribution. Cancer Epidemiol Biomarkers Prev 8:179-183

Wolff MS, Anderson HA 1999 Environmental contaminants and body fat
distribution. Cancer Epidemiol Biomarkers Prev 8:951-952

Baillie-Hamilton PF 2002 Chemical toxins: a hypothesis to explain the
global obesity epidemic. J Altem Complement Med 8:185-192

207

 

49.

50.

51.

52.

53.

54.

55.

56.

57.

Rubin BS, Murray MK, Damassa DA, King JC, Soto AM 2001 Perinatal
exposure to low doses of bisphenol A affects body weight, patterns of
estrous cyclicity, and plasma LH levels. Environmental health perspectives
109:675-680

Newbold RR, Padilla-Banks E, Snyder RJ, Jefferson WN 2005
Developmental exposure to estrogenic compounds and obesity. Birth
Defects Res A Clin Mol Teratol 73:478-480

Naaz A, Zakroczymski M, Heine P, Taylor J, Saunders P, Lubahn D,
Cooke PS 2002 Effect of ovariectomy on adipose tissue of mice in the
absence of estrogen receptor alpha (ERalpha): a potential role for
estrogen receptor beta (ERbeta). Horm Metab Res 34:758-763

Heine PA, Taylor JA, lwamoto GA, Lubahn DB, Cooke PS 2000
Increased adipose tissue in male and female estrogen receptor-alpha
knockout mice. Proc Natl Acad Sci USA 97:12729-12734

Yamasaki K, Sawaki M, Noda S, lmatanaka N, Takatsuki M 2002
Subacute oral toxicity study of ethynylestradiol and bisphenol A, based on
the draft protocol for the "Enhanced OECD Test Guideline no. 407". Arch
Toxicol 76:65-74

Yasuda Y, Kihara T, Tanimura T, Nishimura H 1985 Gonadal
dysgenesis induced by prenatal exposure to ethinyl estradiol in mice.
Teratology 322219-227

Yasuda Y, Konishi H, Matuso T, Tanimura T 1986 Accelerated
differentiation in seminiferous tubules of fetal mice prenatally exposed to
ethinyl estradiol. Anat Embryol (Berl) 174:289-29

Walker AH, Bernstein L, Warren DW, Warner NE, Zheng X, Henderson
BE 1990 The effect of in utero ethinyl oestradiol exposure on the risk of
cryptorchid testis and testicular teratoma in mice. Br J Cancer 62:599-602

Eicher EM, Beamer WG, Washbum LL, Whitten WK 1980 A cytogenetic
investigation of inherited true hennaphroditism in BALB/th mice.
Cytogenet Cell Genet 28: 1 04-1 1 5

208

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

Imaizuml K, Imado N, Minato Y, Wada I, Takeshita M, Okaniwa A 1987
Case report of hennaphroditism in a rat. Nippon Juigaku Zasshi 4921171-
1 173

Kai K, Satoh N, Watanabe A, Shiraiwa K, Sasano H, Furuhama K 2003
Case report of rat true herrnaphroditism: colocalization of oocytes and
granulosa and sertoli cells in the germinal cord. Toxicol Pathol 312290-294

Hotchkiss AK, Vandenbergh JG 2005 The anogenital distance index of
mice (Mus musculus domesticus): an analysis. Contemp Top Lab Anim
Sci 44246-48

Salazar-Martinez E, Romano-Riquer P, Yanez-Marquez E, Longnecker
MP, Hemandez—Avila M 2004 Anogenital distance in human male and
female newborns: a descriptive, cross-sectional study. Environ Health 328

Clark RL, Antonello JM, Grossman SJ, Wise LD, Anderson C, Bagdon
WJ, Prahalada S, MacDonald JS, Robertson RT 1990 External genitalia
abnormalities in male rats exposed in utero to finasteride, a 5 alpha-
reductase inhibitor. Teratology 42:91-100

Habert R, Picon R 1984 Testosterone, dihydrotestosterone and estradiol-
17 beta levels in maternal and fetal plasma and in fetal testes in the rat. J
Steroid Biochem 212193-198

Ryan BC, Vandenbergh JG 2006 Developmental exposure to
environmental estrogens alters anxiety and spatial memory in female
mice. Horm Behav 50:85-93

Sawaki M, Noda S, Muroi T, Mitoma H, Takakura S, Sakamoto S,
Yamasaki K 2003 In utero through lactational exposure to ethinyl estradiol
induces cleft phallus and delayed ovarian dysfunction in the offspring.
Toxicol Sci 75: 402-411

de Kretser DM, Kerr JB 1988 The cytology of the testis. In: Knobil E NJ
ed. The Physiology of Reproduction. New York: Raven Press Ltd; 837-932

Sanderson JT 2006 The steroid hormone biosynthesis pathway as a
target for endocrine-disrupting chemicals. Toxicol Sci 94:3-21

209

68.

69.

70.

71.

72.

73.

74.

75.

76.

Daehlin L, Hammar M, Damber JE, Berg AA, Petersson F 1986 Effects
of oestradiol-17 beta and ethinyl oestradiol on human testicular
steroidogenesis in vitro. Scand J Urol Nephrol 202177-181

Yasuda Y, Kihara 'I’, Nishimura H 1981 Effect of ethinyl estradiol on
development of mouse fetuses. Teratology 23:233-239

Yasuda Y, Ohara I, Konishi H, Tanimura T 1988 Long-term effects on
male reproductive organs of prenatal exposure to ethinyl estradiol. Am J
Obstet Gynecol 1 59:1246-1250

Andrews P, Freyberger A, Hartmann E, Eiben R, Loof I, Schmidt U,
Temerowski M, Folkerts A, Stahl B, Kayser M 2002 Sensitive detection
of the endocrine effects of the estrogen analogue ethinylestradiol using a
modified enhanced subacute rat study protocol (OECD Test Guideline no.
407). Arch Toxicol 76:194-202

Kaneto M, Kanamori S, Hishikawa A, Kishi K 1999 Epididymal sperm
motion as a parameter of male reproductive toxicity: sperm motion,

fertility, and histopathology in ethinylestradiol-treated rats. Reprod Toxicol
13(4): :279-289

Eddy EM, Washbum TF, Bunch DO, Goulding EH, Gladen BC,
Lubahn DB, Korach KS 1996 Targeted disruption of the estrogen
receptor gene in male mice causes alteration of sperrnatogenesis and
infertility. Endocrinology 13724796-4805

Lindzey J, Wetsel WC, Couse JF, Stoker T, Cooper R, Korach KS
1998 Effects of castration and chronic steroid treatments on hypothalamic
gonadotropin-releasing hormone content and pituitary gonadotropins in
male wild-type and estrogen receptor-alpha knockout mice. Endocrinology
1 3924092-4101

Franchimont P, Chari S, Demoulin A1975 Hypothalamus-pituitary-testis
interaction. J Reprod Fertil 442335-350

Hayes FJ, Seminara SB, Decruz S, Boepple PA, Crowley WFJ 2000
Aromatase inhibition in the human male reveals a hypothalamic site of
estrogen feedback. J Clin Endocrinol Metab 85:3027-3035

210

 

77.

Li H, Papadopoulos V, Vidic B, Dym M, Culty M 1997 Regulation of rat
testis gonocyte proliferation by platelet-derived growth factor and estradiol:

identification of signaling mechanisms involved. Endocrinology 13821289-
1298

211

CHAPTER 6

Effects of Gestational and Lactational Exposure of Female Mlce to 17a-
Ethynyl Estradiol on Reproduction.

6.1 ABSTRACT

Previously, in our diethylstilbestrol (DES) study with mice (1), fecundity,
body weight, litter size, litter weight, and the percentage of male offspring were
significantly decreased in the 10 pg DES/kg bw/d treatment group. The aim of
the present study was to examine the effects of gestational and lactational
exposure to 17a-ethynyl estradiol (EE2) on reproduction of dams. Ethynyl
estradiol is a widely used estrogenic component of oral contraceptives (2).
Pregnant CS7BL/6 mice, bred with DBA/2 males, were gavaged with EE2 (0, 0.1,
1.0, or 10 pglkg body weight per day (bw/d)) from gestational day (GD) 12 to
postnatal day (PND) 20. Average pup weight in the 1 and 10 pg EE2/kg bw/d
treatment groups on PND 1 was significantly decreased (P < 0.01) without a
change in litter weight on PND 1. Four-day and 21-day pup survival rates were
significantly decreased in the 10 pg EE2/kg treatment group (P < 0.01). The
percentage of male offspring was not changed by gestational and lactational
exposure to EE2. Our results suggest that gestational and lactational exposure
to EE2 treatment decreased perinatal body weight and survival rate of offspring

through weaning.

212

 

6.2 INTRODUCTION

There have been human and ecological health concerns about a
pharmaceutical estrogenic compound and its metabolites present in drinking
water supplies due to the inability of sewage treatment facilities to remove them
from the waste stream. Ethynyl estradiol is a widely used estrogenic component
of oral contraceptives (2). Each year, there are approximately 2 million women in
the United States (US) and the United Kingdom (UK) exposed to 0.4 to 2.0 pg
EE2/kg bw/d during undetected early pregnancy (2). EE2 has been known as an
environmental estrogenic modulator (EEM) because it and its metabolite are
excreted in the urine and are found in effluents from wastewater treatment
facilities (3-5). Approximately 17 to 29% of sulfate or glucuronide metabolites of
EE2 ingested as an oral contraceptive are excreted in the urine (3, 4). In March,
2008, the Associated Press reported that pharmaceuticals, including estrogenic
compounds such as EE2, had been detected in the drinking water supplies of 24
major US metropolitan areas supplying 41 million Americans (6). The
concentration ranges of EE2 in sewage treatment plant effluents were in the
range of 0.4 to 15 ng/l and 300 to 1800 ng/l in the UK and US, respectively (7-9).
Concentrations of EE2 were measured at 0.48 pgll in Las Vegas Wash and at
0.52 pg/l in Las Vegas Bay (10). In addition, the concentrations of EE2 in
surface water and rivers are about 5 to 50 pg/ml (11). Based on the US.
Environmental Protection Agency’s (USEPA) drinking water exposure estimates,
women of childbearing age (15 to 44 years of age) could be exposed to 6.3 x 10'6

to 2.5 x 10'2 pg EE2/kg bw/d through water consumption, and children younger

213

 

than one year of age could be exposed to 2.1 x 10'5 to 8.3 x 10'2 pg EE2/kg bwld
(12, 13). Therefore, potential adverse reproductive effects of exposure to EE2
through consumption of drinking water in women and wildlife need to be
investigated.

Environmental estrogenic modulators have been linked to a variety of
adverse effects on reproductive health in both humans and wildlife.
Approximately 8 to 12% of all pregnancy losses in humans are the result of
endocrine factors (14). Since exposure to estrogen during pregnancy plays a
key role in regulating female reproductive process, exposure to EEMs during a
critical period of fetal and neonatal development could permanently disrupt
fertility of the offspring. For example, Yasuda et al. (15) demonstrated that daily
oral gavage of mice with EE2 (0.02, 0.2, and 2.0 mglkg bw) from GD 11 to GD 17
decreased average fetal body weight and increased the number of fetal deaths in

a dose-related manner.

6.3 MATERIALS AND METHODS
Animals

EIeven-week-old virgin C57BL/6 females and seven- to eight-week-old
DBA/2 males were obtained from Charles River Laboratories (Portage, MI, USA).
All mice were housed in polycarbonate cages with cellulose fiber chips (Aspen
Chip Laboratory Bedding, Northeastern Products, Warrensberg, NY, USA) as
bedding. The animal room was maintained at a humidity of 30 to 40% and a

temperature of 23 °C. Lighting in the room was on 12-h light/dark cycle. All

214

animals were given free access to deionized water in glass bottles with rubber
stoppers and AlN-76A rodent feed (Research Diets, New Brunswick, NJ, USA)
ad libitum. This diet is a casein and sucrose-based open-forrnula purified diet
with non-detectable concentrations of the estrogenic isoflavones genistein and

diadzein (16).

Treatment

All mice were housed two per cage on arrival and acclimatized for at least
three days before mating. Each pair of females was housed with a male within a
week of arrival. Females were separated from the male following evidence of
pregnancy and housed individually and randomly assigned to one of treatment
groups. Corn oil and Wet-ethynylestradiol were obtained from Sigma Chemical
Co. (St. Louis, MO, USA). Pregnant females were treated by daily gavage with
Wan-ethynylestradiol (0, 0.1, 1.0, 10 pglkg bw/d) in 0.1 ml corn oil from GD 12 to
PND 20 (Figure 6-1). On the day of parturition (PND 0), animals were not
gavaged to avoid cannibalism of pups by the dams. The dose of test chemical
was adjusted each day to the dam’s body weight, which was measured between

09:00 and 12:00 before dosing.

Reproductive performances
Body weights of females were recorded prior to mating and on PND 1, 7,

14, and 21. Fecundity [(number of pregnant females giving birth to live

215

Pairing female (057BL) and male (DBA) BlflhliBGDZI Wea Ing

Mating Gestation Lactation
F-O IIIIIllllllllllIIIIIIIIIIIIIIIIIIIII

Offspring
F.1

Treatment period
Gestaflonal day 12 Postnatal day 20
- Pregnant females were treated by gavage with EE2 (0,
0.1, 1.0, 10 pglkg maternal bwlday) from gestational
day 12 to postnatal day 20

 

 

 

m We hg

F-OIIIIIIIIIIIIIIIIIIIIII
Ofispflng
F-1
Age Day 0 Day? Day 14 Day 21

Fecundity Body weights

Body weights Organ weights

Litter size Litter size

Litter weight Survival rate of pups

Survival rate of pups Sex ralio

Figure 6-1. Schedule of treatments and parameters measured in females.

young/number of pregnant females) x 100, %] and body weight gain (difference
between pre-mating body weight and final body weight on PND 21) were
calculated. The ratio of the number of pups born live and stillborn pups was
recorded on PND 0. Litter weight and average pup weight were recorded on
PND 1. Litter size was recorded daily for the pup survival analysis. Sex of the

pups was determined on PND 21 and the sex ratio (% of male pups) was

216

calculated. Dams were euthanized and their organ weights were measured on

PND 21.

Statistical analysis

SAS version 8.2 (SAS Inc, Cary, NC, USA) was used for all data analyses.
The litter was considered as the experimental unit. All data were tested for
normality by the Shapiro-Wilk test and residual plot. The MIXED procedure of
SAS was used for analysis, where treatment was a fixed effect and dam was a
random effect. Organ weights were adjusted by body weights at time of
necropsy. Litter size on PND 0 and sex ratio on PND 21 were included as a
covariate due to significant correlations with average pup weights. Dunnett’s test
was used for comparisons between control and treatment groups. Statements of

significance are based on a P value less than 0.05.

6.4 RESULTS
Fecundity

In EE2-exposed females, there was a decreasing trend in fecundity in the
0.1 and 10 pglkg bwld treatment groups (4% and 16%, respectively) when
compared to the control group (Table 6-1). In our previous DES study, fecundity

was significantly decreased in the 10 pglkg bwld treatment group (34%) (Table 6-

2)(1).

217

Body weights and organ weights

Body weights in the our previous DES study were significantly decreased
in the 0.1 and 10 pg DES/kg bw/d treatment groups on PND 7 and 20 (Table 6-
2)(1), whereas body weights and weight gain were not affected by EE2 exposure
from gestation through lactation (Table 6-1). Correspondingly, there were no
differences in liver and thymus weights of dams on PND 21 regardless of body

weight-adjustment.

Reproductive performance

The number of live and stillborn pups was not significantly changed by
EE2 treatment when compared to the control group. However, there was a trend
toward an increase in the number of stillborn pups in EE2-exposed groups (Table
6-1). Average pup weight in the 1.0 and 10 pg EE2/kg bwld treatment groups on
PND 1 was significantly decreased (P < 0.01) without a change in litter weight on
PND 1. Pup body weight in the 1.0 and 10 pg EE2/kg bwld treatment groups
was depressed compared to controls. Body weight was negatively correlated
with litter size (P < 0.05). Sex ratio was positively correlated with average pup
weight (P < 0.05). Four-day and 21-day pup survival rates were significantly
decreased in the 10 pg EE2/kg treatment group (P < 0.01). In our previous study,
the high dose of DES decreased average pup weight on PND 1 and litter size on
PND 0 and 20 (Table 6-2)(1). The percentage of male offspring was not
changed in the EE2 treatment, in contrast to the lower percentage of males in the

10 pg DES/kg treatment of our previous DES study. Overall, gestational and

218

lactational exposure to GEN did not affect any reproductive parameters (Table 6-

3)(17).

219

.ww H CNOE 2.69.: 0.53"“ ummg ..O r505 95 mm:_w> =<

 

 

0.0 H 0.~0 0.0 H 0.00 0.~ H 0.0.. 2.0 H 0.~0 8.0... 008.00
:00 H 0.00 0.2 H 02 DH H 0.00 0.0 H 0.00 0.90300 00.. .600
:02 H 0.0 .0 H 0.20 0... H N~0 0.0 H 3.0 .0323 0:0 .021.
:30 H 0~2 :30 H ~02 00.0 H 03 3.0 H 00.. 0.0 .2 0200 £0.03 ...... 00223
...00.0 H 20.2 .000 H 00.. 00.0 H 03 00.0 H 20.. 0.0 .0 :20. £0.03 00.. 000.24.
100.0 H 00.0 2.00.0 H 00.0 00.0 H D; 00.0 H 00.0 .0 .r 020. £0.03 0:0 000.0><
00.0 H 00.0 2.0 H .~. 3 00.0 H 30 ~00 H 00.2 .0 .H 020. £0.03 .20...
0.0 H ~.v 0.0 H 2.0 0.0 H ~.0 0.0 H ~.0 :~ 020. an.» .30...
0.0 H 0.0 0.0 H 0.0 0.0 H 0.0 0.0 H 0.0 . A0 020. 0N? .03...
0.0 H 0.0 0.0 H 0.0 0.0 H 0.0 .020 H 0.0 .0 0200 00:0 €00.50 ho ..0nE:z
0.0 H 0.0 0.0 H _..0 0.0 H 0.0 0.0 H 0.0 A0 0200 00:0 0>__ ho .0nE:z
00.0~ H 00.0 00.0~ H 00.02 00.00 H 00.00 E- H v~..0 0.0.5 000...... 00.00.00.
00.0 H 2.. 00.0 H 00.. 00.0 H 0: 00.0 H ~02 0.00 .9: 0900.2
0000 H 00.00 00.~0 H 2.02 030 H 00.00 .0. 20 H ~02 .000 005.3»
2005 2.0H 8.2 30:02 2.0H 20.. arms:
E 020 2.0.0; 0005
00.0 H ~10 00.0 H -.~ ~00 H 0: 00.0 H 00.0 0500 20.02.
04.0 H S0~ 00.0 H 00.0~ 00.0 H 20.5 00.0 H 0v.0~ 282..
«0.0 H 00.0~ 00.0 H 0~.0~ 30 H 00- 0.0 H H0- 3020
00.0 H 0v0~ 00.0 H 00.0~ 00.0 H ..0.0~ 00.0 H 0~.0~ 520
00.0 H 00.0~ 20.0 H ~0.0~ ~40 H 0~.0~ 20.0 H 0~.0~ 520
00.0 H 00.0N 00.0 H 00.0.. 00.0 H 00.00 00.0 H 00.00 0:_00E-0.0
20.25 .600
02 0.00 0.00 ~00 03.0008".
2 3 3 2 0.2... 200.30 30:52
00 00 9 t 00.... “00:00.0 .3 .3052
2 0.2 2.0 0

$0200.00. 3.02.8 $03.32

 

 

0000000. 50:25 N_. 30 000000000 Ea... 66000.0 30050.8: 5.2. 000000 00.080. 0o 02.00.0200 0200202000 .70 0.00..

220

...0.0 v Q: 60.0 v n... ”0:90 .0008 0.0.. 2.0.050 2.0005005 ..
0.00 0.0508 020
.o\o .000 x .0 020 :o 03.0 00:0 .0 .0>E:E..~ 020 :o 02.0 00:0 .0 008:2. 0
00 .000 x .0 020 02.0 00:0 .0 .3050? 020 :0 02:“. 0:0 .0 .3802.
H~ 02. 00 000. x00 .0. 8.00.2 M
.0 020 00 000 .00.. .0. 0900.2
.000.... me=2 0. 0000.00: .3 0...... .0 £0.03 >08 .0. 00.09.90.”
...N 020 :0 2.0.03 .62. .00... .000 £0.03 >000 0:..0E0.0 0002000 0000.005
.00 .000 x .00.0E0. 0.0.00.0 .0 0053.059. 02. o. 5.5 00.20 00.0.00. E00020 .o .0952. M

0.08 2-0 2000

221

.3. o v 0.: .00. o v 0... 0:90 .0008 E9. 00.0.00 2.0005005 .

.00 0.0580 .020

..x. .03 x 8 020 00 02.0 00:0 .0 .0>.0:0.FN 020 00 02.0 00:0 .0 .3052.

0

..x. 03 x 8 020 02.0 00:0 .0 .0250? 020 00 02.0 0:0 .0 .000.:2.0

.FN 020 00 £0.03 >000 .00.. 000 0.0.0.5 >000 0020.000 0003.00 0000.00.00

0
.00 02 x 00.000. 00000.0 .0 .00E:0.00:0> 02. 0. 00.0 00.20 00.0.00. E00000 .0 .000.:2.0
.mm H 000.0 0000.0 0.0:00 .000. .0 000.0 0.0 00:.0> _.<

 

 

.00 H F. 3 m... H 0.00 F... H 0.; F. v H 0. 0v 00.0.0 0.00.00

0.0 H 0.8 0... H 0.3 0.0 H 0.5 3‘ H :0 02.0 .0223 0:0 2.030

0... H 0.8 0... H 0.3 ...0 H 3.0 N... H .00 03.0 .0203 0:0 0.30.0

00.0 H 00F 00.0 H 0.1 00.0 H :1 00.0 H 00F .0 F nwzn: £0.03 0:0 000.0><

:Fnd H 00.0 00.0 H 0N.FF 0..... H mmNF 00.0 H 0m.NF .0. F n20. 2.0.0.... .00...

:00 H 0.0 0.0 H 0.0 .0 H 0.0 0.0 H 0.0 :0 020. an.» .00...

:0... H 0.0 0.0 H 0.0 .0 H 0.0 0.0 H 0.0 ... 020. on.» .25
00... H 00.0 00.0 H 2.0 E... H 8.0 00.0 H 5.2 00.00 000.5
:00... H R0... 0v... H 00.00 .000 H :00 00.0 H 3.00 00020
0..... H 00.00 00.0 H 00.00 .00 H :00 :3 H 8.00 3020
.3... H 00.8 00.0 H 00.00 .00... H 3.00 .0... H 5.00 .020
.00... H 00.00 00.0 H 0.00 «to H .000 00.0 H 0000 .020
~00 H 00.9 00.0 H «0.9 00.0 H 00.9 3.0 H 00.3 05.0.0-0...

0.0.2: 230

.000 .00 0.0. 0.00 02.00300

2 N. o. E 0.2... 2:030 .o .3052

0F 0F 9 0F 00.0. 30000.0 .0 000.0:2

0F 0.. F.0 0

02.0.00 008.... N. .00 .mcoumwj0 0008.00.30... ....

0.00.9.0... .2330003020

 

.s 000000 00.0000. .0 0000000000 0200:0900”. 0-0 0.00...

222

 

.52.. 33:. 3 cmuzmcm new b99093 MimamficomN >595... .5 53, 83983.8 s 32.85 mm; Ema

Sun .9958 Hoza

3 .03 x 8 02¢ co m>__m maaa ho 525553 02a :0 m>__m 83a .0 .wnsszv o

..x. do? x 8 02m m>__m 83a ho 3583:? 021 :o w>=m can “_o 5953

n

..x. .02 x AmmfiEE Emcmma ho 89:35:33 02. 2 5.5 9.35 $563 €9.35 *0 35:55 m

.mw H 23.: 23am “mam. 8 :99: 9m 82.? __<

 

 

5.0 H 33 5.0 H 8.2. 8.“ H 3% is H 8.3 «I. H 8.8 no.2. .523.
mm...” H 8.3 new H 8. 5 :5 H 5.3 «on H 83 «3 H 8.8 use .32..» ea :2...“
S.» H 8.8 5.» H 2. a 8.” H 8% :8 H 8.5 t.» H 8.8 ass .33...» 9:. 283.
So H 84 8.0 H m: 8.0 H 93 85 H o: 8.0 H v? a .. oz... 292., ea $202
56 H 3d 3.0 H m; Be H 8.: «3 H 8.2 NS H 8. : a .52“: £925 5::
No H 3 3 H 3 3 H 3 3 H 3 9o H 3 E“ nzn: on» an...
E H to 3 H 3 2. H No 3 H 3 ed H 2 3 oz... can as...
md H 3 9o H I no H 3 ed H o.» no H E 3 oza. 3.» 3::
QE 2: m3 8. new «£2.83.
3 5 a S S 22:. 233.. 33:2
2 3 3 2 NH 8.... 2.29.... .o .352

S 3 ...... to a

$529.55. £2£50

 

 

.5398. 3:95 N? >8 .mcozflwmm E9; EQmEmm 5? ommmzwm mmfiEmz ho 8cm§otwa w>=o=uoawm .m-o 29m...

223

6.5 DISCUSSION

The present study was the third study of a series of experiments to
examine effects of gestational and lactational exposure to EEMs (DES, GEN,
and EE2, respectively) on reproduction in mice. Average pup weights on PND 1
were decreased in the high doses (1 .O and 10 pglkg bwld) of EE2, as were pup
survival rates on PND 4 and PND 21 in the high dose group compared to the
control group. These results suggest that maternal exposure to EE2 during the
gestational and lactational periods may alter body weights and survival rates of
offspring at an early stage of life.

Gestational exposure to EEMs at low-doses may alter fecundity. In the
present study, we observed an approximate 16% decrease in fecundity in female
mice exposed to 10 pg EE2/kg bwld. This result is consistent with our previous
DES study in that the high dose (10 pglkg bw/d) of DES resulted in a 34%
decrease in fecundity, whereas the phytoestrogen GEN did not affect fecundity (1,,
17, 18). The decrease in fecundity induced by EEM exposure during gestation
may be due to an altered uterine environment. It has been demonstrated that
estrogen administration (5 mglanimal by intramuscular injection on gestational
day 9 and 10), before the conceptus secretes estrogen on gestational day 12,
alters the pattern of insulin-like growth factor (IGF) gene expression through the
nuclear factor kappa B (NF-KB) system. This desynchronizes the uterine
environment for conceptus implantation resulting in embryonic loss in pigs (19).
Yasuda et al. (15) demonstrated that daily oral gavage of mice with EE2 (0.02,

0.2, and 2.0 mg/kg bw) from GD 11 to GD 17 increased the number of fetal

224

 

deaths in a dose-related manner. These results suggest that gestational
exposure to EEMs may lead to embryonic loss due to alteration of the uterine
environment. A possible reason for different effects between synthetic estrogens

(DES and EE2) and phytoestrogens (GEN) on fecundity could be that estrogenic

transactivation of GEN is 104 to 105 times less potent in the luciferase reporter
gene assay and in the cell proliferation assay in estrogen response cell lines (20,
21).

Litter size on PND O in the 1.0 pg EE2/kg bwld treatment group was
increased, while it was decreased in the 10 pg DES/kg bw/d treatment group (P
< 0.01) (18). The increase in litter size in the 1.0 pg EE2/kg bwld treatment
group may be a result of increased fetal retention. An increase in litter size after
gestational exposure to EE2 was also reported by Edgren et al. (22) in rats
treated with 1.25 pglkg maternal bwld. Litter weight on PND 1 was significantly
decreased in the 10 pg DES/kg treatment group (P < 0.05) without changing
average pup weight on PND 1 (1 , 18).

Gestational and lactational exposure to EE2 decreased average pup
weights on PND 1, while exposure to DES and GEN treatment did not.
Decreased perinatal body weights corresponded to decreased fetal body weights
in primates maternally exposed to estrogen due to reduced placental blood flow
during late pregnancy (23, 24). Yasuda et al. (15) demonstrated that daily oral
gavage of mice with EE2 (0.02, 0.2, and 2.0 mglkg bw) from GD 11 to GD 17

caused a dose-dependent decreased in average fetal body weight.

225

 

The 10 pg EE2/kg bw/d treatment decreased survival at 4 and 21 days of
age, while DES and GEN treatment did not affect pup’s survival (1, 17, 18). In
the present study, body weight-adjusted maternal liver weights were significantly
decreased in the high EE2 treatment group (Table 6-1). Pup survival rate was
positively correlated with the dam’s liver weight and litter size PND 0 (P < 0.05)
and negatively correlated with thymus weight (P < 0.01). These results are
consistent with other studies that indicate that estrogen induces cytochrome
P450 in the liver, thus increasing liver weight (25, 26). Therefore, the hepatic
metabolism of estrogenic compounds by the dam could enhance the pup’s
survival rate. Contrary to the protective effect of the dam’s hepatic metabolism of
estrogenic compounds, estrogenic compounds suppress the dam’s immune
system and increase thymus weight (27).

Interestingly, the percentage of males was not changed in the EE2 and
GEN treatment groups, but it was slightly greater compared to the control group
in the 1.0 pg DES/kg bwld treatment group (P < 0.05) (18). There was a large
and significant decrease (32%; P < 0.05) in the percentage of males in the 10 pg
DES/kg treatment group. This result is consistent with a study that indicated that
the sex ratio at birth-was decreased at doses 0.02 and 0.2 pg DES/kg bw.
Administered from GD 11 to 17 in CF-1 mice (P < 0.01) (28). Furthermore, it is
known that EEMs have estrogenic and antiestrogenic properties, thus altering the
sex ratio via changing parental biological factors. For example, high serum
concentrations of 2,3,7,8-tetrachlorochlibenzo-p-dioxin (TCDD) in fathers

exposed during the Seveso accident in 1976 were significantly associated with a

226

decline in sex ratio up to eight years after the incident in two areas of highest
contamination (Meda and Seveso) (P = 0.008) (29). The effect of TCDD on the
sex ratio was detected at serum concentrations lower than 20 ng/kg bw in the
fathers who were exposed to TCDD (30). However, since pregnant females in
our studies and in the study of Palanza et al. (28) were randomly assigned to
treatment groups and administered compounds after conception, this precludes a
direct effect on the fertilization of eggs by XX- or XY-bearing sperm. However,
published studies addressing possible association or effects of EEMs on
mammalian sex ratios have still produced contradictory results because of
unknown mechanisms. Further studies are needed to confirm the effects of
EEMs on sex-selection and associated biological mechanisms.

In conclusion, the present study indicates that gestational and lactational
exposure of female mice to 10 pg EE2/kg bwld leads to a decrease in offspring
body weight and survival. Considering the environmental exposure
concentrations of EE2 through both drinking water and the diet, further studies
are needed to investigate potential adverse effects of EE2 on reproduction in the

human and wildlife.
Acknowledgements

This work was supported by a grant from US EPA (R827-402-01-0) to KC,
PMS, and T2.

227

6.6 REFERENCES

1.

Fielden MR, Halgren RG, Fong CJ, Staub C, Johnson L, Chou K,
Zacharewski TR 2002 Gestational and lactational exposure of male mice
to diethylstilbestrol causes long-term effects on the testis, sperm fertilizing
ability in vitro, and testicular gene expression. Endocrinology 143:3044-
3059

Thayer KA, Ruhlen RL, Howdeshell KL, Buchanan DL, Cooke PS,
Preziosi D, Welshons WV, Haseman J, Vom Saal FS 2001 Altered
prostate growth and daily sperm production in male mice exposed
prenatally to subclinical doses of 17alpha-ethinyl oestradiol. Hum Reprod
162988-996

Maggs JL, Grimmer SF, Orme ML, Breckenridge AM, Park BK,
Gilmore IT 1983 The biliary and urinary metabolites of [3H]17 alpha-
ethynylestradiol in women. Xenobiotica 13:421-431

Williams MC, Goldzieher JW 1980 Chromatographic patterns of urinary
ethynyl estrogen metabolites in various populations. Steroids 36:255-282

Ranney RE 1977 Comparative metabolism of 17alpha-ethynyl steroids
used in oral contraceptives. J Toxicol Environ Health 3:139-166

Donn J, Mendoza M, Pritchard J 2008 AP Probe Finds Drugs in Drinking
Water. In: The Associated Press. New York

Aheme GW, Briggs R 1989 The relevance of the presence of certain
synthetic steroids in the aquatic environment. Pharm Pharmacol 41:735-
736

Tabak HH, Bloomhuff RN, Bunch RL 1981 Steroid hormones as water
pollutants, ll. Studies on the persistence and stability of natural urinary
and synthetic ovulation-inhibiting hormones in untreated and treated
wastewaters. Dev lnd Microbiol 22:497-519

Williams RJ, Johnson AC, Smith JJ, Kanda R 2003 Steroid estrogens
profiles along river stretches arising from sewage treatment works
discharges. Environ Sci Technol 37:1744-1750

228

 

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

Roofer P, Snyder S, Zegers RE, Rexing DJ, Fronk JL 2000 Endocrine-
disrupting chemicals in a source water. J AWWA 92:52-58

Fent K, Weston AA, Caminada D 2006 Ecotoxicology of human
pharmaceuticals. Aquat Toxicol 76:122-159

US EPA 2000 Estimated per capita water ingestion in the United States.
In. Washington DC: US EPA, Office of Water 1301; 40

US EPA 2001 National primary drinking water regulations; arsenic and
clarifications to compliance and new source contaminants monitoring; final
rule. In. Washington DC: US EPA

Arredondo F, Noble LS 2006 Endocrinology of recurrent pregnancy loss.
Semin Reprod Med 24:33-39

Yasuda Y, Kihara T, Nishimura H 1981 Effect of ethinyl estradiol on
development of mouse fetuses. Teratology 23:233—239

Odum J, Tinwell H, Jones K, Van Miller JP, Joiner RL, Tobin G,
Kawasaki H, Deghenghi R, Ashby J 2001 Effect of rodent diets on the
sexual development of the rat. Toxicol Sci 61 :1 15-127

Fielden MR, Samy SM, Chou KC, Zacharewski TR 2003 Effect of
human dietary exposure levels of genistein during gestation and lactation
on long-term reproductive development and sperm quality in mice. Food
Chem Toxicol 41 :447-454

Fielden MR 2002 Reprodictive and genomic effects of gestational and
lactational exposure to estrogenic endocrine disruptors in male mice. In:
Department of biochemistry and molecular biology. East Lansing:
Michigan state university; 3-57, 99-138

Geisert RD, Ross JW, Ashworth MD, White FJ, Johnson GA, DeSiIva
U 2006 Maternal recognition of pregnancy signal or endocrine disruptor“.
the two faces of oestrogen during establishment of pregnancy in the pig.
Reprod Suppl 62:131-145

229

 

20.

21.

22.

23.

24.

25.

26.

27.

28.

Shapiro S, Slone D 1979 The effects of exogenous female hormones on
the fetus. Epidemiol Rev 1:110-123

Mussman HC 1975 Drug and chemical residues in domestic animals. Fed
Proc 34:197-201

Edgren RA, Clancy DP 1968 The effects of norgestrel, ethinyl estradiol,
and their combination (ovral) on the young of female rats treated during
pregnancy. Int J Fertil 13:209-214

Beydoun SN, Abdul-Karim RW, Haviland ME 1974 The regulatory effect
of estrogens on fetal growth. Ill. Placental deoxyribonucleic acid,
ribonucleic acid, and proteins. Am J Obstet Gynecol 120:918-921

Shepherd RW, Stanczyk FZ, Bethea CL, Novy MJ 1992 Fetal and
maternal endocrine responses to reduced uteroplacental blood flow. J Clin
Endocrinol Metab 75:301-307

Sundstrom SA, Sinclair JF, Smith EL, Sinclair PR 1988 Effect of 17
alpha-ethynylestradiol on the induction of cytochrome P-450 by 3-
methylcholanthrene in cultured chick embryo hepatocytes. Biochem
Phannacol 37:1003—1008

Jager W, Correia MA, Bomheim LM, Mahnke A, Hanstein WG, Xue L,
Benet L2 1999 Ethynylestradiol-mediated induction of hepatic CYP3A9 in
female rats: implication for cyclosporine metabolism. Drug Metab Dispos
27:1505-1511

Salem ML, Matsuzaki G, Kishihara K, Madkour GA, Nomoto K 2000
Beta-estradiol suppresses T cell-mediated delayed-type hypersensitivity
through suppression of antigen-presenting cell function and Th1 induction.
Int Arch Allergy Immunol 121:161-169

Palanza P, Parmigiani S, vom Saal FS 2001 Effects of prenatal
exposure to low doses of diethylstilbestrol, o,p'DDT, and methoxychlor on
postnatal growth and neurobehavioral development in male and female
mice. Horm Behav 40:252-265

230

 

29.

30.

Figa-Talamanca I, Tarquini M, Lauria L 2003 Is it possible to use sex
ratio at birth as indicator of the presence of endocrine disrupters in
environmental pollution?]. G Ital Med Lav Ergon 25:52-53

Mocarelli P, Gerthoux PM, Ferrari E, Patterson DGJ, Kieszak SM,
Brambilla P, Vincoli N, Signorini S, Tramacere P, Carreri V, Sampson
EJ, Turner WE, Needham LL 2000 Paternal concentrations of dioxin and
sex ratio of offspring. Lancet 355:1858—1863

231

CHAPTER 7
7.1 CONCLUSIONS

The aims of the present study was to examine and to compare the effects
of maternally administrated three different EEMs, DES, EE2, and GEN, at human
exposure related concentration during in utero and lactation on sperm
production, sperm quality, and sperm fertilizing ability in male offspring (F-1) and
on superovulation and egg fertilizing ability in female offspring (F-1).

This study demonstrated that in utero and lactational exposure to EEMs at
human exposure related concentrations may have persistent or latent effects in
sperm production in male offspring. The high doses (10 pglkg) of DES and EE2
decrease sperm production in middle-aged male offspring. This result suggests .
that in utero and lactational exposure to synthetic and potent EEMs may
decrease the total population of germ cells through affecting directly on the fetal
testis and/or indirectly on the fetal hypothalamic-pituitary axis.

On the other hand, in utero and lactational exposure to the EEMs could
persistently alter ovarian function. The low dose (0.1 pglkg) of DES increases
the number of ovulated eggs in postpubertal female offspring and the high dose
(10 pglkg) of EE2 decreases fertilizing ability of eggs in peripubertal female
offspring. However, in utero and lactational exposure to a phytoestrogen, GEN
at human exposure related concentration may have no health risk to reproduction.

Additionally, this study demonstrated that gestational and lactational
exposure to EEMs at human exposure related concentrations decreases body

weights of offspring from early postnatal period through lactation. Taken together,

232

our results indicate that potential effects on reproduction of EEMs may not be
predictive as results of different adsorption, metabolism, disposition, excretion,
potency, duration of exposure and dose of each EEMs. Collectively, it is
important to consider and to examine potential effects of an EEM both in vivo as
well as in vitro studies. In vivo assessment of EEMs involves multigenerational
studies, could provide the entire facts essential for health hazard assessment
required because degree of adverse effects would be different with age as well

as effects of EEMs may be persistent and/or latent.

233