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EFFECTS OF ESTROGENIC COMPOUNDS ON THE FATHEAD MINNOW
(PIMEPHALES PROMELAS)

By

Stephanie R. Miles-Richardson

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Pathology

1997

ABSTRACT

EFFECTS OF ESTROGENIC COMPOUNDS ON THE FATHEAD MINNOW
(PIMEPHALES PROMELAS)

By

Stephanie Miles-Richardson

The purpose of this study was to assess the usefulness of the fathead
minnow (Pimephales promelas) as a pathologic bioindicator of environmental
estrogen exposure by characterizing the gross and histologic response of the
secondary sex characteristics and gonads. Compounds selected included 17 [3-
estradiol (E2), the endogenous estrogen, and environmentally relevant
compounds, 4-nonyl phenol (NP) and nonylphenol ethoxylate (NPE). A
reversibility study was conducted to determine if gross and histologic lesions
attributed to estrogen exposure were reversible once exposure was discontinued
and a field study was conducted to assess the environmental usefulness of this
model. Sexually mature male and female fathead minnow breeding sets were
exposed to several concentrations of E2, NP, or NPE via a proportional flow-
through diluter to characterize lesions that might be observed with estrogenic
compounds. Fish were exposed to E2 for 14 days in a definitive study. To
simulate more environmentally relevant concentrations, fish were exposed to
0.0625, 0.1, 0.125, 0.25, 0.5, 1, 2, or 10nM E2. Males had does-dependent
atrophy of their secondary sex characteristics, the fatpads and nuptial breeding

tubercles. Histologic testicular lesions included the presence of numerous

Sertoli cells and germ cell syncytia. Ultrastructurally. Sertoli cells had large
phagolysosomes distended with necrotic spermatozoa and myelin figures. The
ovaries contained a preponderance of primary follicles with few secondary and
Graafian follicles. To determine if testicular lesions caused by E2 were
reversible, males exposed to 10nM E2 for 10 days were examined histologically
and Ultrastructurally. Testes were examined on the final day of exposure and
over a period of 4 months. Sixteen weeks after exposure to E2, testicular lesions
were reversed. Sexually mature breeding sets were exposed to 0.1, 0.3, 1, 3, or
10 pg NP and NPE for 42 days each. Although NP is a weak estrogen agonist,
lesions in males were similar to those observed in the E2 studies. Exposure to
0.1 or 0.3 pg NP caused atrophy of the male breeding tubercles. Males exposed
to 1, 3, or 10 pg NP had seminiferous tubules containing intraluminal
macrophages and occasional germ cell syncytia. Sertoli cell cytoplasm was
distended with myelin figures and residual bodies. The effects of wastewater
treatment plant effluents known to contain E2 and NPE, as well as other
synthetic estrogens such as ethynylestradiol and 4-tert-octylphenol (OP) was
examined. Two replicates of caged fathead minnows were place in 6 area
wastewater treatment sites and 2 reference sites for 4 weeks. One wastewater
treatment site which had a histologic response of the testes consisting of Sertoli
cell proliferation, was significantly different from other sites. Results of these
studies suggest that the gross and histologic responses of fathead minnows can

be used as bioindicators of exposure to environmental estrogens.

To Burnell and Brandon

ACKNOWLEDGMENTS

I would like to extend special thanks to my academic advisor, Dr. Jim
Render, and my major professor, Dr. John Giesy for their encouragement and
dedication in assisting me in the completion of this research. I also extend my
gratitude to the other members of my guidance committee, Dr. Scott Fitzgerald,
and Dr. Tom Bell for their suggestions, support and contributions to my research
program. I would also like to acknowledge Drs. Stuart Sleight and Bill Helferich
for their contributions to my research project.

A project of this nature could not have been successfully undertaken
without the collaboration and goodwill of the members of my research team at
the Aquatic Toxicology Laboratory. Led by the vision of Dr. Giesy, our research
team shared not only technical expertise, but also friendship and mutual respect.
I am indebted to Drs. Vince Kramer, Sue Pierens, Alan Blankenship, and Alex
Villalobos. I would also like to thank Krista Nichols, Erin Snyder, Shane Snyder
and Dan Villeneuve for their assistance, collaboration, and camaraderie.

I would like to acknowledge Ralph Common and Donna Craft of the
Department of Pathology, Electron Microscopy Laboratory. I appreciate their
time, effort, and technical expertise. I extend my gratitude to Sharon Thon of

the Animal Health Diagnostic Laboratory for her creative input and generous

V

offering of her time and assistance in this project. I would like to acknowledge
the financial support of this research by the National Institutes of Health (NIH)
Public Health Service (Award/Grant #5 T32 ESO7146), the National Institutes of
Environmental Health Sciences (NIEHS) Superfund Basic Research Program
(ESO4911), the Chlorine Chemistry Council (CCC) of the Chemical
Manufacturers Association (CMA), and Michigan State Univerisity’s College of
Veterinary Medicine and Department of Urban Affairs.

I must thank my family and friends who have consistently supported me
throughout my academic endeavors. Without a such a support network, my
achievements would remain merely dreams. I would like to thank my parents,
Dr. and Mrs. Benny M.Miles of Grambling, LA for giving me the courage and
tenacity to follow my dreams and my parents-in-law, Mr. and Mrs. Burnell
Richardson, Sr., and my grandmother-in-law, Mrs. Mildred Richardson of
Winnsboro, LA for being patient and supportive while I did. I thank my little
brother Benjamin, my Godparents, Jack and Valena Lane, and my cousin Jerel
for being avid “front row supporters” throughout my endeavors.

I also would like to acknowledge the support and encouragement of my
extended family, Melise Huggins, Raynald Higgins, Susan Billups VVIley,
Devonne Johnson Washington, Minnie Hoey, Robert Killins, Dr. Lisa Metoyer
Harris, Dr. Ellyne Mary Brown, and Dr. Steve Andrews. I would like to thank my
mentor, Dr. Fred Tippet of Tuskegee School of Veterinary Medicine for his

consistent support through the years. I am certainly indebted to my

vi

undergraduate institution, Grambling State University, for equipping me with the
tools and desire to continue my academic pursuits.

Finally, I would like to thank my husband Burnell for his unwavering
support and my son Brandon for being a source of inspiration as I realized my

dreams.

vii

TABLE OF CONTENTS

Page

LIST OF TABLES .................................................................................... xi
LIST OF FIGURES ................................................................................ xii
LIST OF ABBREVIATIONS ................................................................... xix
INTRODUCTION ..................................................................................... 1
LITERATURE REVIEW ........................................................................... 5
Reproductive anatomy and physiology of fish .............................. 5
Anatomy and differentiation of the gonads ....................... 5

The testes and spermatogenesis ...................................... 7

The ovarian follicle .......................................................... 10

Reproductive endocrinology ....................................................... 15

The pituitary gland .......................................................... 15

Gonadal hormones ......................................................... 16

Testicular hormones ....................................................... 17

Ovarian hormones .......................................................... 17

Sexual dimorphism .......................................................... 18

Reproductive cycles ........................................................ 19
Environmental estrogens ............................................................ 22
Estrogenic compounds .................................................... 22

Dichloro diphenyl trichloroethane ................................... 23
Diethylstilbestrol .............................................................. 26

Alkylphenol ethoxylates .................................................. 28

Natural estrogenic compounds ....................................... 30

Mechanism of action ....................................................... 31

Measures of estrogenicity ............................................... 33

Human health implications .............................................. 36

viii

CHAPTER 1: Effects of waterborne exposure of 17 B-estradiol on
the secondary sex characteristics and gonads of fathead minnows
(Pimephales promelas).

Abstract ....................................................................................... 39
Introduction ................................................................................. 40
Materials and Methods ................................................................ 41
Study design ................................................................... 41
Gross examination and specimen collection ................... 44
Gonad scoring criteria ..................................................... 45
Statistical analysis .......................................................... 46
Results ........................................................................................ 46
Fish survival .................................................................... 46
Gross examination .......................................................... 46
Histologic lesions ............................................................ 50
Ultrastructural findings .................................................... 58
Discussion ................................................................................... 58

CHAPTER 2: Lack of persistence of 17 B-estradiol- induced
testicular lesions in fathead minnows.

Abstract ....................................................................................... 66
Introduction ................................................................................. 67
Materials and Methods ................................................................ 68
Study design ................................................................... 68
Gross examination and specimen collection ................... 68
Gonad scoring criteria ..................................................... 70
Results ........................................................................................ 44
Fish survival .................................................................... 70
Gross examination .......................................................... 70
Histologic and Ultrastructural findings ............................. 71
Discussion ................................................................................... 71

CHAPTER 3: Effects of waterborne concentrations of 4-nonyl
phenol and nonylphenol ethoxylate on the secondary sex characteristics
and gonads of fathead minnows.

Abstract ....................................................................................... 83
Introduction ................................................................................. 84
Materials and Methods ................................................................ 85
Study design ................................................................... 85
Gross examination and specimen collection ................... 86
Gonad scoring criteria ..................................................... 87
Statistical analysis .......................................................... 88
Results ........................................................................................ 89

ix

Fish survival .................................................................... 89

Gross examination .......................................................... 89

Histologic lesions ............................................................ 89

Discussion ................................................................................... 99
CHAPTER 4: Application of the fathead minnow as a bioindicator

of suspect environmental estrogens in wastewater treatment effluent: a
field study.

Abstract ..................................................................................... 1 15
Introduction ............................................................................... 116
Materials and Methods .............................................................. 119
Study design ................................................................. 119
Gross examination and specimen collection ................. 119
Gonad scoring criteria ................................................... 120
Statistical analysis ........................................................ 121
Results ...................................................................................... 121
Fish survival .................................................................. 121
Gross examination ........................................................ 122
Histologic lesions .......................................................... 122
Discussion ................................................................................. 122
SUMMARY AND CONCLUSIONS ....................................................... 136
RECOMMENDATIONS ........................................................................ 139
APPENDICES
Appendix A
Fathead minnow necropsy form .................................... 141
Appendix B
Clinical pathology of fathead minnows during exposure
to xeno-estrogens ........................................................ 142
Appendix C
Recipe for resin used in tissue preparation for electron
microscopy ................................................. ° .................. 143
Appendix D
Histologic examination of fathead minnows during
exposure to xeno—estrogens .......................................... 144
LIST OF REFERENCES ...................................................................... 146

Table

LIST OF TABLES

Page

Summary of experimental designs of four 17 B-
estradiol (E2) exposure studies. .................................... 43

Survival of fish and testicular severity scores of
male fathead minnows exposed to 17 B-estradiol
(E2) ................................................................................. 47

Survival of fish and ovarian follicular stages in
females exposed to OnM, 2nM, or 10 nM 17 B-
estradiol .......................................................................... 48

Medians and ranges of the diameter of tubercles
and the length of fatpads of male fathead minnows
exposed to 17 B-estradiol (E2). ....................................... 51

Histologic scores and measurements of fatpads
and tubercles of males in 17 B-estradiol reversal
study ............................................................................... 72

Survival of fish and testicular severity scores of
males exposed to nonylphenol ........................................ 90

Survival of fish and percentage of ovarian follicular
stages of females exposed to nonylphenol ..................... 91

Survival of fish and testicular severity scores of
males exposed to nonylphenol ethoxylate ...................... 92

Survival of fish and percentage of ovarian follicular

stages in females exposed to nonylphenol
ethoxylate ........................................................................ 93

xi

Table Page

10. Measurements of the diameter of tubercles and the
length of fatpads of male fathead minnows
exposed to nonylphenol .................................................. 94

11. Measurements of the diameter of tubercles and the
length of fatpads of male fathead minnows
exposed to nonylphenol ethoxylate ............................... 108

12. Measurements of the diameter of tubercles and
the length of fatpads of male fathead minnows
exposed to wastewater effluent ..................................... 123

13. Testicular severity scores of males exposed to
wastewater treatment effluent. ...................................... 127

14. Percentage of ovarian follicular stages in females
exposed to wastewater treatment plant effluent ............ 131

xii

Figure

LIST OF FIGURES

Page

Sexually mature fathead minnows. Females are

lighter and have a slender, tapered head devoid of
tubercles. Males have a short, blunt head with

breeding tubercles on the snout (arrow) and a

dorsally located fatpad (arrowhead) ................................ 49

Three sexually mature male fathead minnows in 17
B-estradiol (E2) treatment groups, from left to right:
Control, 2nM E2, and 10nM E2. Note atrophy on

nuptial breeding tubercles of the two treated fish

(right). x2.0 ...................................................................... 52

Relationship between average tubercle diameter

and treatment groups; Cont (control), Solv (solvent
control), 2nM 17 B-estradiol and 10nM 17 (3-

estradiol. Bar denoted by (A) is statistically

different from bar denoted by (B). ................................... 53

Relationship between average fatpad size and

treatment group of males exposed to 17 B-estradiol

(E2) with no statistical difference between Cont

(control), Solv (solvent control), 2nM E2, or 10 nM

E2 .................................................................................... 54

Photomicrographs of testes from fathead minnows
exposed to 2 and 10nM 17 B-estradiol (E2). a)

Sertoli cell proliferation (arrowheads) is present in

testis of a 2nM E2-treated fish. Hematoxylin and

eosin. x 350. b) Greater magnification shows a

germ cell ......... syncyntia (arrow). Hematoxylin and eosin.
x 700. .............................................................................. 55

xiii

Figure

10.

11.

12.

Page
Photomicrograph of testis from a fathead minnow
exposed to 10 nM 17 B-estradiol (E2). Note marked
Sertoli cell proliferation (arrowheads) and a germ
cell syncytia (arrow). Hematoxylin and eosin.
x350. ............................................................................... 56

Relationship between average testicular severity

score and treatment group. Bar denoted by letter

(A) indicates that Cont (control) and Solv (solvent

control) are statistically different from 2nM or 10nM

17 B-estradiol denoted by letter (B). ............................... 57

Photomicrograph of an ovary from a female

fathead minnow exposed to 10nM 17 B-estradiol.

Note preponderance of primary and secondary

follicles and a single atretic follicle (arrow).

Hematoxylin and eosin. x 175. ........................................ 59

Relationship between average percentage of each

stage of ovarian follicular development and

treatment group with no statistical differences

between Cont (control), Solv (solvent control), 2nM

17 B—estradiol or 10 nM 17 B—estradiol ........................... 60

Transmission electron micrographs of testes from

fathead minnows exposed to (a) 2nM 17 B-estradiol (E2)
and (b) 0nM E2. a) Notice the Sertoli cell cytoplasm is
distended by phagolysosomes that contain necrotic
spermatozoa, myelin figures and membranous debris
(arrows) b) Spermiation yields a small residual body
(arrow) that is normally phagocytized ............................... 62

Photomicrograph of testis from a fathead minnow
exposed to 10 nM 17 B-estradiol, week 4 post -
exposure. Note the germ cell syncytium present in
the lumen of the seminiferous tubule (arrow).
Hematoxylin and eosin. x 780 ......................................... 73

Transmission-electron micrograph of the testis from
a fathead minnow exposed to 10nM 17 B-estradiol,
week 2 post-exposure. Note a cluster of
spermatozoa in the Sertoli cell cytoplasm in
varying degrees of degeneration (arrows). x 10,499 ...... 74

xiv

Figure
13.

'14.

15.

16.

17.

Page
Photomicrograph of testis from a fathead minnow
exposed to 10nM 17 B-estradiol, week 16
post-exposure Note a single Sertoli cell
characterized by a vesiculate nucleus and a
prominent nucleolus (arrow). Hematoxylin and eosin.
x 780. ................................................................................. 75

Transmission electron micrograph of testis from a
fathead minnow exposed to 10nM 17 B-estradiol,
week 2 post-exposure. Note the phagocytized
germ cells in varying stages of digestion (arrows).
x 21,907. ......................................................................... 76

Transmission electron micrograph of testis from a

fathead minnow exposed to 10nM 17 B-estradiol,

week 3 post-exposure. Note Sertoli cell

distended with degenerate spermatozoa, myelin

figures, and residual bodies. x 17,092. ........................... 77

Transmission electron micrograph of testis from a
fathead minnow exposed to 10nM 17 B-estradiol,
week 5 post-exposure. Note Sertoli cell with
residual body in cytoplasm (arrow). x 26,068 .................. 78

Photomicrographs of testes from fathead minnows
exposed to 10nM 17 B-estradiol (E2) at (a) Week 6

post E2- exposure and (b) Week 16 post E2-exposure

a) Note the large number of Sertoli cells lining seminiferous
tubules (arrows) and a germ cell syncytia (arrowhead) as
well as a monomorphic population of germ cells filling the
lumen of tubules. Hematoxylin and eosin. x 312.

b) Week 16 post E2-exposure. Note occasional Sertoli
cells and several stages of germ cells. Hematoxylin and
eosin. x312. .................................................................... 80

Figure
18.

19.

20.

21.

22.

23.

Page
Relationship between tubercle diameter and nonylphenol
(NP) exposure concentration. (1) Experiment I. There
is a statistical difference in the tubercle diameter
between exposure groups 0.1 and 0.3 mgNP/L.
(2) Experiment ll. Although there is no statistical
difference in any of the exposure groups, note a
similar trend in the diameter of tubercles in
experiment I and II. ........................................................... 95

Relationship between the average testicular severity

score and nonylphenol (NP) exposure concentration.

(1) Experiment I. There is no statistical difference in

the severity score between any concentration of NP.

(2) Experiment. ll. Note that Solv (solvent control),

0.1, 0.3, 1 and 3 pgNP/L are not statistically

different (denoted by bar with letter A). Fish

exposed to 10 pgNPIL had scores which were

statistically different from both controls, 0.1 or 0.3

pgNP/L. ............................................................................ 97

Photomicrograph of testis from a fathead minnow

exposed to 3pg nonylphenol/L. Note the germ cell syncytia
and necrotic spermatozoa near Sertoli cells (arrows).
Hematoxylin and eosin. x 1560 ........................................ 98

Transmission electron micrograph of testis from a
fathead minnow exposed to 3pg nonylphenol/L .
Note the Sertoli cell distended with necrotic
spermatozoa and myelin figures. X 14,820 .................... 101

Transmission electron micrograph of testis from a
fathead minnow exposed to 10pg nonylphenol/L.
Note the presence of numerous myelin figures and
residual bodies. x 15,088. .............................................. 103

Transmission electron micrograph of testis from a
fathead minnow exposed to mpg nonylphenol/L.
Note the spermatozoa- filled macrophages in the
lumen of a seminiferous tubule. x 8,185. ........................ 105

Figure
24.

25.

26.

27.

28.

29.

30.

Page
Transmission electron micrograph of testis from a
fathead minnow exposed to 3pg nonylphenol/L.
Note a Sertoli cell containing 4 spermatozoa and a
single residual body (arrow). x 20,158. ......................... 107

Relationship between tubercle diameter and
concentration of nonylphenol ethoxylate (NPE) with
no statistical difference between exposure groups. ...... 109

Relationship between the fatpad size and
concentration of nonylphenol ethoxylate (NPE) with
no statistical difference between exposure groups ........ 110

Relationship between average tubercle diameter

and nonylphenol ethoxylate (NPE) exposure
concentration. There was no statistical difference
between any concentrations. ........................................ 111

Relationship between average tubercle diameter
and wastewater treatment plant effluent at field
sites. There was a statistical difference between
the Limnology pond and all other sites which were
not different from one another. (PT= Portland;
WM= Williamston; OW= Owosso; DL= Delta
Township; BV= Bellevue; EA= Eaton Rapids;

RS= Reference site; L|= Limnology pond) .................... 124 -

Relationship between average fatpad size and

wastewater treatment plant effluent at field

sites. Statistical differences are denoted by

letters A, B, and C where the same letter indicates

no difference. (PT=Portland; WM=Williamston;
OW=Owosso; DL=Delta Township; BV= Bellevue;

EA= Eaton Rapids; RS= Reference site; Ll= Limnology
pond) .............................................................................. 125

Photomicrograph of gill from a fathead minnow. Note
branchitis characterized by blunting and fusion of gill
lamellae and intralesional protozoa consistent with
Ichthyophthin'us multifiliis. Hematoxylin and eosin.

x 624. ............................................................................ 126

xvii

Figure
31.

32.

33.

34.

35.

Page
Photomicrograph of testis from a fathead minnow from
the Portland site. Note numerous Sertoli cells lining
seminiferous tubules. Hematoxylin and eosin. x
499 ................................................................................ 128

Relationship of testicular severity score and

wastewater treatment plant effluent at field sites.
Statistical differences are denoted by letters A, B,

and C where the same letter indicates no

difference. (PT=Portland; WM= Williamston;
OW=Owosso; DL=Delta Township; BV= Bellevue;

EA= Eaton Rapids; RS= Reference site; L|=

Limnology pond). .......................................................... 129

Photomicrograph of testis from a male fathead minnow
at the Eaton Rapids site. Note the normal appearance of
seminferous tubules. Hematoxylin and eosin. x 62 ........ 130

Photomicrograph of ovary from a fathead minnow
at the Portland site. Note the preponderance of
primary follicles. Hematoxylin and eosin. x 121. ............ 132

Photomicrograph of ovary from a female at the

Eaton Rapids site. Note a relatively equal ratio of

primary (P), secondary (S) and Graafian (G)

follicles. Hematoxylin and eosin. x 62 ............................ 133

xviii

AhR
APE
BV

CAT

cc
CC
cERE
CMA
DDD
DDE
DDT
DES
DT
DNA

EC1
EP,
EQ
' ER

ERE
FSH

hERE
L-cells
LH

Ll
MCF-7

#9"-
mg/L
mm

N/A
nM
nM

LIST OF ABBREVIATIONS

adenine nucleotide

aryl hydrocarbon receptor

alkylphenol ethoxylate

Bellevue

chloramphenicol acetyltransferase
cytosine nucleotide

cubic centimeter

control

consensus estrogen response element
Chemical Manufacturers Association

1 ,1-dichloro-2,2-bis(p-chlorophenyl)ethane: TDE
dichloro diphenyl dichloroethylene
dichloro diphenyl trichloroethylene
diethylstilbestrol

Delta Township, Lansing, Michigan
deoxyribonucleic acid

17 p-estradiol

compounds with estrogen activity

potency of compounds with estrogen activity
estrogen equivalent

estrogen receptor

Eaton Rapids, Michigan

estrogen response element

follicle stimulating hormone

guanine nucleotide

human estrogen response element

CH3 mouse fibroblasts

luteinizing hormone

Limnology pond, Michigan State University
estrogen receptor positive human breast cancer cells
microgram per literl parts per billion
milligrams per liter

millimeter

sample size

not applicable

nanomolar

nanomolar

xix

NP

NP-l
NP-ll
NPE
OP
o,p’DDT
o,p’DDE
OW
PAH
PAS
PCB
PCDD
PCDF
PO

ppb

ppm
RF

SC

TCDD
VTG
WM
YES

nonylphenol

nonylphenol experiment I
nonylphenol experiment ll
nonylphenol ethoxylate
4-tert-octylphenol

ortho, para prime dichloro diphenyl trichloroethylene
para, para prime dichloro diphenyl dichloroethylene
Owosso, Michigan

polycyclic aromatic hydrocarbon
periodic acid-Schiff
polychlorinated biphenyl
polychlorinated dibenzo-p-dioxin
polychlorinated dibenzofuran
Portland, Michigan

parts per billion

parts per million

reference site

solvent control

thymidine nucleotide
2,3,7,8-tetrachlorodibenzo-p-dioxin
vitellogenin

Williamston, Michigan

yeast estrogen screen

INTRODUCTION

Insecticides, drugs, natural compounds, and industrial effluents may
contain chemicals or chemical mixtures with estrogenic activity (Sharpe and
Skakkebaek, 1993). Environmental pollutants that have estrogenic action
include some organochlorine insecticides, such as chlordecone (Eroschenko,
1985; Eroschenko and Palmer, 1980) o,p’- dichloro diphenyl trichloroethylene
(DDT) and its derivatives or metabolites (Fry and Toone, 1981) some phthalate
plasticizers (Jobling et al. 1995), and some industrial chemicals such as
nonylphenol (Jobling and Sumpter, 1993b; Soto et al. 1991), bisphenol A
(Hileman, 1997; Krishnan et al. 1993) and polychlorinated biphenyls (PCBs)
(Eroschenko, 1985). Although the chemical structures of these are different
from steroidal estrogens, some mimic estrogenic compounds by binding with
estrogen receptors (ERs) in the reproductive tracts of laboratory animals
(Eroschenko, 1982).

The ERs bind natural estrogens with high affinity and specificity but also
binds exogenous compounds such as organochlorine insecticides and synthetic
chemicals (Thomas and Smith, 1993; Korach et al. 1991; Klotz et al. 1996).
While these exogenous synthetic estrogens bind ER with a lesser affinity, at

environmentally relevant concentrations, they can elicit estrogenic effects

(Purdom et al. 1994; Shore et al. 1993; Guillette, Jr. et al. 1994; Gross et al.
1994; Holm et al. 1993) Environmental estrogens can mimic the natural
estrogen, 17(3 -estradiol (E2) and disrupt the endocrine system of aquatic
animals, wildlife, and human beings (Gray and Metcalfe, 1997; MacLatchy and
Van Der Kraak, 1995; Lye et al. 1997; Eroschenko, 1981; Jobling et al. 1996).
These compounds may affect the reproductive performance of males (Sharpe
and Skakkebaek, 1993) and females (Gray and Metcalfe, 1997). Potentially,
sexual dysfunction and alterations in gonads can occur and lead to reproductive
impairment (Gimeno et al. 1995; Jobling et al. 1996; Russell et al. 1990).

For the past 50 years, the use of environmental chemicals, including
chemicals which are believed to be estrogenic, has increased sharply (Dibb,
1995) There has also been an increase in the incidence of breast cancer
Western Europe and the US. (Hileman, 1994; Houghton and Ritter, 1995).
Some epidemiological studies have shown an increase in dichloro diphenyl
dichloroethylene (DDE), a metabolite of the insecticide dichloro diphenyl
trichloroethylene (DDT), and polychlorinated biphenyls (PCBs) in breast adipose
tissue and serum taken from breast cancer patients (Houghton and Ritter, 1995).
The increase in breast cancer, reported increase in testicular and prostrate
cancer, and a worldwide decrease in sperm counts (Carlsen et al. 1992) are
speculated to be linked to the use of estrogenic environmental chemicals
(Hileman, 1994).

Attention has been focused on the effects of estrogenic compounds on

the embryonic development of various species of wildlife (Rolland et al. 1995;

Colbom et al. 1993). Reproductive failure of western gulls in the Channel
Islands of California has been linked to the contamination of eggs by DDT in the
early 1970’s (Fry and Toone, 1981). Contamination of wildlife with estrogenic
xenobiotics has altered embryonic sexual development and as a result,
significantly reduced subsequent reproductive success (Colborn, 1994; Colborn
et al. 1993). Experimental exposure of laboratory mice during critical periods of
development has caused teratogenic or carcinogenic effects (McLachIan and
Newbold, 1985). Alligators in Lake Apopka, Florida have a lower rate of hatching
and males have smaller than normal penises (Guillette, Jr. et al. 1994; Guillette,
Jr. et al. 1995).

A biomonitor is needed to screen for estrogenic compounds. Since the
primary source of environmental contamination is water, a fish, as a part of the
food chain, may be an ideal biomonitor. A fish commonly used in bioassays is
the fathead minnow, a ubiquitous and robust species with obvious sexual
dimorphism. Males have prominent secondary sex characteristics which
distinguish sexually mature males from females. These characteristics make it
an ideal fish to use as a bioindicator of estrogenic compounds. Bioindicators
need to be calibrated to ecologically-relevant responses, such as reproductive
efficiency. Once these responses are correlated with E2, the severity of gross
and histologic responses can be used to monitor the potential exposure of fish.
Once biochemical or physiological changes are correlated with histologic

changes, these changes may serve as a predictor of histologic lesions.

This purpose of this study was to use the fathead minnow as a
bioindicator of exposure to environmental estrogens. Objectives of this study
were:

1) to characterize the gross and histologic lesions in male and female fathead
minnows to an endogenous estrogen, E2;

2) to determine if the lesions observed in fathead minnows exposed to E2 are
reversible once exposure has been discontinued;

3) to characterize the gross and histologic lesions in male and female fathead
minnows caused by exposure to the environmentally relevant compounds, NP
and NPE;

4) to test the validity of using the fathead minnow as a field biomonitor of

environmental exposure to suspect estrogens in wastewater effluent.

LITERATURE REVIEW

REPRODUCTIVE ANATOMY AND PHYSIOLOGY OF FISH

Anatormr and Differentiation oLt_he Gonacg Fish have one pair
of bilateral gonads suspended, closely associated with the kidneys. The gonads
are suspended from the dorsal portion of the body cavity by the mesovarium in
females and the mesorchium in males (Hibiya, 1982; Moyle and Cech, Jr. 1988;
Adkins-Regan, 1987; Matty, 1985a). During ontogenesis, the undifferentiated
gonad is a long thin transparent structure (van den Hurk and Slof, 1981) which
forms along the genital ridge and contains primordial germ cells (Hibiya, 1982).
Primordial germ cells are large and ovoid, with a large spheroidal nuclei each
containing a large nucleolus (van den Hurk and Slof, 1981; Hibiya, 1982). The
primordial germ cell nuclei is surrounded by follicle cells and the interstitium
contains capillaries, fibroblasts, collagen fibers, and small clusters of interstitial
cells characterized by round nuclei with one or two nucleoli (van den Hurk and
Slof, 1981). Cartilaginous fishes, as well as other vertebrates, have a
corticomedullary primordium, but teleostean, or bony fish, have primordial tissue
which resembles cortical tissue. The lack of corticomedullary differentiation
makes it difficult to determine at which point in development testicular or ovarian

differentiation has begun (Moyle and Cech, Jr. 1988) but eventually, the

primordial germ cells in the gonads differentiate into testes and ovaries (Hibiya,
1982). During gonadal differentiation, germ cells in ovaries often differentiate
before testes (Nagahama, 1987; Brusle, 1986). The period of differentiation of
the gonads varies in different species. In mammals such as humans and pigs,
for example, differentiation occurs during prenatal life (vom Saal et al. 1992).
Compared to mammals, teleostean gonadal differentiation begins surprisingly
late and takes remarkably long to complete (Nagahama, 1987). For example, in
grass carp (Ctenopharyngodon idella) and rainbow trout (Salmo gairdnen)
gonadal differentiation begins 50 days or more after hatching (Jensen and
Shelton, 1983; van den Hurk and Slof, 1981; van den Hurk et al. 1982) and in
Mugil auratus it begins at the end of the first year (Brusle and Brusle, 1978). In
some species, the undifferentiated stage can last for several years (Adkins-
Regan, 1987), therefore, differentiation and sexual maturity may occur at the
same point in time. With the exception of salmon (Salmo salar and
Oncorhynchus kisutch) which undergo sexual differentiation as early as 27 days
post-hatching (Laird et al. 1978) and mature in 2 to 4 years, species with
relatively early gonadal differentiation also reach sexual maturity early (Adkins-
Regan, 1987).

At the onset of sex differentiation, testes are thin, well-vascularized
structures. Testicular weight increases dramatically. For example, in rainbow
trout (Salmo gairdnen), mean testicular weight increases from 6 pg to
approximately 37 mg as the testes develop over 350 days (van den Hurk and

Slof, 1981). Once sexually mature, during the spawning season, the previously

transparent testes become smooth, white structures that account for
approximately 12% of the weight of a fish. As the ovaries develop, they are
characterized histologically by their lamellar structure and the presence of
groups of up to 8 germs cells containing nuclei in meiotic prophase (van den
Hurk and Slof, 1981). The large, oval, granular appearing yellowish structures
may be 30% to 70% of a fish’s weight_(Moyle and Cech, Jr. 1988).

The Testes and Spermatogenesis. While the testes of different
species of fish may vary in complexity, they are generally similar. Spermatozoa
are formed from spematogonia, much like that which occurs in mammalian
species (Hoar, 1969; Eddy and O'Brien, 1994; Hibiya, 1982; Lofts, 1987).
Spermatogenesis involves a proliferation of spermatogonia through repeated
mitotic divisions and growth to form primary spematocytes which, through
meiosis, form secondary spermatocytes. The division of the secondary
spematocytes produces the sperrnatids which ultimately undergo spermiation
and metamorphose into the motile and potentially functional spermatozoa or
sperm (Hoar, 1969; Hibiya, 1982) In some species, particularly elasmobranchs
and viviparous teleosts, spermatogenesis involves packaging of sperm into
spermatophores which are transferred to the female (Hoar, 1969).

Spermatogenesis occurs within testicular units. These units may be in
small sacs, ampullae, lobules, or tubules (Hoar, 1969; Lofts, 1987). Although
these units have similarities with mammalian seminiferous tubules, the testicular
structure of the fish differs from mammals in that thin tubular components with

fibrous walls lack a pennanent'germinal epithelium and communicate with the

efferent duct system except in cyclostomes (Lofts, 1987). For this reason, the
units are usually called Iobules which accommodate clones of developing germ
cells ensheathed in fibrous capsules known as germinal cysts (Lofts, 1987).

In fishes spermatogenesis occurs within cysts. A germinal cyst consists
of a primary spermatogonium surrounded by one or more flattened follicular
cells which form an ensheathing membranous wall (Lofts, 1987). In contrast to
higher vertebrates, the seminiferous tubule of the teleost does not have a
permanent germinal epithelium (Lofts, 1987; Hibiya, 1982; Hoar, 1969). Nests
of spermatogonia proliferate from the resting germ cells near the margin or basal
region of the seminiferous tubule (Lofts, 1987; Hibiya, 1982). The seasonal
recrudescence of mitotic activity in these cells repopulates the seminiferous
elements with germinal cysts (Lofts, 1987). Primary spermatogonia, the largest
germ cells, often have irregularly shaped nuclei and each divides repeatedly to
produce a uniform cluster of daughter cells with rounded nuclei (Lofts, 1987).
Within this clone of secondary spermatogonia each cell is interconnected by
means of cytoplasmic bridges (Grier and Flechon, 1975; Grier, 1976; Lofts,
1987) which remain throughout the subsequent meiotic division (Lofts, 1987).
In some species, these cytoplasmic bridges persist through formation of
spermatids. Within a given cyst, cells develop at a similar rate which may be
because the intercelluar bridges help maintain synchrony during division within
the individual cysts (Lofts, 1987). Spermatogonial cysts are transformed into
primary spermatocystic cysts through meiosis and then through subsequent

synchronous division are transformed into secondary spermatocystic cysts.

Secondary spermatocystic cysts become greatly enlarged post-meiotic
sperrnatid cysts (Lofts, 1987). Eventually, the sperm-filled spermatocystic cysts
liberates the spermatozoa into the Iobular lumina which become filled with an
amorphous mass of spermatozoa (Lofts, 1987) Spermatozoa are ultimately
released and pass out through efferent sperm ducts (Lofts, 1987; Hoar, 1969)

The main sperm duct, or vas deferens, arises from the posterior
mesodorsal surface of the elongated testis and leads to the urinogenital papilla.
It may be traced anteriorly for a variable distance in a connective tissue groove
of the testis along with the spermatic blood vessels and nerves (Hoar, 1969). In
many teleosts, the paired testes fuse posteriorly and vasa deferentia are
combined into a single sperm duct (Hoar, 1969). Within the body of the testis,
the main sperm ducts give rise to smaller ducts (vasa efferentia) which penetrate
ventrally and laterally to form a drainage system. In some species, for example
poeciliids, these tubules are extremely short, while in others they form an
extensive system of seminiferous tubules which can be followed almost to the
periphery of the organ (Hoar, 1969; Hibiya, 1982). Testes of the poeciliid type
are sometimes referred to as “acinar” while those with the extensive duct system
are called “tubular” (Hoar, 1969).

Sertoli, or cyst cells, and interstitial, or Leydig’s cells, are
nonspermatOQenic cells in the testis (Hibiya, 1982; Matty, 1985a; Fawcett, 1975;
Fawcett, 1977). Sertoli cells exist with the germinal cells and are believed to
play a role in the supplying of nutrients (Hibiya, 1982; Fawcett, 1975; Fawcett,

1977). During spermatogenesis, follicular cells which surround the primary

10

spermatogonium change from flattened cells into a more glandular form, and
take on the appearance and Ultrastructural characteristics of Sertoli cells (Lofts,
1987; Fawcett, 1975; Fawcett, 1977). Then, as the spermatids undergo
spermiogenesis and mature into spermatozoa, the sperm heads embed into their
adluminal surface. Ultrastructural examination has shown that tight-junctional
complexes join Sertoli cells, forming an effective blood-testis barrier during
spermiogenesis (Abrahan et al. 1980; Fawcett, 1975; Fawcett, 1977; Lofts,
1987). The interstitial stromal tissue of the testis contains clusters of cells
which, morphologically and ulstrastructurally, resemble mammalian Leydig cells
(Lofts, 1987) and secrete sex steroids (Hibiya, 1982). Interstitial cells are either
arranged in a pattern similar to the vertebrate pattern where Leydig cells are
grouped between the seminiferous tubules or lobules or the cells occur in the
walls of the seminiferous tubules and are known as the tubule-boundary cells
(Matty, 1985a).

The Ovarian Follicle. The ovary undergoes massive change in size
from a structure which occupies nearly all of the body cavity to a thin. threadlike
organ containing a few immature oocytes. As in higher vertebrates, the oocyte
with surrounding cells forms a follicle. These follicles develop from the germinal
epithelium and become surrounded by and embedded in connective tissue
stroma. During follicular development, an epithelial layer equivalent to the
mammalian granulosa surrounds the egg, and surrounding the granulosa is a
fibrous theca. Corpora lutea, or atretic follicles, can form both before and after

ovulation (Matty, 1985a; Ball and Baker, 1969). Atresia of a maturing follicle can

11

occur with the breakdown of the egg and a mass of cells referred to as a
preovulatory corpus luteum can form. An ovulated follicle can also form a luteal
mass. It is uncertain whether these corpora lutea have an endocrine function in
fish (Matty, 1985a; Ball and Baker, 1969). The fish ovary is generally a pair of
sac-shaped organs covered with an ovarian wall and consisting of an ovarian
cavity, and numerous ovarian lamellae where oogenesis takes place (Hibiya,
1982). It may be a hollow sac or a solid body, bilobed or single, suspended in
the body cavity by a vascularized mesovarium (Matty, 1985a). In some fish
there is no oviduct, so the eggs are discharged into the coelomic space and
escape through an abdominal pore. The majority of teleosts have a sac type of
ovary, with the sac ending posteriorly in an oviduct (Matty, 1985a). While the
ovarian structure varies, ranging from an expanded mesentery or mesovarium to
a complex hollow organ, the functions of this organ are to produce eggs, store
sperm, serve as a site for fertilization, and provide nourishment for the
development of young (Hoar, 1969).

The ovarian cavity connects with the oviduct, and the oviduct from each
bilateral ovary join together to lead to the genital pore (Hibiya, 1982). An ovary
with such a structure is a cystovarian type (Hibiya, 1982; Hoar, 1969). While
teleosts generally have cystovarian ovaries, salmon and trout have a pocket-like
ovary. The semicystovarian or pocket-like ovary has a funnel-shaped
transporting groove leading to the genital pore (Hibiya, 1982; Groman, 1982).
The most simple type of ovary, the gymnovarian type, hangs down releasing

ovulated eggs into the body cavity (Hibiya, 1982). This type is found in the eel.

12

Ovarian follicles develop in association with the germinal epithelium
which covers the surface of the ovary as an extension of the peritoneum referred
to as the mesovarium (Hoar, 1969; Hibiya, 1982). This germinal epithelium also
lines the cavity of the hollow teleost ovary or cystovarian ovary (Hibiya, 1982).
Ovarian follicles are supported by a rich vascular connective tissue stroma. the
tunica albuginea, which is located just under the germinal epithelium (Groman,
1982; Hoar, 1969). The internal lining of the hollow teleost ovary is thrown into a
complex series of ovigerous folds that almost obliterate the cavity. Eggs,
discharged from mature ovarian follicles, pass into the peritoneum or into the
cavity of the ovary, containing rich elastic tissue and smooth muscle (Hoar,
1969).

Early in development, oogonia arise from primordial sex cells associated
with the germinal epithelium and become surrounded by a layer of small
epithelial cells to form the ovarian follicle. The connective tissue near this nest
of cells forms a distinct theca. In some species, the theca assume a very active
role during follicular development (Hoar, 1969; Matty, 1985a) . As the follicle
differentiates, the ova become mature. The ovarian epithelial cells increase in
size and number and form a glandular granulosa. The theca become more
distinct and may be divided into an interna and an externa (Hibiya, 1982; Hoar,
1969). Maturing ova separate from the granulosa by a noncellular membrane,
the zona pellucida (Groman, 1982; Hoar, 1969).

Oogenesis involves the proliferation of oogonia on the ovarian lamella

(Groman, 1982; Hoar, 1969). Initially, the oogonia are large cells containing a

13

large nucleus with a single nucleolus. After multiplication, the oogonia become
considerably smaller and develop into primary oocytes (Hibiya, 1982; Groman,
1982).

Three stages of oocyte development are morphologically distinct.
Chromosomes initially appear thread-like and are distributed throughout the
nucleus during the leptotene stage. During the zygotene stage, the
chromosomes assemble at one side of the nucleus and the nucleolus localizes
adjacent to the nuclear membrane at the opposite end of the nucleus. As the
primary oocyte enters the pachytene stage, the nucleolus moves to the central
part of the nucleus. At this stage, the nucleus increases in size as the
chromosomes become slender and evenly distributed throughout the nucleus.
As the nucleoli become smaller, they move to the periphery of the nucleus and
arrange themselves on the inner side of the membrane. At this time, the cell
body becomes very large and the cytoplasm. becomes basophilic. Follicular
cells surrounding the oocyte become distinguishable in this diplotene‘stage
(Hibiya, 1982). The basophilic cytoplasm becomes acidophilic as yolk
substances accumulate, thus marking the onset of vitellogenesis, or the time
when the yolk vesicles, yolk globules, and oil droplets coalesce (Hibiya, 1982;
Hoar, 1969). The eosinophilic, periodic acid-Schiff (PAS) -positive yolk vesicles
containing glycoprotein eventually become the cortical alveoli and eventually the
perivittelin space. The eosinophilic, weakly PAS-positive yolk globules contain
lipoprotein and carbohydrates (Hibiya, 1982). The oil droplets, containing

glycerides and a small amount of cholesterol, appear as circular vacuoles with

14

paraffin embedded tissue stained with hematoxylin and eosin stain. The oil
droplets stain black when stained with osmic acid. Although the appearance of
the three kinds of yolk substances differs among species, the yolk vesicles
always appear earlier than the yolk globules (Hibiya, 1982).

As accumulation of yolk substances become conspicuous, follicle cells
are proliferating and squamous thecal cells form two layers, the outer and inner
theca membranes, outside the follicle cell layer (Hibiya, 1982; Chieffi and
Pierantoni, 1986). As vitellogenesis commences, the egg membrane becomes
clear, PASapositive, and differentiates into outer and inner layers. At this stage,
the zona radiata, a thick zone of granulosa cells surrounding the oocyte,
becomes apparent. The thickness of the egg membrane decreases just before
full maturation. With the completion of vitellogenesis, movement of the germinal
vesicle, fusion of yolk globules and grouping of oil droplets occurs (Hibiya,
1982). After the germinal vesicles moves near the zona radiata and micropyle,
the first meiotic division is completed and the first polar body is released. Then,
the second meiotic division starts and an egg, arrested in rnetaphase, is
ovulated (Groman, 1982; Hibiya, 1982; Hoar, 1969). Once spawning has
occurred, the ovary is composed of many postovulatory follicles, immature
oocytes and unspawned mature eggs. Follicular cells in the postovulatory
follicles, hypertrophy, and become phagocytic (Hibiya, 1982).

Many of the oocytes become atretic in the course of maturation.
Degeneration is first recognized in the yolk of the peripheral portion of the

oocytes. Follicular cells take part in the absorption of denatured and unspawned

15

eggs (Hibiya, 1982). In fish that spawn many times in one season, several
oocyte groups in different stages of maturation can be recognized in the matured
ovary (Hibiya, 1982; Chieffi and Pierantoni, 1986).

The follicular epithelium of fish has specific functions (Chieffi and
Pierantoni, 1986). The granulosa is responsible for deposition of yolk in the
developing ova and for removing yolk before ovulation. Since the number of
ovarian follicles that develop exceed the number of ova released (Hoar, 1969),
some ova are resorbed during different stages of development (Hoar, 1969;

Chieffi and Pierantoni, 1986).

flEPRODUCTIVEiLIDOCRINOLQGY

The Pituitary Gland. The pituitary gland is composed of the
neurohypophysis, or posterior pituitary, and the adenohypophysis, or anterior
pituitary. The neurohypophysis arises from a downgrth from the floor of the
diencephalon, and the adenohypophysis originates as an ectodermal upgrowth
(i.e., Rathke’s pouch) from the roof of the embryonic buccal cavity (Wingstrand,
1966; Ball and Baker, 1969). The two parts of the pituitary gland surround a
mesodermal rudiment between them which gives rise to their intrinsic blood
supply (Green and Maxwell, 1959; Ball and Baker, 1969).

The adenohypophysis is divided into the pars distalis, which produces
most of the adenohypophysial hormones, and the pars intermedia. The
neurohypophyseal stalk is virtually absent in most fish, and the pituitary is

located clOse to the ventral surface of the hypothalamus. In a few teleosts, the

16

neurohypophysial stalk is extremely long (Wingstrand, 1966; Ball and Baker,
1969). The neurohypophysis is suspended by the hypophysial stalk which
extends from the ventral region of the hypothalamus and contains an extension
of the third ventricle (Ball and Baker, 1969). At the distal end of the stalk, there
is an enlargement, the neurohypophysial lobe or core, which forms the middle of
the gland (Wingstrand, 1966). The stalk contains axonal fibers of
neurosecretory cells which have cell bodies located in the hypothalamus. The
neurohypophysial core consists largely of the endings of these fibers
interspersed with cells called “pituicytes” (Ball and Baker, 1969). The
neurohypophysis functions as a general a storage-release center for materials
which are actually synthesized in the hypothalamus and then transported to the
neurohypophysial core along the neurosecretory axons (Ball and Baker, 1969).
G_orlaga_l Hormones. The most active gonadal hormones of higher
vertebrates are testosterone produced by the interstitial or Leydig cells of the
testis, 17 B—estradiol (E2) and its derivatives from the ovarian follicle, and
progesterone from the corpus luteum (Hoar, 1969). These compounds originate
from acetate via cholesterol, as the parent sterol, to testosterone through
intermediate steps involving progesterone; the estrogens are derivatives of
testosterone or closely related molecules and from the terminal part of the
biosynthetic chain (Hoar, 1969). Similarly, in fishes, androgens and estrogens
are present in the gonadal tissues and blood of many different species (Hoar,

1969)

17

Testfiflar hormones. Testosterone is present in the testes of
teleosts. Testosterone, androstenedione, and androsterone and also 11-
ketotestosterone have been isolated from blood and plasma of a number of fish.
In both sexes, testosterone occurs in the plasma both in the free form and also
conjugated with glucoronic acid (Matty, 1985a). Progesterone has been isolated
from the gonads of teleosts but not from the plasma (Matty, 1985a). While
progesterone is important in the metabolic pathway of steroid production in
teleosts, its absence from plasma probably indicates that it has no biological role
as a hormone in the male fish (Matty, 1985a). Androgens are synthesized
shortly after the onset of gonadal sex differentiation (Matty, 1985a; Hoar, 1969).
Testosterone influences the development of the gonads, secondary sex
characteristics and sexual behavior in the male and can be produced by Leydig
cells, modified cells of the seminiferous tubules, from the spermatogenic
epithelium or by organs other than the testes (Matty, 1985a). The origin of
testosterone from the interstitial or Leydig cells is well known in mammals (Matty,
1985a). While these cells may vary in their prominence, they are always present
as a source of testosterone. The second source of testosterone comes from the
Sertoli cells, which are associated with the basement membrane of the
seminiferous tubules. Sertoli cells are also thought to be involved in the growth
and maturation of the mammal sperm. They also phagocytize unused or
degenerate sperm (Matty, 1985a; Fawcett, 1975; Fawcett, 1977).

Mn homones. For over five decades, it has been known that

extracts of fish ovaries and eggs contain substances which behave similarly to

18

those of estrogenic steroids (Matty, 1985a; Hoar, 1969; Ball and Baker, 1969).
The granulosa cells, certain thecal cells, and the preovulatory and postovulatory
corpora lutea all produce steroids (Matty, 1985a). The theca cells, which
participate in the formation of the corpus luteum in some mammals, probably
secrete progesterone and perhaps estrogen (Hoar, 1969). The major ovarian
steroid, E2 has been identified in several species of teleosts. Estrone and
estriol have also been identified (Matty, 1985a).

Sexual Dimorphism. While internal differences between the sexes
are generally obvious in mature fish, it is often difficult to distinguish between
sexes externally. Fish which spawn a large number of eggs (i.e., mass
spawnwers) often show virtually no sexual dimorphism based upon dichromatism
(i.e., color differences), even when spawning (Moyle and Cech, Jr. 1988). On
the other hand, fish with internal fertilization, are sometimes permanently and
obviously dimorphic or dichromatic.

Sexual dimorphism in fish is based on several features, including body
size. The males of egg-laying species of fish are often larger than the females,
but in live-bearing fish, the female is larger than the male, or at least, the female
achieves a larger size (Moyle and Cech, Jr. 1988). When the female is larger,
the male usually has brighter coloration. This sexual dichromatism is usually a
seasonal phenomenon because bright coloration increases the possibility for
reproductive success (Moyle and Cech, Jr. 1988). Dichromatic fish often have
temporary secondary sexual structural modifications. For example, many

spawning salmon have hooked mouths, or kypes and humps (Moyle and Cech,

19

Jr. 1988). Other examples include breeding tubercles and contact organs which
are present on males of at least 25 families of fish (Moyle and Cech, Jr. 1988).
Breeding tubercles are tiny, keratinized bumps that grow on the fins, head, and
body scales during the breeding season. Contact organs are similar bumps that
have an internal core of bone. Tubercles assist the males in maintaining contact
and in stimulating the females during spawning. Contact organs assist the
males in defending their territories and nests (Moyle and Cech, Jr. 1988).

Not much is known about the role of hormones in the development of the
pronounced and numerous secondary sex characteristics of teleostean fish.
Treatment of adult females with androgens, or treatment of adult males with
estrogen often causes complete reversal of these characteristics. This implies
that sexual dimorphism is a product of activational, rather than organizational,
hormonal influence (Adkins-Regan, 1987; Liley and Stacey, 1983). In each of
the major groups of fishes, development of the secondary sex characteristics
prior to breeding has been shown to depend on gonadal steroids. Studies have
shown that following the removal of the gonad, secondary sex characteristics
fail to develop or regressed (Hoar, 1969). Differentiation of these characteristics
may be initiated or stimulated by a wide variety of natural and synthetic
androgens and estrogens (Hoar, 1969).

Reproductive Cycles. In fishes, reproduction is almost always a
seasonal or cyclical occurrence. Reproduction takes advantage of seasons
which offer the greatest opportunities for survival and development of progeny,

particularly in seasonally unstable environments (Hoar, 1969). Even when

20

environmental conditions are relatively stable and eggs or young are produced
regularly throughout the year, ther'e may still be a cycle of gonadal maturation
imposed by the energy demands of maturing eggs or young (Hoar, 1969).

In the few species where the histologic appearance of the testis has been
recorded throughout an annual cycle of seasons, all spermatogenetic stages
have been noted in all seasons. Marked changes occur in the relative areas of
the different spermatogenetic zones with seasonal variation of testicular weight
(Lofts, 1987). The spermatogenietic cycle of teleosts is commonly
discontinuous, and incorporates a postnuptial resurgence of high mitotic activity
in stem spermatogonia to rapidly restock the spent lobules with germinal cysts
(Lofts, 1987). At this point, proliferation of spermatogonia ceases, but the cysts
already formed continue to mature and eventually discharge mature
spermatozoa into the Iobular lumina (Lofts, 1987). In most northern teleostean
species, the annual cycle is of the “postnuptial” pattern with fully developed
spermatozoa formed before the onset of winter. These spermatozoa are stored
for several months, either in the lobules or sometimes in the efferent ducts,
before being discharged the following spring (Lofts, 1987). As a result, at the
time of spawning, the lobules are primarily filled with large numbers of
spermatozoa (Lofts, 1987). In some species, however, the cycle becomes
halted at an intermediate stage through the winter, and the final maturation into
spermatozoa resumes during the following spring. In this type of cycle, there is
no protracted period of sperm storage, and spermatozoa are only produced

shortly before spawning. This type of cycle is referred to as “prenuptial”. In

21

these species, a post-spawning period of hypothalamic refractoriness to
environmental stimulation imposes a spermatogenetically quiescent break
before gonad recrudescence begins (Baggerman, 1972; Egami and Hosokawa,
1973; Kaya, 1973). Then, once this has finished the degree of spermatogenetic
advancement reached before the onset of winter often depends on the
environmental temperature (Lofts, 1987; Yamamoto, 1969).

Cycles of gonadal development frequently alter many aspects of
metabolism, as well as, behavior and reproductive physiology. Cycles of active
feeding, with storage of fat and long periods of starvation, are characteristic of
many species (Yamamoto, 1969). Electrolyte metabolism may change
seasonally in species which inhabit the ocean waters of relatively constant
salinity and in the euryhaline and anadromous forms. The basic cycle is
probably the one imposed by the seasonal nature of reproduction, and these
regular changes in metabolism are secondary (Yamamoto, 1969).

The endocrine system forms the major link between the environment and
the organs concerned with reproduction. Changing environmental conditions,
operating through the sensory system and specific centers in the brain, trigger
neurosecretion which in turn regulates the activities of the pituitary gland
(Yamamoto, 1969; Hoar, 1969). Pituitary hormones have direct effects on
gametogenesis, metabolism, and behavior and also regulate the development of
the gonadal endocrine tissues (Hoar, 1969). Gonadal hormones take over

some of these pituitary responsibilities and carry on the coordination of events in

22

the production of gametes, sexual behavior, fertilization, and sometimes parental
care (Hoar, 1969).
EMIRONMENTALfESTROGENS

Memo Com‘munds. Since the 19405, manufactured chemicals
have been widely used (Dibb, 1995). Most chemicals are relatively safe,
however, others have been identified as being able to either mimic the natural
estrogen or to interfere with the various systems that regulate the body’s
production of estrogen and other sex-linked hormones (Dibb, 1995). Such
compounds are often referred to as “environmental estrogens” or “environmental
endocrine disrupters”. These chemicals include classical organochlorines such
as o,p’- DDT and its metabolite DDE (Fry and Toone, 1981), Kepone
(Eroschenko and Palmer, 1980) and many of the PCBs (Eroschenko, 1985).
Recent reports indicate that chemicals originating from the plastics and
detergent industry, such as alkylphenols (APEs) (Jobling and Sumpter, 1993b;
Soto et al. 1992) and bisphenol-A (Krishnan et al. 1993; Hileman, 1997) are
also estrogenic (Dibb, 1995; Jobling et al. 1995; Houghton and Ritter, 1995;
Copeland et al. 1986; Sharpe, 1995; Hileman, 1994; Hileman, 1995; Colborn et
al. 1993). Organochlorines are persistent, lipophilic compounds and are
widespread in the environment. This class of chemicals consists of agricultural
pesticides and industrial compounds, most of which were extensively used in the
past (Colbom et al. 1993). While many of these compounds are no longer used

in this country, they can accumulate in lipid-rich tissues as a result of their

23

lipophilicity and lengthy half-lives of several years (McLachIan and Arnold, 1996;
Colborn et al. 1993).

Dichloro diphenyl trichloroethane. The chlorinated hydrocarbon,
dichloro diphenyl trichloroethane (DDT) and its metabolite, DDE have been
shown to be estrogenic (Hileman, 1994). This compound was one of the first
environmental chemical agents to be banned in the United States (McLachIan
and Arnold, 1996). First synthesized in 1874, DDT took on its modern role in
the late 1930’s after Paul Mueller recognized its potential as an insecticide
(McLachIan and Arnold, 1996). Hailed as the new war weapon of the Allies
during the Second World War in Europe, DDT was used copiously on soldiers
because of its ability to kill the lice responsible for the spread of typhoid (Sharpe,
1995). It was also widely used in the tropics to control mosquitoes, and in many
countries as a household fly spray. Because of the beneficial nature of this
compound, Mueller was award the 1948 Nobel Prize for Medicine and
Physiology (McLachIan and Arnold, 1996). Decades later, after its commercial
use in agriculture and in public health programs to control vectors of disease as
malaria, typhus, yellow fever, encephalitis, and chagas, DDT was found to be
potentially disastrous and banned in the United States in 1972 (Guillette, Jr. et
al. 1996). As recently as 1990, DDT was still being produced in Italy, India,
Indonesia, and China (Guillette, Jr. et al. 1996).

The compound, DDT, along with its metabolite DDE gained notoriety 30
years ago in North America when they were blamed for collapsed egg-shells that

led to marked declines in the population of bald eagles, brown pelicans, and

24

peregrine falcons (Line, 1996). Early in the 1960s, scientists had noticed a
decrease in certain bird populations in Europe which was eventually linked to
the use of various pesticides, including DDT. By the early 19705, DDT was
banned for use in the United States, Canada, and many European countries
(McLachIan and Arnold, 1996) largely because of its environmental persistence
and its accumulation and concentration in food chains (Sharpe, 1995) even
though it is still used in developing countries for malarial control (Sharpe, 1995).

The estrogenic activity of DDT has been well characterized. The
compound binds to the ER protein and has a similar mechanism of action of the
endogenous hormone, E2 (Ireland et al. 1980). The affinity of estrogen
receptors for DDT compound is very weak and reported to be 1/2,000- 1/10,000
that of E2 (Houghton and Ritter, 1995).

Evidence of the estrogenic action of DDT can be demonstrated by its
effects on humans and wildlife. In 1975, a spill of Kepone used in the
manufacture of pesticides was linked to a lowered sperm count in men exposed
to the chemical (McLachIan and Arnold, 1996). Subsequent studies
demonstrated that Kepone was a weak estrogen with no structural similarity to
estradiol (McLachIan and Arnold, 1996). A spill of the pesticide Dicofol, which
occurred in Lake Apopka, Florida in 1980, led to various adverse reproductive
findings in native alligator populations. Dicofol contains DDT and its metabolites
1,1-dichloro-2,2-bis(p-chlorophenyl) ethane (DDD) and DDE. Studies conducted
at this site revealed reduced hatchability and viability of eggs, increased

neonatal mortality, higher plasma estradiol in female juvenile alligators, reduced

25

plasma testosterone in males, and small phalluses (Guillette, Jr. et al. 1994;
Guillette, Jr. et al. 1995; Gross et al. 1994).

Otherevidence of the effect of DDT on wildlife exists throughout the
scientific literature. Correlations have been found between drastic declines in
the population of raptors and fish-eating birds and environmental exposure to
organochlorine pesticides, particularly DDT and DDE. The eggshells of these
birds were thinner than normal and DDE was found in the wings of mallards and
wild ducks (Heath et al. 1969). Between 1950 and 1970 offshore California was
subjected to massive contamination by the discharge of up to 1.9 million
kilograms of commercial DDT from the Los Angeles sewer system (Fry and
Toone, 1981). This resulted in high concentrations of DDT in fish, sea lions, and
seabirds. This compound has also been reported to as the cause of breeding
failure in brown pelicans, double-crested cormorants (Fry and Toone, 1981) and
bald eagles (Colborn et al. 1993). The reproductive problem was presumably
due to egg shell thinning and cracking. In Black-footed albatross of Midway
Island, eggshell thinning occurred in a number of bird at levels of 2-3 ppm, thus
causing reduced reproductive productivity because of these contaminants (Line,
1996). Mallard ducks fed DDE at 10 and 40 ppm induced eggshell thinning, and
eggshell cracking and an increased embryo mortality rate. Impaired reproduction
which is less severe than that caused by DDE is caused by 000 and DDT
(Heath et al. 1969).

Several studies have been conducted that examine the effects of DDT on

fish reproduction. Sublethal concentrations of DDT (2.0 mg/kglweek) fed to

26

sexually mature yearling brook trout led to the production of fewer mature ova
(Macek, 1968). Mortality of sac fry from eggs of treated fish occurred around 15
weeks old coinciding with the period of maximum utilization of yolk fat.
Insecticide residues released when yolk was being absorbed are believed to
result in the observed mortality (Macek, 1968). A genetically all-male sexually
undifferentiated population of the common carp used to detect the effects of the
xenoestrogen, 4-tert-pentylphenol, demonstrated oviduct and primordial germ
cell development in exposed male fish at environmentally realistic
concentrations. Males also had poorly developed testis and impaired
spermatogenesis (Gimeno et al. 1996).

Diethylstilbestrol. Diethylstilbestrol (DES) has been used to
study the effects of estrogen in the environment. This synthetic estrogen was
used by women since 1948 to prevent spontaneous abortions but 'in 1971 was
banned (Colbom et al. 1993). Girls born of mothers who took DES were found
to have reproductive organ dysfunction, abnormal pregnancies, a reduction in
fertility, immune system disorders, and periods of depression when young adults
(Colborn et al. 1993; Wilcox et al. 1995; McLachlan and Newbold, 1985; Stilman,
1982). These young women also had an increased rate of vaginal clear-cell
adenocarcinomas, a cancer typically found in much older women (Colbom et al.
1993).

The sons of women treated with diethylstilbestrol during pregnancy had
several reproductive lesions. Epididymal cysts, hypotrophic testes, and capsular

induration of the testes were the most common lesion observed. Analyses of

27

spen'natozoa revealed abnormalities in the sperm count, sperm motility, and ‘
sperm morphology (Gill et al. 1977; Wilcox et al. 1995).

Laboratory studies have substantiated effects observed in persons
exposed to DES during the critical period of sexual differentiation (Colborn et al.
1993; Newbold, 1995; Stilman, 1982; McLachlan and Dixon, 1976; Jones and
Hajek, 1995; Santii et al. 1994). Classic DES studies show that DES has
profound effects on the reproductive tracts of mice following critical periods of
sexual development (McLachlan and Newbold, 1985; McLachlan and Dixon,
1976; McLachlan and Bullock, 1975) Treated female mice were shown to have
oophoritis, ovarian cysts, and numerous changes in the oviduct. Mice exposed
to the equivalent amount of DES that was prescribed to women had retarded
oviductal morphogenesis resulting in oviductal malformations. In treated mice,
histological differentiation of the ovary was arrested. Fimbriae were minimally
developed and ampullae had an irregular lining of columnar cells and had
mucosal folds. Epithelium of the oviduct was hyperplastic and abnormal
glandular structures extended through the muscularis (McLachlan and Dixon,
1976; McLachlan and Bullock, 1975; McLachlan and Newbold, 1985). These
lesions resembled salpingitis isthmica nodosa in women, a condition that had
been previously related to ectopic tubal pregnancy and infertility.

Male mice prenatally exposed to DES had retention of Mullerian duct
remnants (McLachlan and Newbold, 1985). These Mullerian remnants
differentiated into structures homologous to the oviduct and uterus. The

Mullerian remnants were often enlarged and cystic and shared supporting

28

connecting tissue with adjacent male structures (Newbold et al. 1987). Lesions
observed in the epididymis included sperm granulomas, epididymal cysts,
hyperplasia, and adenoma of the epididymal duct (Newbold et al. 1987).
Akylphenol ethoxylates. Alkylphenol ethoxylates (APEs) are
nonionic surfactants comprising approximately 6% of total surfactant production
and 25% of total nonionic surfactant production in the United States (Nimrod and
Benson, 1996; McLachlan and Korach, 1995). Alkylphenols such as nonylphenol
(NP) and related compounds are found widely in surface waters and aquatic
sediments (Ahel et al. 1987; Naylor, 1992; Jobling and Sumpter, 1993b; Naylor
et al. 1992). They are the products of the microbial breakdown during sewage
treatment of a number of industrial surfactants, the APEs (Giger et al. 1984;
Naylor, 1992). Alkylphenols are used in the formulation of certain plastic
products, many of which are used in laboratory supplies (Korach et al. 1991). It
has been estimated that 60% of the more than 300,000 tons of APEs ends up in
the aquatic environment after sewage treatment as short chain alkylphenol
polyethoxylates(Giger et al. 1984; Kvestak et al. 1994). These compounds
function as surfactants because of their alkylphenol hydrophobe and a para-
substituted long chain of repeating ethylene oxide units as the hydrophilic moiety
(Nimrod and Benson, 1996; Talmage, 1997). Nonylphenol ethoxylates (NPEs)
are the most common form of the APEs, constituting 82% of production (Nimrod
and Benson, 1996). Approximately 204 million kilograms of APE surfactants
were sold in the United States in 1990, most of which were NPEs (Naylor et al.

1992). Surfactants, such as NPE are used in a variety of applications including

29

textile manufacturing, pulp and paper manufacturing, polymers, pharmaceuticals,
household products, and various industrial and institutional cleaning applications
(Naylor et al. 1992; Blackburn and Waldock, 1995). These compounds are used
in water, and as a result are found in wastewaters. NPEs and octylphenol
ethyoxylates are also used as spermicides (Nimrod and Benson, 1996).

Municipal wastewaters contain concentrations of APEs ranging from 0.3
to 3 mg/L (Ahel et al. 1994a; Giger et al. 1987). In wastewaters from wood pulp
mills, however, concentrations can be up to ten times higher (Naylor, 1992; Holt
et al. 1992). However, primary degradation of NPEs, nonylphenol
polyethoxylates, and APEs in sewage treatment plants is very efficient (Kvestak
and Ahel, 1995; Giger et al. 1987). But since these compounds can form stable
intermediates, their final degradation efficiency is significantly lower (Ahel et al.
1994b). Some estimate that approximately 65% of NPEs found in wastewater
influent are ultimately released into the environment. The release occurs
primarily as persistent, lipophilic, biotransformation products. As result,
nonylphenolic compounds have been found in rivers (Naylor, 1992; Ahel et al.
1994b; Macromini et al. 1990; Kvestak et al. 1994; Kvestak and Ahel, 1994),
coastal waters (Macromini et al. 1990), and estuaries (Kvestak et al. 1994;
Kvestak and Ahel, 1994).

Several investigations have been conducted to determine the effects of
APEs and related compounds on aquatic species. Alkylphenols, particularly
those with alkyl chains ranging from 6 to 12 carbon atoms, are toxic to salmon

and shrimp (McLeese et al. 1981). Adult male rainbow trout exposed to 30 pglL

30

of NP, 4-tert-octylphenol (OP), and NPEs had significant induction of
vitellogenin, an estrogen-responsive yolk protein (Jobling et al. 1996). During
the 3—week study, rainbow trout males had increased vitellogenin concentrations
along with decreased growth of testes. Histologic examination of the testes
revealed varying degrees of spermatogenic inhibition compared to controls. A
field study demonstrated that caged carp and rainbow trout placed in sewage
effluent had increased plasma vitellogenin concentrations (Purdom et al. 1994).
Natural Estrggenic Compounds. Several different classes of
chemicals in the food supply exhibit both estrogenic and antiestrogenic activity
(Safe, 1995). Over 50 species of plants have estrogenic activity (Verdeal and
Ryan, 1979). These compounds fall into 1 of 3 classes of compounds (Verdeal
and Ryan, 1979; McLachlan, 1980; Martin et al. 1978; Mayr et al. 1992).
lsoflavones, the most common naturally-occurring isofavonoids, contain some
compounds which are estrogenic. An example is genistein,which has a structure
with some resemblance to that of estradiol and diethylstilbestrol (Verdeal and
Ryan, 1979; Martin et al. 1978; Safe, 1995). Genistein is approximately 50 times
less potent than E2. Other common isoflavones which are weakly estrogenic
include daidzein, biochanin A, and formononetin (Verdeal and Ryan, 1979).
Another class of isoflavonoids are coumestans, which are found in alfalfa and
clover. The third class of estrogenic compounds, which include mycotoxins (i.e,
zearalenone) are derived from resorcyclic acid lactones. Some believe that
increased consumption of phytoestrogens are another cause of reproductive

impairment, but this is unlikely since phytoestrogens are quickly metabolized

31

and excreted (Hileman, 1995). The estrogenic activity of phytoestrogens in fish
has been investigated in sturgeon (Pelissero et al. 1993). When phytoestrogens
such as daidzen, biochanin A, genistein, equol, and coumestrol were injected
into the peritoneal cavity, estrogen- responsive hepatic vitellogenesis was
induced (Pelissero et al. 1993).

Mechanism of Acti_og. Endogenous and environmental estrogens are
believed to function in the same way to convert the ER from an inactive state to
an active state (Stancel et al. 1995; Katzenellenbogen, 1995; Smith and Toft,
1993). Estrogens are lipophilic and can diffuse through cell membranes,
cytoplasm, and nuclear envelope to interact with the nuclear receptor (Gorski et
al. 1996). Transcription of estrogen-regulated genes is mediated by high-affinity
binding of estrogens to a specific nuclear protein ER (Klinge et al. 1992). The
ER is one of a family of the steroid receptor superfamily with two highly
conserved regions. These regions are the ligand binding domain, and the DNA
(deoxyribonucleic acid) binding domain and they include two zinc-fingers that
interact with DNA to modulate gene transcription (O'Malley and Tsai, 1992;
Klinge et al. 1992). The binding of E2 to ER initiates a series of steps. During
activation, there is a structural change that includes dimerization of the ER
complex which binds to specific DNA sequences called estrogen response
elements (EREs), located upstream of the promoter of estrogen regulated genes
(Klinge et al. 1992; Smith and Toft, 1993; Carson-Jurica et al. 1990; Clark, 1979;
Mueller, 1986). The EREs are DNA sequences of nucleotides that enable the

receptor to bind to target genes (Stancel et al. 1995; Smith and Toft, 1993). The

32

activated receptor bound to the ERE then interacts with transcription factors to
enhance transcription of proteins.

At one time, it was believed that compounds with a structural similarity to
estradiol would bind to the ER. It has been observed, however, that compounds
with markedly different structures can compete for the traditional estrogen
binding site on the estrogen receptor and elicit varying degrees of responses
(Duax and Griffin, 1985). A phenolic ring appears to be necessary to promote
high affinity for the estrogen receptor and this ring is believed to mimic the
steroid A ring (Duax and Griffin, 1985).

Recent work has suggested that EREs of different genes may be
differentially activated by the complex of the ER with any given estrogen, and the
complexes formed between the ER and different estrogens may show selective
patterns of ERE activation (Stancel et al. 1995). Most studies of EREs have
used the consensus ERE (c-ERE) originally identified from the vitellogenin
genes of xenopus and chickens (Stancel et al. 1995; Burch et al. 1988; Klein-
Hitpass et al. 1988). The c-ERE has the palindromic sequence,
GGTCAnnnTGACC, that binds the estrogen receptor as a homodimer, confers
estrogen inducibility to target genes, and is located in the 5’- flanking region of
the endogenous genes (Burch et al. 1988; Klein-Hitpass et al. 1988; Kumar and
Chambon, 1988). Recently, EREs in other genes regulated by estrogens have
been identified (Stancel et al. 1995). Many of these EREs have different
sequences and are present in different locations than the c-ERE. These

findings suggest that the toxicity of environmental estrogens could involve a

33

number of factors and could occur via several related but distinct mechanisms
(Stancel et al. 1995).

While the phenotypic alterations resulting from exposure to DDT are
thought to be mediated by the estrogen receptor, effects caused by exposure to
DDE may have a completely different mechanism of action. The major
metabolite, p,p’ DDE has little ability to bind to the estrogen receptor but inhibits
androgen receptor binding, androgen-induced transcriptional activity, and
androgen action in developing, pubertal and adult male rats (Stancel et al.
1995). This suggests that abnormalities in male sex development induced by
p,p’ -DDE and related environmental chemicals may be mediated at the level of
the androgen receptor (Kelce et al. 1995; Sharpe, 1995) rather than at the
estrogen receptor.

Metabolism of endogenous and exogenous estrogens occurs by
oxidation, reduction, and conjugation, in order to increase their water solubility
so that they are more readily excreted (Metzler, 1985).

Measures of Estrmenicig. Estrogenicity was first defined as a
physiological response to a compound that induces estrus in vivo (Hertz, 1995;
Korach and McLachlan, 1995). Thus, the measure of estrogenicity has involved
the use of a uterotrophic assay where the response evaluated is an estrogen-
induced increase in wet weight and tissue mass (Korach and McLachlan, 1995).

In recent years, other measures have been developed. A useful ,
measure of the real or perceived impact of dietary estrogens can be obtained by

estimating “estrogen equivalents”, which compare the relative dietary impacts of

34

various classes of estrogenic chemicals (Safe, 1995). The uptake of
environmental or dietary chemicals that elicit common biochemical and/or toxic
responses can be estimated by using an equivalency factor approach in which
estrogen equivalents (EQs) in any mixture are equal to the sum of the
concentration of the individual compounds with estrogenic activity (EC1)
multiplied by their potency (EP1) relative to an assigned standard such as E2 or
DES (Safe, 1995). A similar approach has been used to determine the TCDD
(2,3,7,8- tetrachlorodibenzo-p—dioxin) equivalents of various mixtures containing
halogenated hydrocarbons (Safe, 1995; Safe, 1990). Halogenated aromatics
including polychlorinated dibenzo—p—dioxins (PCDDs), dibenzofurans (PCDFs),
TCDD, and PCBs are an important class of organochlorine pollutants that elicit a
diverse spectrum of biochemical and toxic responses (Safe, 1995). These
chemicals act through the aryl hydrocarbon receptor (AhR)-mediated signal
transduction pathway. The AhR agonists, like TCDD, have been characterized
as antiestrogens (Safe, 1995).

Because of the need to rapidly screen for environmental estrogens,
several in vitro assays have been developed. The E-SCREEN is a cell
proliferation assay which utilizes estrogen-responsive MCF-7 breast cancer cells
(Soto et al. 1992; Soto et al. 1995; Soto and Sonnenchein, 1984). The E-
SCREEN assay compares the cell yield after 4 to 6 days of culture in medium
supplemented with 5 to 10 % charcoal-dextran stripped human serum in the
presence or absence or estradiol and various concentrations of estrogenic

compounds. Other proliferation assays have been developed using MCF-7 cells

35

and genetically transformed mouse L-cells. In the mouse L-cell line, hormone
activity is measured by the expression of the bacterial chloramphenicol
acetyltransferase (CAT) gene under the control of an estrogen-responsive
element (Mayr et al. 1992). Another assay involves the use of vitellogenin
(VT G), an estrogen-inducible protein found in oviparous vertebrates (Ho, 1987).
This protein is the precursor to egg yolk, and usually occurs in females but can
be induced in exposed males (Copeland et al. 1986; Mommsen and Walsh,
1988). Because the liver of fish contains large numbers of estrogen receptors, 3
large amount of vitellogenin is synthesized when stimulated by estrogen (Weis
and Weis, 1986; Pottinger, 1986). Exposure of male fish to various
concentrations of estrogenic compounds has shown pronounced dose-response
VTG induction demonstrating that fish are sensitive to estrogens in the water
(Bromage and Cumaranatunga, 1988; Sumpter and Jobling, 1995). In order to
use \fl'G as a biomarker for estrogens, antibodies must be developed which are
able to recognize VTG regardless of species (Heppell et al. 1995). While a
universal assay for vitellogenesis has not been developed, a significant amount
of research has focused on vitellogenesis in several species of fish including
rainbow trout (LeGuellec et al. 1988; Chen, 1983; Sumpter, 1985; Copeland et
al. 1986; Elliot et al. 1979; Carragher and Sumpter, 1991; MacLatchy and Van
Der Kraak, 1995) tilapia (Kishida and Specker, 1993), carp (Folmar et al. 1996),
catfish (Sundararaj and Nath, 1981), flounder.(Emmersen and Petersen, 1976),

sea trout (Maisse et al. 1991), salmon (So et al. 1985) and goldfish.

36

Reporter gene constructs have been developed to screen for estrogens.
Novel recombinant cell lines have been developed to determine estrogen
agonist activity . An example is the yeast estrogen screen (YES), developed by
expressing the human ER and two estrogen response elements linked to the
lacZ gene in yeast. The YES screen includes a reporter gene containing an
ERE, so that the presence of an estrogen bound receptor is indicated in a
colorimetric fashion. The transformed yeast cell turns blue to indicate the
presence of natural or synthetic estrogens (McLachlan and Arnold, 1996; Arnold
et al. 1996b; Arnold et al. 1996a). The YES screen has demonstrated that
combinations of environmental chemicals may have synergistic activity (Arnold
et al. 1996a).

Studies have also been undertaken to demonstrate the use of receptor
binding as a possible screening method. Estrogens that bind to the mammalian
ER were tested for their ability to bind to the hepatic ER spotted seatrout
(Thomas and Smith, 1993; Smith and Thomas, 1990). While chlordecone bound
to the ER, other estrogenic compounds like DDT and PCB mixtures did not bind
to the seatrout ER. This study demonstrated that the seatrout ER assay may be
useful to screen compounds with estrogenic activity in teleosts (Thomas and
Smith, 1993).

flu—min Heajh lmfiations. Environmental estrogens have also
been linked to adverse human health effects. Since 1940, the incidence of
female breast cancer and endometriosis, a previously rare condition, has risen

in Western Europe and the United States (Safe, 1995; Hileman, 1994). Studies

37

have shown that women with hormone-responsive breast cancer have a higher
concentrations of DDE in their body when compared to women with benign
breast disease (Adlercreutz, 1990). Experimental evidence shows that
compounds, such as some chlorinated organics, polycyclic aromatic
hydrocarbons (PAHs), triazine herbicides, and pharmaceuticals increase
estrogen production and metabolism and thus function as xenoestrogens (Davis
et al. 1993). Epidemiologic studies have found that breast fat and serum lipid of
women with breast cancer contain significantly higher concentrations of some
chlorinated organics when compared to results from women without cancer
(Davis et al. 1993; Houghton and Ritter, 1995). Organochlorines such as DDT
and DDE have weak estrogenic activity, as measured by receptor binding
assays (Houghton and Ritter, 1995). These organochlorines, which induce
cytochrome P450 in humans, may act by altering the metabolism of estradiol to
produce metabolites with prolonged estrogenic activity (Houghton and Ritter,
1995; Davis et al. 1993). Sperm counts in men worldwide have fallen about
50% since 1940 (Hileman, 1994; Sharpe, 1993) and over the same time period,
the incidence of testicular cancer and prostate cancer have increased (Hileman,
1994; Carlsen et al. 1992). In Finland, sperm counts and testicular cancer rates
have remained constant (Hileman, 1995) but this may be explained because
their drinking water comes from unpolluted groundwater rather than rivers or

lakes (Hileman, 1995). If declining human sperm counts is a real trend,

38

exposure to estrogenic or antiestrogenic chemicals may be a possible cause

(Hileman, 1995).

CHAPTER 1:

EFFECTS OF WATERBORNE EXPOSURE OF 17 B-ESTRADIOL ON THE
SECONDARY SEX CHARACTERISTICS AND GONADS OF FATHEAD

MINNOWS (PIMEPHAliES PROMELAS)

ABSTRACT

Environmental contaminants with estrogenic activity have recently
received much attention because of their potential effects on the reproductive
efficiency of humans and animals. This study was conducted with the
endogenous estrogen 17 B-estradiol (E2) to establish the histologic response of
the fathead minnow as a bioindicator species. Sexually mature fathead minnows
were exposed for 14 days to waterborne concentrations of 0.0625, 0.1, 0.125,
0.5, 1, 2, 10, 100, or 1000nM E2. Exposure to E2 caused atrophy of the
prominent male secondary sex characteristics, the fatpads and nuptial breeding
tubercles. Histologic lesions observed in the testes included proliferation of
Sertoli cells and germ cell syncytia consisting of necrotic aggregates of various
stages of cells in the spermatogenic sequence. Ultrastructurally, seminiferous
tubules had large phagolysosomes filled with necrotic spermatozoa, myelin

figures and cellular debris. Females had ovaries filled with increased numbers

39

40

of primary follicles and fewer secondary and Graafian follicles. These findings
support the hypothesis that compounds which mimic E2 have the potential to
alter the reproductive system of sexually mature fish. Results of this study also
support the idea that gross changes and histologic lesions in fathead minnows
may be useful as in vivo bioindicators of environmental exposure to estrogenic

compounds.

INTRODUCTION

Some environmental compounds have been found to contain some level
of estrogenic activity (Sharpe and Skakkebaek, 1993). Included are compounds
such as organochlorine insecticides (Eroschenko, 1985; Eroschenko and
Palmer, 1980), ortho, para prime dichloro diphenyl trichloroethylene (o,p'-DDT)
along with its metabolites (Fry and Toone, 1981), and nonylphenol (NP) (Jobling
and Sumpter, 1993b; Soto et al. 1991). These, and other estrogenic compounds
are believed to mimic the action of E2 by binding with the estrogen receptor, as
has been demonstrated in studies with laboratory animals (Eroschenko, 1982).
Exogenous estrogenic compounds such as organochlorine insecticides and
other synthetic chemicals can bind the estrogen receptor much like natural
estrogens (Thomas and Smith, 1993; Korach et al. 1991; Klotz et al. 1996) at
environmental concentrations that are sufficient to elicit biological effects
(Thomas and Smith, 1993; Jobling and Sumpter, 1993b; Purdom et al. 1994;
Gross et al. 1994; Lech et al. 1996). Environmental estrogens can mimic

endogenous E2 and disrupt the endocrine system of aquatic animals, wildlife

41

(Eroschenko, 1981; Jobling et al. 1996) and potentially human beings. Estrogen-
induced alterations in gonads can lead to reproductive impairments that may
adversely affect individuals and populations (Gimeno et al. 1995; Jobling et al.
1996; Jobling and Sumpter, 1993b; Russell et al. 1990).

Since E2 and other estrogenic compounds are present in the environment
(Shore et al. 1993), there is a need for a sensitive fish species to be used as a
bioindicator. The fathead minnow has very prominent sexual dimorphism and is
therefore an ideal fish species to use as a bioindicator. Mature males are easily
differentiated from females based on the presence of prominent male secondary
sex characteristics (Duda and Buttner, 1993). When sexually mature, males
develop several rows of nuptial tubercles on the rostrum and a prominent fatpad
on the dorsal aspect of the head. In contrast, females are smaller and generally
lighter in color with a tapered head. The gross features of mature males can be
of potential use in screening for effects of potentially estrogenic compounds in
laboratory studies and in field monitoring. The purpose of this study was to
characterize the effects of the prototypic estrogen, E2, on the secondary sex

characteristics and gonads of sexually mature male and female fathead

 

minnows.
MATERIALS AND METHODS
Study design. Fathead minnows (Pimephales promelas) were

obtained from the United States Environmental Protection Agency, Newtown, OH

or reared in the Michigan State University Aquatic Toxicology Laboratory. In a

42

series of four E2 experiments with two replicates each, 172 sexually mature male
and 153 sexually mature female 6-8 month old fathead minnows were placed in
breeding sets in 40 liter aquaria containing one or two clay flower pots (7.5 cm
diameter) to be used as breeding tiles (Table 1). The photoperiod was
maintained at 16 hours light to 8 hours dark and the continuously flowing,
aerated, dechlorinated tap water was maintained at 25-26 °C. The temperature
and dissolved oxygen content of the water were monitored daily. The fish were
fed a mixture of 0.5 grams of Purina Trout Chow® (Purina Mills, Earth City,
MO) and 0.5 grams of TetraMin® flake food (Tetra Sales, Blacksburg, VA) . The
dry food was supplemented with 0.145 grams of brine shrimp (Artemia nauplir).
Four exposure studies, each lasting 14 days, were conducted in a proportional
flow-through diluter (Ace Glass, lnc., Vineland, NJ). Concentrations of E2 were
initially determined by increasing environmental levels 10-fold (Shore et al.
1993). In all experiments, E2 (Sigma Chemical, St. Louis, MO) was dissolved in
0.05% ethanol. The first experiment had E2 concentrations of 10, 100, and 1000
nM and the second experiment had concentrations of 2 and 10nM. To simulate
more environmentally relevant levels of estrogen exposure and to determine an
approximate no effect level, a third E2 experiment was conducted with 0.0625,
0.125, 0.25, 0.5, 0.1, 0.5nM, and 1nM E2. The fourth E2 experiment was
designed to bracket the concentrations of previous experiments, including
concentrations in which effects were seen and those which were environmentally

relevant. All experiments included 2 replicates each and 2 controls, one with

43

Table 1. Summary of experimental designs of four 17 B-estradiol (E2)
exposure studies.

 

Experiment Concentration Total Numbers Endpoint(s) *
nM E2 Males Females

I A0 15 30 HxT
B0
10
100
1000

n A0 58 35 FP; TB; OP;HxT
E’0 HxO; UxT

m A0 71 62 FP; TB; HxT
B0 HxO; UxT

IV A0 28 26 FP; TB; HxT; on

A0= control
B0= solvent (ethanol) control

* FP= fatpad; TB= tubercles; OP= ovipositior; HxT= testicular histology
HxO= ovarian histology; UxT= Ultrastructural examination of testes

44

the solvent ethanol and one control containing only water. The health status of
the fish was monitored during the exposure periods. Fish that were found to
have lost their righting reflex were euthanized with an overdose of Finquel®
(Argent Chemical Laboratories, Redmond, VA).

Gross examination and specimen collection. At the termination of
the study, fish were euthanized with an overdose of F inquel® . Gross secondary
sex characteristics of males were measured prior to histologic examination of the
testes. The height of male fatpad and the diameter of nuptial breeding tubercles
were measured using a 27 mm diameter 6X comparator/magnifier and reticle
(Edmund Scientific Company, Barrington, NJ). Any additional lesions were
noted on the fathead minnow necropsy form (Appendix A). Blood was collected
from the caudal vein (Appendix B) with a heparinized microhematocrit tube for
analysis of plasma vitellogenin (Knights, 1980) and testes were collected for
histopathology and electron microscopy. For electron microscopic examination,
two 5 mm transverse sections were cut from the abdomen of males just cranial
to the pelvic fin. Sections were immersed in 4% glutaraldehyde for 12 hours, at
which point 0.5-1mm sections of testes were dissected with forceps and a
dissecting microscope. The testes were then rinsed in 0.1 M phosphate buffer,
postfixed in osmium tetroxide, and embedded in resin (Appendix C). Thick
sections (1pm) were cut with an ultramicrotome and stained with toluidine blue
and thin sections (800 nm) were stained with uranyl acetate and lead citrate and
examined using a Phillips 301 transmission electron microscope (Phillips

Electronic Instruments, Inc., Roselle, IL). Remaining portions of fish not collected

45

for ultrastructural examination were preserved in Bouin’s solution (Appendix D).
Fish collected for histologic examination were injected intra-abdominally with
0.5 cc of Bouin’s fixative, and the entire fish was immersed in Bouin’s solution for
48 hours. The fish were rinsed by agitating the carcasses in 30 mls of 70%
ethanol for 30 seconds. They were then stored in this solution until trimming.
The fish were trimmed in serial transverse sections, processed and embedded
sequentially in paraffin (SurgiPath, Inc., Richmond, IL). Tissues were sectioned
at 5pm with a microtome, and stained with hematoxylin and eosin for
examination with light microscopy.

gm scoring criterig. Testicular lesions were evaluated according to
the severity of relative or absolute Sertoli cell proliferation and the percentage of
seminiferous tubules affected. Scores ranged from 0 to 4 as follows:

0 = no Sertoli cell proliferation

1= < 25% Sertoli cell proliferation

2= 2 25% < 50% Sertoli cell proliferation

3= 2 50% < 75% Sertoli cell proliferation

4: >75% Sertoli cell proliferation
Degenerative changes including germ cell syncytia, mineralization of
spermatozoa, or necrotic spermatozoa were indicated by (+).

In females, ovaries were evaluated by counting fifty follicles in the ovary
of each female and calculating the percentage of each stage of follicular

development observed. Ovaries were staged according to the following criteria:

46

primary - large nucleus, abundant basophilic cytoplasm and no yolk
vesicles
secondary- presence of numerous eosinophilic yolk vesicles
Graafian- large follicle bordered by an amphiphilic egg membrane
with a central core of eosinophilic yolk protein

atretic- degenerative follicle with shrunken, irregular border

Statistical analysis. Treatment effects were examined by a non-
parametric two-way ANOVA (SAS Institute, Carey, NC; PROC GLM) conducted
on the ranks of the responses. Significant treatment effects showed no
significant interaction. Terms were subsequently examined by Tukey’s HSD
multiple range test. Unless otherwise stated, effects were determined to be
statistically significant at the 0.05 level of type I error (alpha) and 0.2 of type II
error (beta).

RESULTS

Fish spirvival. Fish survival during the exposure studies was at or near
100% except in the second E2 experiment where survival of males and females
were 46 and 80%, respectively (Table 2,3). Most of those fish that died during
the study had no gross or histologic lesions. When present, however, lesions
consisted of moderate to severe bilateral subcutaneous hemorrhage and fin
base hemorrhage. The cause of these lesions was undetermined.

_(_3ross expminaflap. Sexually mature male fathead minnows
exposed to E2 had a more feminine appearance than unexposed males (Figure

1). Although the range of the size of secondary sex characteristics varied,

47

Table 2. Survial of fish and testicular severity scores of male fathead
minnows exposed to 17 B-estradiol (E2).

 

 

 

 

 

 

Experiment Concentration c:N Survival Severity Score (0—4)

nM E2 % mean median range_

I A0 3 100 0 0 00

B0 3 100 0.3 0 0-1

10 4 83 3.5 4 24

100 2 86 3 DMA 24

1000 3 80 3.3 3 34

ll A0 15 100 0.1 0 02

B0 16 100 0.3 0 0-2

2 14 93 3.2 4 14

10 13 46 3.7 4 24

111 A0 3 100 0 0 0-0

E’0 4 75 0 0 00

0.0625 3 100 1 1 02

0.125 3 100 2.7 2 24

0.25 3 67 2 2 2-2

0.5 3 67 3 1 14

1 3 67 3 4 24

2 3 100 3.7 4 34

IV A0 6 100 0.33 0 0-2

30 6 100 0.5 0 0-3

0.1 5 100 1.4 2 0-3

1 5 80 2.6 3 2-3

10 6 50 4 4 44

A0= control

B0=solvent(ethanol) control
CN=sample size
°N/A= not applicable

48

Table 3. Survival of fish and ovarian follicular stages in females
exposed to 0nM, 2nM, or 10 nM 17 B-estradiol.

 

Concentration CN Survival % Follicular Stage*
nM % P S G A

 

mean mean mean mean
(ragge) (range) (rifle) (range)_

A0 8 100 46.5 18.5 34 0.75
(34-66) (10-36) (20-54) (02)
B0 5 100 38 17.8 41.2 1.6
(22-66) (6-34) (28-50) (0-6)
2 4 100 71 10 12 5.5
(36-92) (4-26) (0-34) (4-10)
10 5 80 93.2 4 0 2.8

(88-96) (0-10) (0-0) (0-4)

 

* p: primary; 3: secondary; G= Graafian; A=atretic

A0= control
B0= solvent (ethanol) control
CN= sample size

49

 

Figure 1. Sexually mature fathead minnows. Females are lighter and have a
slender , tapered head devoid of tubercles. Males have a short, blunt head with
breeding tubercles on the snout (arrow) and a dorsally located fatpad
(arrowhead).

50

males exposed to greater concentrations of E2 had lower median values when
fatpads and tubercles were measured (Table 4). This corresponded to the
atrophy of breeding tubercles observed in fish exposed to 2 or 10 nM E2
(Figures 2, 3). Males exposed to 100 nM also resembled female fish due to
dramatic atrophy of breeding tubercles. Mortalilty in the 1000nM exposure
group precluded evaluation of gross effects. The fatpad was atrophied in fish
exposed to 100nM E2, but there was no significant difference among fish
exposed to 10nM E2 or less (Figure 4).

There was no difference in the size of the ovipositor in control females or
those exposed to 2 or 10 nM E2 and no gross changes were observed in
females exposed to any other E2 concentrations.

Histologic lesions. Exposure of males to E2 caused morphologic
alterations in the seminiferous tubules. Sertoli cells, normally present in sparse
numbers, appeared more numerous after exposure to E2 (Figure 5a ) There was
an alteration in the maturation sequence of spermatozoa as indicated by a
monomorphic population of germ cells. Occasional intratubular germ cell
syncytia (Figure 5b) composed of aggregates of various stages of the germ cell
maturation sequence, and necrotic spermatozoa were present in affected
seminiferous tubules. Histologic changes were observed in males exposed to
0.5nM, 1nM, 2nM, and 10nM E2. Sertoli cell proliferation and degenerative
changes were dose—dependent (Figure 6). There was a significant difference in
the mean, median, and range of testicular severity scores between the controls

and the 2 and 10nM E2 exposures (Table 2, Figure 7). Exposure to the greatest

Table 4. Medians and ranges of the diameter of tubercles and the length of

fatpads of male fathead minnows exposed to 17 B—estradiol ( E2).

 

 

 

Experiment Concentration CN Tubercles Fatpad
nM E2 Median (mm) Median (mm)

(range) (range)

ll A0 16 0.9 1.1
(0.3-1.4) (0.3-2.1)

B0 17 0.8 1.3
(0.3-1.2) (0.3-2.2)

2 16 0.4 0.8
(0.3-0.7) (0.3-1.5)

10 14 0.4 0.8
(0.3-1.0) (0.3-1.5)

lV A0 6 0.8 1.7
(0.5-1.0) (1.0-2.5)

E’0 6 0.8 1.2
(0.6-1 .2) (0.4-2.0)

0.1 5 0.7 1.6
(0.6-0.8) (1.3-2.7)

1 6 0.5 1.6
(0.5-0.6) (0.4-2.6)

10 6 0.3 1

(0.2-0.4) (0.7-1.3)

 

A0=control
BO= solvent (ethanol) control
CN= sample size

52

 

Figure 2. Three sexually mature male fathead minnows in 17 B-estradiol (E2)
treatment groups, from left to right: Control, 2nM E2, and 10nM E2. Note
atrophy on nuptial breeding tubercles of the two treated fish (right). X2.0

53

 

1.2

 

 

 

 

 

 

 

Average Tubercle size (mm)

Cont Solv 2 10
Treatment (nM)

Figure 3. Relationship between average tubercle diameter and treatment
groups; Cont (control), Solv (solvent control), 2 nM 17B—estradiol and 10 nM 17
B-estradiol. Bar denoted by (A) is statistically different from bar denoted by (B).

54

1.6
1.4
1.2

 

 

 

 

0.8
0.6
4 0.4
0.2

Fatpad (mm)

 

 

 

Cont Solv 2 10

Treatment (nM)

Figure 4. Relationship between average fatpad size and treatment group of
males exposed to 17B-estradiol (E2) with no statistical difference between
Cont (control), Solv (solvent control), 2nM E2, or 10 nM E2.

 

Figure 5. Photomicrographs of testes from fathead minnows exposed to 2 and
10nM 17 B-estradiol (E2). a) Sertoli cell proliferation (arrowheads) is present in
testis of a 2nM E2-treated fish. Hematoxylin and eosin. x 350. b) Greater
magnification shows a germ cell syncytia (arrow). Hematoxylin and eosin. x
700.

56

 

Figure 6. Photomicrograph of testis from a fathead minnow exposed to 10 nM
17 B-estradiol (E2). Note marked Sertoli cell proliferation (arrowheads) and a
germ cell syncytia (arrow). Hematoxylin and eosin. x 350.

57

Score (04)
N 90 p.
01 00 0| .5 01

A

 

P
GUI—‘UIN

 

Cont Solv 2 10
Treatment (nM)

Figure 7. Relationship between average testicular severity score and
treatment group. Bar denoted by letter (A) indicates that Cont (control) and
Solv (solvent control) are statistically different from 2nM or 10nM 17B—estradiol
denoted by letter (B).

58

concentrations of E2 caused partial to total occlusion of the lumina of
seminiferous tubules due to severe Sertoli cell proliferation.

Female fish exposed to E2 had ovaries which contained a preponderance
of primary follicles with a few secondary and Graafian follicles. Females
exposed to higher concentrations of E2 had ovaries containing several atretic
follicles and no mature or Graafian follicles (Table 3, Figure 8) as compared to
unexposed female fish, however this was not statistically significant (Figure 9).

Ultrastructural findings. Electron microscopic alterations in the
testes consisted of Sertoli cells with distended cytoplasm containing one to
several large phagolysosomes. There were necrotic spermatozoa, remnants of
spermatids, myelin figures, and cellular debris (Figure 10a) present within the
phagolysosomes in exposed fish in comparison to the testes of unexposed male
fish (Figure 10b).

DISCUSSION

Fathead minnows were selected for study because they are fish species
often used in toxicologic bioassays (Duda and Buttner, 1993) and because they
are an easily accessible and commonly used bait fish. As such, there is a great
deal of background information available on their culture, maintenance, (and
breeding conditions and on biochemical, physiological, and reproductive
responses to chemicals. This species of fish is particularly appropriate for the
study of potential endocrine modulating compounds such as environmental

estrogens because'of the strong sexual dimorphism.

59

 

Figure 8. Photomicrograph of an ovary from a female fathead minnow exposed
to 10nM 17 B-estradiol. Note preponderance of primary and secondary follicles
and a single atretic follicle (arrow). Hematoxylin and eosin. x 175.

0')
O

 

 

 

 

 

 

 

100%

80%
8: \Atnetic
t6 Q
5 60% E Graafian
5 ll Secondary
0
If. 40% l Primary
C
°\° 20%

0%
Figure 9. Relationship between average percentage of each stage of

ovarian follicular development and treatment group with no statistical
differences between Cont (control), Solv (solvent control), 2nM 17B—estradiol
or 10 nM 17 B—estradiol.

61

Figure 10. Transmission electron micrographs of testes from fathead minnows
exposed to (a) 2nM 17 B-estradiol (E2) and (b) 0nM E2.

a) Notice the Sertoli cell cytoplasm is distended by phagolysosomes that
contain necrotic spermatozoa, myelin figures and membranous debris
(arrows).

b) Spermiation yields a small residual body (arrow) that is normally
phagocytized.

‘- —vvw—

 

62

 

63

Male fathead minnows exposed to 100nM or 10nM concentrations of E2
had a reduction in the prominence of the secondary sex characteristics,
particularly the breeding tubercles, and were difficult to differentiate from
females without histologic examination of the gonads. Males exposed to lesser
concentrations of E2 had similar atrophy of tubercles that was dose-dependent.
These results suggest that atrophy of male breeding tubercles may be useful as
an indicator of environmental estrogen exposure.

Males exposed to the greatest concentrations of E2 also had atrophy of
the fatpad, however this was not a consistent finding in fish exposed to lesser
concentrations. Since the fatpad is composed of a mucinous matrix containing
mucous cells, there may have been dehydration due to the use of ethanol as a
carrier for E2 and as a rinse during tissue preservation.

Sertoli cells have many supportive roles with regard to the spermatogenic
maturation sequence. One of the major functions of Sertoli cells is to
phagocytize discarded organelles (residual bodies) and cytoplasm during
spermiation. Sertoli cells also phagocytize germ cells that degenerate in the
normal course of spermatogenesis or as the result of the adverse effect of some
deleterious agent or condition (Hunter and Donaldson, 1983; Fawcett, 1977;
Fawcett, 1975). The presence of multiple Sertoli cells with distended cytoplasm
filled with degenerate spermatozoa suggests an increase in necrosis or
apoptosis in E2-exposed male fathead minnows. Additionally, ultrastructural

examination revealed a great number of primary and secondary spermatocytes

64

lying along the basement membrane of the seminiferous tubules which suggests
an arrest in the germ cell maturation sequence.

The predominant histologic and ultrastructural lesions observed in males
exposed to E2 were changes in Sertoli cell number. Seminiferous tubules,
normally lined by sparse numbers of Sertoli cells with indistinct boundaries,
contained numerous Sertoli cells. While it is not clear if the increased number
was relative or absolute, this response was most pronounced in fish exposed to
greater concentrations of E2. This is in contrast to mammals, where Sertoli cells
cease to divide during pubertal development (Russell et al. 1990; Russell and
Peterson, 1984; Fawcett, 1975; Fawcett, 1977; Kretser et al. 1983) and the
number of adult Sertoli cells are believed to remain stable throughout life. This
apparent proliferation of Sertoli cells has not been documented in mammals and
needs to be further documented in fish.

The histologic lesions in males may result from an arrest in maturation
with subsequent degeneration of spermatozoa which may be caused by an
alteration in the hypothalamic-pituitary axis. Hypothalamic control of pituitary
secretion in fish is similar to that in mammals (Matty, 1985b). With the secretion
of follicle stimulating hormone and luteinizing hormone suppressed by E2,
spermatogenesis may not proceed normally (Junqueira et al. 1986a; Stone,
1994; Russell et al. 1990). If the spermatogenic sequence becomes arrested,
germ cells that no longer progress along the maturation sequence will ultimately
degenerate. Since Sertoli cells play a role in phagocytosis and clearing residual

bodies under normal circumstances, the proliferative response observed in E2-

65

exposed fish may be an extension of their normal role and may have occurred
due to the increased need to clear the arrested germ cells.

Ovarian morphologic changes observed in female fish were also most
likely the result of a disruption of the pituitary-hypothalamic axis. Numerous
primary follicles may be attributed to the inhibitory action of E2 on FSH, since
the maturation of late primary and secondary follicles is dependent on adequate
levels of FSH (Junqueira et al. 1986a).

Results from this study suggest that histologic alterations in male and
female fathead minnows, and gross morphologic alterations in males may be
useful indicators of environmental estrogen contamination. Further study needs
to be done to determine if the estrogenic effects on a population of fish can be
linked to an observation of male feminization and, particularly, to determine
whether or not the changes are likely to persist once exposure has been

discontinued.

CHAPTER 2:
LACK OF PERSISTENCE OF 17 B-ESTRADIOL- INDUCED TESTICULAR

LESIONS IN MALE FATHEAD MINNOWS

ABSTRACT

Environmental contaminants with estrogenic activity have the potential to
alter the reproductive system of humans and animals. A previous study
demonstrated that male fathead minnows exposed to 17 B-estradiol (E2) had
gross atrophy of the fatpad and breeding tubercles and histologic alterations
consisting of an increase in the number of Sertoli cells and germ cell syncytia
composed of necrotic aggregates of various stages of germ cells in the
spermatogenic sequence. The purpose of this study was to determine if
testicular lesions observed in male fathead minnows exposed to E2 would
persist once exposure was discontinued. Sexually mature male and female
fathead minnows were exposed to a single concentration of 10nM E2 via a
proportional flow-through system for 10 days. At the end of the exposure period,
subsamples of male fathead minnows were collected for histologic and
ultrastructural examination. The testes were examined on the final day of
exposure and testes from fish were collected and examined over a period of 4

months to determine if lesions would regress once exposure was discontinued.

66

67

Sixteen weeks after exposure to E2 was discontinued, testicular lesions were

reversed.

INTRODUCTION

Many environmental compounds, including organochlorine insecticides
(Eroschenko, 1985; Eroschenko and Palmer, 1980), ortho, para prime dichloro
diphenyl dichloroethylene and its metabolites (Fry and Toone, 1981), and
nonylphenol (Jobling and Sumpter, 1993b; Soto et al. 1991) have the ability to
mimic the endogenous estrogen, E2. Exogenous estrogenic compounds such as
organochlorine insecticides and other synthetic chemicals can bind the
estrogen receptor much like natural estrogens (Thomas and Smith, 1993;
Korach et al. 1991; Klotz et al. 1996) at environmental concentrations sufficient
to elicit biological effects (Thomas and Smith, 1993; Jobling and Sumpter,
1993b; Purdom et al. 1994; Gross et al. 1994; Lech et al. 1996). Estrogen-
induced alterations of gonads can lead to reproductive impairments that may
adversely affect individuals and populations (Gimeno et al. 1995; Jobling et al.
1996; Jobling and Sumpter, 1993b; Russell et al. 1990).

A previous study demonstrated germ cell syncytia and Sertoli cell
proliferation in sexually male fathead minnows exposed to 10nM E2 for 14 days.
Exposure caused atrophy of the prominent male secondary sex characteristics,
the fatpad and breeding tubercles. These lesions suggest a possible impairment
in reproductive efficiency. It is not known, however, whether the gross and

histologic lesions induced persist once E2-exposure is discontinued. The goal of

68

this study was to determine if the gross and histologic lesions induced by E2 in

male fathead minnows were reversible once exposure to E2 was discontinued.

MATERIALS AND METHODS

Study Design. Fathead minnows (Pimephales promelas) were
reared in the Michigan State University Aquatic Toxicology Laboratory. Fifty
male and 50 female sexually mature fathead minnows were placed in a 420 liter
aquarium containing 8 breeding tiles. Females were included in the experiment,
as in previous studies to ensure that males were sexually virile. The endogenous
estrogen, E2, was delivered via a proportional delivery system for 10 days. The
photoperiod was maintained at 16 hours light to 8 hours dark. Continuously
flowing, aerated tap water was maintained at a temperature of 25-26 °C. Fish
were fed a mixture of a 1:1 mix of Purina Trout Chow (Purina Mills, Earth City,
MO) and TetraMin flake food (Tetra Sales, Blacksburg, VA). At the end of the
exposure period, the tanks were flushed, the flow rate of tap water was
increased, and 16 liters of dechlorinated tap water was replaced in the tanks
daily for 5 days. The health status of the fish was monitored. Fish that were
found to have lost their righting reflex were euthanized with an overdose of
Finquel® (Argent Chemical Laboratories, Redmond, VA).

Gross examination and specimen collection. The fish were euthanized
with an overdose of Finquel®. All gross lesions were noted on the fathead
minnow necropsy form ( Appendix A). Gross secondary sex characteristics of

males were measured prior to histologic examination of the testes. The height of

69

male fatpad and the diameter of nuptial breeding tubercles were measured
using a 27 mm diameter 6X comparator/magnifier and reticle (Edmund Scientific
Company, Barrington, NJ). Testes were collected from 26 males on the final day
of the exposure study (hereafter referred to as week 0), and at 1, 2, 3, 4, 5, 10,
12, 14 and 16 weeks after exposure was discontinued. For electron microscopic
examination, transverse sections of the fish containing the testis were cut and
preserved in 4% glutaraldehyde for 12 hours. Several sections of testis, each
0.5-1 mm, were dissected and rinsed in 0.1 M phosphate buffer, postfixed in
osmium tetroxide, and embedded in resin (Appendix C). Thick sections
measuring 1pm, were cut on a ultramicrotome and were stained with toluidine
blue and thin sections, each 800 nm, were stained with uranyl acetate and lead
citrate for examination using a Phillips 301 transmission electron microscope
(Phillips Electronic Instruments, Inc., Roselle, IL). Remaining portions of fish not
collected for electron microscopic examination and other fish collected for
histologic examination were immersed in Bouin’s solution (Appendix D) after the
abdomen was injected with 0.5 cc of the fixative. After 48 hours, the fish were
rinsed by agitating the carcasses in 30 mls of 70% ethanol for 30 seconds. They
were stored in this solution until trimming. The fish were trimmed in serial
transverse sections, processed and embedded sequentially in paraffin
(SurgiPath, Inc., Richmond, IL). Tissues were sectioned at 5pm with a
microtome, and stained with hematoxylin and eosin for examination using a light

microscope.

70

Gonagd Scorim Criteria. Testicular lesions were evaluated
according to the severity of relative or absolute Sertoli cell proliferation and the
percentage of seminiferous tubules affected. Scores ranged from 0 to 4 as
follows:

0 = no Sertoli cell proliferation

1: < 25% Sertoli cell proliferation

2= 2 25% < 50% Sertoli cell proliferation

3= 2 50% < 75% Sertoli cell proliferation

4: >75% Sertoli cell proliferation
Degenerative changes including germ cell syncytia, mineralization of

spermatozoa, or necrotic spermatozoa were indicated by (+).

RESULTS

Fish sprvival. Survival rates for males and females during the
exposure period were 74% and 78%, respectively. .During the 10 day
exposure, four fish lost their righting reflex and were subsequently euthanized.
Other dead fish were noted to have non-specific moderate to severe bilateral
and multifocal subcutaneous hemorrhage while still others had no gross lesions.

Gross gayminatioi Exposure of male fathead minnows to 10 nM
E2 induced gross changes in male secondary sex characteristics consisting of
atrophy of both the fatpad and breeding tubercles. For approximately 3 months
post E2-exposure, the secondary sex characteristics remained at a reduced size.

Fatpad measurements averaged between 0.1603 mm through week 14. By

71

week 16 post E2-exposure, the average linear measurement of the fatpad was
1.1 mm. The average of the diameter of breeding tubercles remained between
0.2-0.36 mm until week 16 post E2-exposure when tubercle diameter was
measured at 0.76 mm (Table 5).

Histologic and ultrastructural findings. The testes of 26 male
fathead minnows were examined for tissue changes using the light microscope
and testes were collected from 12 males for ultrastructural examination.
Samples were collected on the final day of exposure and collection continued at
intervals for 16 weeks. There was an apparent increase in the number of Sertoli
cells and occasional germ cell syncytia (Figure 11,12) in early subsamples
compared to final samples collected at week 16 post-E2 exposure (Figure 13)
when Sertoli cell numbers were fewer and germ cell syncytia were absent. The
distended Sertoli cell cytoplasm contained necrotic spermatozoa, residual
bodies, and myelin figures within phagolysosomes (Figure 14, 15, 16).
Morphologic changes of Sertoli cells became less pronounced from week 0 to

week 16 post-exposure (Figure 17 a, b).

DISCUSSION
Exposure to E2 caused gross atrophy of the secondary sex
characteristics and morphologic changes in Sertoli cells including an apparent
increase in Sertoli cell number. Atrophy of the fatpad and nuptial breeding

tubercles probably occurred due to the inhibition of luteining hormone (LH) by

72

Table 5. Histologic scores and measurements of fatpads and tubercles of
males in 17 B-estradiol reversal study.

 

AWeek Histologic Score Secondary Sex Characteristics (mm)

 

(mean) Fatpad Tubercles

'30 3.7 0.3 0.2

1 3.3 0.2 0.27
2 3.6 0.16 0.27
3 3.3 0.2 0.29
4 3 0.3 0.36
5 4 0.3 0.26
10 4 0.3 0.26
12 2 0.3 0.36
14 4 0.2 0.26
16 0 1.1 0.76

 

A Post 17 B-estradiol exposure
3 Final day of exposure

73

 

Figure 11. Photomicrograph of testis from a fathead minnow exposed to 10
nM 17 B-estradiol, week 4 post -exposure. Note the germ cell syncytium present
in the lumen of the seminiferous tubule (arrow). Hematoxylin and eosin. x 780.

I c . I N Im\.. .u a. u ..¢.r1 . I0. I ..
«wk-RH that! age... 9954455935: z... in: . ...omt...'tar. .e. . 5.... 591:1” v.99... ox .139 2. .9...

74

 

Figure 12. Transmission electron micrograph of the testis from a fathead
minnow exposed to 10nM 17 B-estradiol, week 2 post-exposure. Note a cluster
of spermatozoa in the Sertoli cell cytoplasm in varying degrees of degeneration
(arrows). x 10,499

75

0.,

o
5E6:
n..- a .

Figure 13. Photomicrograph of testis from a fathead minnow exposed to 10nM
17 B—estradiol, week 16 post-exposure Note a single Sertoli cell characterized by
a vesiculate nucleus and a prominent nucleolus (arrow). Hematoxylin and eosin. x
780.

 

76

 

Figure 14. Transmission electron micrograph of testis from a fathead minnow
exposed to 10nM 17 B-estradiol, week 2 post-exposure. Note the phagocytized
germ cells in varying stages of digestion (arrows). x 21,907.

77

' .4."
ft?!
- ’r-y“ ,

 

Figure 15. Transmission electron micrograph of testis from a fathead minnow
exposed to 10nM 17 B-estradiol, week 3 post-exposure. Note Sertoli cell
distended with degenerate spermatozoa, myelin figures, and residual bodies. x

17,092.

78

 

Figure 16. Transmission electron micrograph of testis from a fathead minnow
exposed to 10nM 17 B-estradiol, week 5 post-exposure. Note Sertoli cell with
residual body in cytoplasm (arrow). x 26,068.

79

Figure 17. Photomicrographs of testes from fathead minnows exposed to

10nM 17 [3-estradiol (E2) at (a) Week 6 post E2- exposure and (b) Week 16 post
E2-exposure.

a) Note the large number of Sertoli cells lining seminiferous tubules
(arrows) and a germ cell syncytia (arrowhead) as well as a monomorphic
population of germ cells filling the lumen of tubules. Hematoxylin and
eosin. x 312.

b) Week 16 post E2—exposure. Note occasional Sertoli cells and several
stages of germ cells. Hematoxylin and eosin. x312.

80

 

81

E2, since the development and maintenance of secondary sex characteristics is
dependent on testosterone, which is regulated by LH (Junqueira et al. 1986a).

Sertoli cell lesions were probably the result of a similar alteration in the
hypothalamic-pituitary axis. It is well documented in mammals that Sertoli cell
numbers do not increase after sexual maturity (Russell et al. 1990; Fawcett,
1975; Fawcett, 1977; Goss, 1966). To date, studies have not been undertaken
to determine if the same is true in fish. In this study and in a previous E2 study,
Sertoli cells appeared more numerous. With morphologic evaluation alone, it
was not possible to determine whether this increase was absolute or relative. It
is possible that the apparent increase may have been due to an increased need
to clear degenerate spermatozoa since phagocytosis is a normal function of
Sertoli cells (Russell et al. 1990; Fawcett, 1977; Fawcett, 1975). Degeneration
of germ cells may have occurred do to an arrest in the spermatogenic maturation
sequence resulting from the negative feedback of E2 on LH and follicle
stimulating hormone, both of which are necessary for spermatogenesis to
proceed normally (Junqueira et al. 1986a; Eddy and O'Brien, 1994) Alternatively
or additionally, Sertoli cells may have appeared to be more numerous because
of a reduction in the germ cell population.

Other lesions observed included germ cell syncytia, residual bodies and
myelin figures. Germ cell syncytia, also known as symplasts, are thought to
result from an opening of intercellular bridges between the cells (Russell et al.
1990). Although the cause of the bridge opening is unknown, it may be related

to the destabilization of the cytoskeletal apparatus maintaining the integrity of

82

the bridge (Russell et al. 1981). Residual bodies are composed of the remnants
of the extruded cytoplasm of a maturing spermatozoa (Russell et al. 1990) and
contain large aggregates of RNA and clustered, tightly packed organelles. They
resemble necrotic germ cells except that they are present for a short time
(Russell et al. 1990) and typically in fewer numbers. The presence of numerous '
myelin figures indicate that a degenerative process was occurring since these
form when hydrophilic phospholipids are released from degenerating
membranes (Jones and Hunt, 1983).

While lesions observed in males appeared dramatic, results of this study
demonstrate that E2-induced gross and histologic lesions in the testes are
reversible. Atrophy, a reduction in the number or size of cells after normal
growth, and hyperplasia, an increase in cell numbers, are typically reversible
lesions (Thomson, 1978). Therefore, it is not surprising that after exposure was
discontinued, the secondary sex characteristics became more prominent over
time, nor was it unexpected to find that Sertoli cell lesions reversed. If observed
testicular lesions are indicative of reproductive efficiency and secondary sex
characteristics are gross indicators, it is conceivable that male fish exposed to
environmental estrogens will not have permanent reproductive impairment.
However, because of the time it takes for testicular lesions to reverse, a resulting
decrease in reproductive efficiency may potentially lead to a temporary reduction

in the normal reproductive capacity of the exposed fish.

In I
“.I{ 'I
I’ll

III
III!
1'1
llr
I‘

III

 

I III III
III
IlIlIIIII

CHAPTER 3:
EFFECTS OF WATERBORNE CONCENTRATIONS OF 4-NONYLPHENOL
AND NONYLPHENOL ETHOXYLATE ON THE SECONDARY SEX

CHARACTERISTICS AND GONADS OF FATHEAD MINNOWS

ABSTRACT

Fathead minnows were exposed to 4-nonylphenol (NP) and nonylphenol
ethoxylate (NPE) to test the utility of the secondary sex characteristics and
gonads as bioindicators of known environmental estrogens. Sexually mature
male and female fathead minnows were exposed for 42 days to waterborne
concentrations of 0.1, 0.3, 1.0, 3.0, or 10pg/L of NP and NPE. Exposure to NP
caused a reduction in the diameter of the breeding tubercles at 0.1 or 0.3
pgNP/L. There was no significant change noted in size of the fatpads. Absolute
or relative Sertoli cell proliferation and germ cell syncytia, consisting of necrotic
aggregates of various stages of germ cells in the spermatogenic sequence, were
observed in the testes of males exposed to 1, 3 or 10 pgNP/L. Electron
microscopy of the testes of NP-exposed males revealed the presence of
phagocytic cells in the lumina of seminiferous tubules. The cytoplasm of Sertoli
cells was often distended with myelin figures and necrotic spermatozoa.

Females exposed to NP had no significant differences in the stages of ovarian

83

84

follicular development. There were no differences in the gonads or secondary
sex characteristics of males or females exposed to any concentration of NPE.
Results from this study suggest that the histologic response of the testes and the
size of the breeding tubercles are sensitive indicators of waterborne exposure to

NP at environmentally relevant concentrations.

INTRODUCTION

In recent years, there has been concern by government, industry, and the
public over the possibility that some synthetic compounds may act as disrupters
of the endocrine system. Particular attention has focused on compounds which
have the ability to mimic the action of endogenous estrogen (Colborn et al. 1993;
Stone, 1994; Raloff, 1994a; Raloff, 1994b). Similar to endogenous estrogen,
these compounds are believed to act through the estrogen receptor regulating
the activity of estrogen responsive genes (Jobling et al. 1996), although other
epigenetic mechanisms are also being investigated. Several different groups of
chemicals are believed to be potentially estrogenic, including a large class of
non-ionic surfactants (Naylor, 1992) and plasticizers (Stone, 1994) which may
accumulate to toxic levels in the environment (Naylor et al. 1992).

Nonylphenol and NPEs are members of a large group of nonionic
surfactants used in a wide variety of applications. These compounds are the
products of microbial breakdown during sewage treatment of a group of
industrial surfactants, the alkylphenol ethoxylates (APEs) (Giger et al. 1984).

Non-ionic surfactants are used in industrial processing, and in household and

85

institutional cleaning products (Naylor et al. 1992; Jobling et al. 1996; Nimrod
and Benson, 1996) and are found in such common products as detergents,
paints, herbicides, and cosmetics (Jobling et al. 1996). Alkylphenol ethoxylates
have been used for over forty years and remain one of the largest volume
groups of surfactants with production in the United States exceeding 450 million
pounds (Naylor et al. 1992).

Alkylphenols such as nonylphenol and related compounds are found
widely in surface waters and sediments (Naylor et al. 1992; Ahel et al. 1987;
Giger et al. 1984). While environmental concentrations of akylphenolic
compounds are well documented, there is geographic variability (Jobling et al.
1996). These compounds, like many other environmental estrogenic chemicals,
are lipophilic and sorptive, thus they have been reported to accumulate in
animals (Ahel et al. 1994a; Ganmo et al. 1989) and sediments (Jobling et al.
1996).

The purpose of this study was to characterize the gross and histological
response of the male and female fathead minnow as bioindicators of waterborne

exposure to the environmentally relevant compounds, NP and NPE.

MATERIALS AND METHODS
Study Design. Forty male and 40 female sexually mature fathead
minnows (Pimephales promelas) reared in the Michigan State University
Aquatic Toxicology Laboratory were randomly selected for study. Replicate

exposure studies were conducted with NP dissolved in ethanol (Experiments l

86

and II) and one study was conducted with NPE. In each study, twenty 16 liter
aquaria were divided by glass partitions into 8 liter chambers for a total of 40
treatment tanks per study. Each treatment tank contained a single breeding tile
constructed of a 7.5 cm clay pot and a breeding pair consisting of one male and
one female fathead minnow. The photoperiod was maintained at 16 hours light
to 8 hours dark with continuously flowing, aerated water maintained at 25-26 C°.
The temperature and dissolved oxygen were monitored every other day. The
fish were fed a mixture of 0.25 grams of Purina Trout Chow (Purina Mills, Earth
City, MO) and 0.25 grams of TetraMin flake food (Tetra Sales, Blacksburg, VA).
The dry food was supplemented with 1 ml of brine shrimp (Anemia nauplii )
Concentrations of both test compounds were 0.1, 0.3, 1, and 10 pglL. There
were two controls, one with water only and one containing 0.00001% ethanol.
The compounds were delivered via a proportional flow-through diluter and the
exposure period was 42 days. The health status of the fish was monitored. Fish
that were found to have lost their righting reflex were euthanized with an
overdose of Finquel® (Argent Chemical Laboratories, Redmond, VA).

Gross examinatign amLspecimen collectm At the end of the
exposure period, the fish were euthanized with an overdose of Finquel®. Any
gross lesions present were noted on the fathead minnow necropsy form
(Appendix A). Gross secondary sex characteristics of males were measured
prior to histologic examination of the testis. The height of the male fatpad and
the diameter of nuptial tubercles were measured using a 27 mm diameter 6X

comparator/ magnifier and reticle (Edmund Scientific Company, Barrington, NJ).

87

Testes were collected from 10 males from the NP study for ultrastructural
examination. Two 5 mm transverse sections were cut from the abdomen of the
fish just cranial to the pelvic fin. Sections were immersed in 4% glutaraldehyde
for 12 hours. After the glutaraldehyde fixation, 0.5 mm sections of testis were
dissected and rinsed in 0.1 M phosphate buffer. These sections were postfixed
in osmium tetroxide, and embedded in resin (Appendix C). Thick sections cut
with an ultramicrotome (1pm) were stained with toluidine blue and thin sections
measuring 800nm were stained with uranyl acetate and lead citrate and
examined with a Phillips 301 transmission electron microscope (Phillips
Electronic Instruments, Inc). Remaining portions of fish not collected for electron
microscopic examination were preserved in Bouin’s solution for histologic
examination (Appendix D) in addition to a total of 80 males and females from the
nonylphenol study, and 90 males and females from the nonylphenol ethoxylate
study. After injecting the abdomen with 5 cc of Bouin’s fixative, the carcass was
immersed in the fixative for 48-72 hours. Fish were then rinsed by agitating the
carcasses for 30 seconds in 30 mls of 70% ethanol and they were stored in that
solution until trimmed. Fish were trimmed in serial transverse sections,
processed and embedded sequentially in paraffin (SurgiPath, Inc., Richmond,
IL). Tissues were sectioned at 5 pm with a microtome and stained with
hematoxylin and eosin for examination with light microscopy.
Gonadscoringcriterig. Testicular lesions were evaluated

according to the severity of relative or absolute Sertoli cell proliferation and the

88

percentage of seminiferous tubules affected. Scores ranged from O to 4 as
follows:

0 = no Sertoli cell proliferation

1: < 25% Sertoli cell proliferation

2= 2 25% < 50% Sertoli cell proliferation

3= 2 50% < 75% Sertoli cell proliferation

4: >75% Sertoli cell proliferation
Degenerative changes including germ cell syncytia, mineralization of
spermatozoa, or necrotic spermatozoa were indicated by (+).

In females, ovaries were evaluated by counting fifty follicles in the ovary
of each female and calculating the percentage of each stage of follicular
development observed. Ovaries were staged according to the following criteria:

primary - large nucleus, abundant basophilic cytoplasm and no yolk

vesicles

secondary- presence of numerous eosinophilic yolk vesicles

Graafian- large follicle bordered by an amphiphilic egg membrane

with a central core of eosinophilic yolk protein

atretic- degenerative follicle with shrunken, irregular border

Statistical pnalvsis. Treatment effects were examined by non-
parametric two-way ANOVA (SAS Institute, Carey, NC; PROC GLM) conducted
on the ranks of the responses. Significant treatment effects showed no
significant interaction. Terms were subsequently examined by Tukey’s HSD

multiple range test. Unless otherwise stated, effects were determined to be

89

statistically significant at the 0.05 level of type I error (alpha) and 0.2 of type II

error (beta).

RESULTS

Fish grvival. Survival rates remained above 80% for males (Table
6) and females (Table 7) in the NP study and 87% or greater in the NPE study
(Table 8, 9). The fish that were found dead had no gross or histologic lesions.

Gross Examination. There was no change in the gross appearance
of the fatpad in males exposed to any concentration of NP or NPE. Males
exposed to 0.1 or 0.3 pgNP/L in the first experiment had significant atrophy of
the breeding tubercles (Table 10, Figure 18) and a similar trend, though not
statistically significant, in the second NP experiment. No gross changes were
observed in females exposed to NP or NPE.

Histologic lesions. Although there were no statistically significant
differences between any of the exposure concentrations in the first NP
experiment, a dose-dependent increase was observed in the severity scores of
the testes of males exposed to 3 or 10 pgNP/L . In the second NP experiment,
a similar trend, which was statistically significant, was noted in the mean,
median, and range of severity scores of males exposed to 1, 3, or 10 pgNP/L
compared to controls and males exposed to 0.1 and 0.3 pg NP/L (Table 6,
Figure 19). In addition to an apparent relative or absolute increase in the number
of Sertoli cells, necrotic spermatozoa and germ cell syncytia were often present

(Figure 20). Electron microscopic examination of NP-exposed males

90

Table 6. Survival of fish and testicular severity scores of males exposed to
nonylphenol. _

 

 

 

 

Experiment Concentration CN Survival Severity Score (0-4)

pg/L % mean median rang_e_

l A0 5 100 2 2 04

E’0 2 50 2 2 2-2

0.1 4 100 1 0 0-4

0.3 3 75 2 2 2-2

1 2 50 1.5 0N/A 03

4 100 2.75 3 2-4

10 4 100 3.75 4 3-4

II C 2 50 0 0 0-0

SC 1 25 1 1 1-1

0.1 1 25 1 1 1-1

0.3 3 75 0 0 0-0

1 2 50 0.5 NIA 0-1

3 3 100 0 0 0-0

10 2 50 2.5 NIA 2-3

A0= control

B0= solvent (ethanol) control
CN= sample size
E’NIA= not applicable

91

Table 7. Survival of fish and percentage of ovarian follicular stages of
females exposed to nonylphenol.

 

Experiment Concentration CN % Follicular Stage*
pg/L P S G A
mean mean mean mean

(range) (range) (range) (range)

 

I A0 6 44 19 32 5
(28-60) (12-28) (14-52) (0-12)

E0 2 41 24 32 3
(38-44) (18-30) (32-32) (0-6)

0.1 2 37 22 41 0
(36-38) (16-28) (3646) (0-0)

0.3 5 54 15 26 6
(48-64) (12-16) (1040) (0-22)

1 4 44 15 39 2
(24-58) (6-22) (28-52) (04)

3 5 58 14 23 2

(24-90) (2-32) (044) (0-8)

10 3 46 17 29 6
(4248) (10-26) (22-38) (4-10)

ll A0 2 34 14 51 1
(32-36) (6-22) (44-58) (0-2)

80 2 37 20 42 1
(3044) (18-22) (3846) (0-2)

0.1 3 48 19 31 1
(44-52) (16-22) (26—32) (04)

0.3 1 58 14 28 0
CNIA NIA NIA NIA

1 1 40 12 48 0
NIA N/A N/A NIA

3 3 37 12 51 0
(3442) (6-16) (50-52) (0-0)

10 2 45 14 41 0

(40-50) (12-16) (3448) (0-0)

 

*P=primary; S=secondary; G=Graafian; A=atretic
A0= control; 30: solvent (ethanol) control;
C3N=sample size; °N/A= not applicable

92

Table 8. Survival of fish and testicular severity scores of males
exposed to nonylphenol ethoxylate.

 

Concentration BN Survival Severity Score (0-4)

 

 

pg/L % mean median rang
A0 7 88 1.7 4 04
0.3 8 100 3.5 4 1-4
1 8 100 3 3 0-4
4 50 3.5 4 2-4
10 8 100 2.5 4 0-4
A0= control

BN= sample size

Table 9.

93

Survival of fish and percentage of ovarian follicular stages in

females exposed to nonylphenol ethoxylate.

 

 

Concentration BN Survival % Follicular Stage*
pg/L % P S G A
mean mean mean mean
(range) (range) (range) (ranggL
A0 10 100 67 10 21 1
(34-88) (2-16) (2-54) (0-2)
0.3 10 100 61 14 23 3
(34-86) (6-1 8) (0-50) (0-8)
1 9 90 61 16 19 4
(40-78) (6-28) (0-50) (0-8)
3 8 80 60 10 25 4
(22-88) (6—18) (0-64) (2-12)
10 6 60 73 12 16 0
(50-92) (8—16) (0-36) (0-0)

 

*P=primary; S=secondary; G=Graafian; A=atretic

A0=control
BN=sample size

Table 10. Measurements of the diameter of tubercles and the length of
fatpads of male fathead minnows exposed to nonylphenol.

94

 

 

 

Experiment Concentration CN Tubercles F atpad
pg/L Median (mm) Median (mm)
(rangg) (range)
I A0 2 0.82 1.15
(0.8-0.83) (1-1.3)
B0 2 0.47 0.65
(0.23-0.7) (0.3-1)
0.1 3 0.52 0.37
(0.3-0.8) (0.2-0.5)
0.3 3 0.72 1.07
(0.67-0.73) (1-1.1)
1 2 0.68 1.05
(0.6-0.77) (1-1.1)
3 5 0.55 0.66
(0.4-0.63) (0.2-1.1)
10 2 0.65 0.85
(0.6-0.7) (0.8-0.9)
ll A0 8 0.68 0.9
(0.4-0.8) (0.5-2)
B0 6 0.58 0.9
(0.2-0.9) (0.3-1.5)
0.1 6 0.4 0.6
(0.2-0.6) (0.2-1.2)
0.3 6 0.76 0.77
(0.5-0.9) (0.2-1.5)
1 6 0.68 0.65
(0.4-0.9) (0.3-1.0)
3 10 0.5 0.8
(0.2-0.6) (0.2-1.7)
10 5 0.66 0.86
(0.6-0.77) (0.3-1.5)

 

A0= control
B0= solvent (ethanol) control
CN= sample size

95

 

(1)

 

 

 

 

Cont Solv 0.1 0.3 1 3 10

 

Average Tubercle size (mm)
S

 

 

 

Cont Solv 0.1 0.3 1 3 10
pg NP/L

Figure 18. Relationship between tubercle diameter and nonylphenol (NP)
exposure concentration.

(1) Experiment I. There is a statistical difference in the tubercle diameter
between exposure groups 0.1 and 0.3 pgNP/L.

(2) Experiment ll. Although there is no statistical difference in any of the
exposure groups, note a similar trend in the diameter of tubercles in
experiment I and II.

96

Figure 19. Relationship between the average testicular severity score and
nonylphenol (NP) exposure concentration.

(1) Experiment I. There is no statistical difference in the severity score
between any concentration of NP.

(2) Experiment. ll. Note that Solv (solvent control), 0.1, 0.3, 1 and 3
pgNP/L are not statistically different (denoted by bar with letter A). Fish
exposed to 10 pgNPlL had scores which were statistically different from
both controls, 0.1 or 0.3 pgNP/L.

 

 

Average Score (0-4)

97

(1)

 

 

 

 

 

 

 

001de 0.1 0.3 1 3 1O

 

Cont Solv 0.1 0.3 1 3 10

pg NPIL

98

 

Figure 20. Photomicrograph of testis from a fathead minnow exposed to 3pg
nonylphenol/L. Note the germ cell syncytia and necrotic spermatozoa near
Sertoli cells (arrows). Hematoxylin and eosin. x 1560.

99

demonstrated that Sertoli cell cytoplasm was distended by residual bodies and
myelin figures (Figures 21, 22). Males had phagocytic cells present in the
lumina (Figure 23) of seminiferous tubules which appeared to aid Sertoli cells in
clearing degenerate spermatozoa (Figure 24). There was no significant
difference in the size of the secondary sex characteristics (Table 11, Figure 25,
26) or in the mean, median, or range of the gonads (Table 8) of males exposed
to any concentrations of NPE. No change was noted in the stages of follicles of
females exposed to any concentration of NP (Table 7, Figure 27) or NPE (Table

9).

DISCUSSION

The results of this study suggest that the fathead minnow, a fish species
commonly used in toxicity bioassays (Duda and Buttner, 1993), may be a useful
bioindicator of environmental estrogens. The compounds used in this study are
common wastewater contaminants which can act as an estrogen agoniSts
(Jobling and Sumpter, 1993a). Although the chemical structures of NP and NPE
are different from the endogenous estrogen, 17 B-estradiol (E2), they may bind
.with estrogen receptors, as has been demonstrated in laboratory studies
(Jobling et al. 1996; Lech et al. 1996; Gray and Metcalfe, 1997)

Development of secondary sex characteristics in fish occurs when the
gonadotropic hormones, follicle stimulating hormone (FSH) and luteinizing
hormone (LH), stimulate the gonads to produce steroids (Moyle and Cech, Jr.

1988). In male fathead minnows, the development of the secondary sex

100

Figure 21. Transmission electron micrograph of testis from a fathead minnow
exposed to 3pg nonylphenol/L . Note the Sertoli cell distended with necrotic
spermatozoa and myelin figures. X 14,820.

 

101

 

102

 

 

Figure 22. Transmission electron micrograph of testis from a fathead minnow
exposed to 10pg nonylphenol/L. Note the presence of numerous myelin figures
and residual bodies. x 15,088.

 

‘1 ,2 2"“

..-- w.-

1‘; >.._._

‘I‘I‘ \l. \l1\l. \lll]\l

ww‘.

*-

‘,.‘-¥\7

 

 

103

 

104

Figure 23. Transmission electron micrograph of testis from a fathead minnow
exposed to 10pg nonylphenol/L. Note the spermatozoa- filled macrophages in
the lumen of a seminiferous tubule. x 8,185.

 

 

 

 

105

 

 

106

Figure 24. Transmission electron micrograph of testis from a fathead minnow
exposed to 3pg nonylphenol/L. Note a Sertoli cell containing 4 spermatozoa and
a single residual body . x 20,158.

 

 

 

 

107

 

108

Table 11. Measurements of the diameter of tubercles and the length of
fatpads of male fathead minnows exposed to nonylphenol ethoxylate.

 

 

 

Concentration BN Tubercles Fatpad
pg/L Median (mm) Median (mm)
(range) (range)
A0 1 1 0.45 0.45
(0.1-0.7) (0.1-0.6)
0.3 7 0.33 0.59
(0.23-0.43) (0.3-1.2)
1 9 0.39 0.39
(0.07-0.6) (0.1-0.7)
3 7 0.41 0.4
(023-053) (0.3-0.6)
10 10 0.41 0.47
(0.17-0.67) (0.2-1.2)
A0= control

E3N=sample size

109

 

 

 

 

 

 

 

 

E
E.
2
U
I.
d)
.0
3
I-
d)
U)
16
L
G)
>
<
Cont 0.3 1 3 10
pgNPE/L
Figure 25. Relationship between tubercle diameter and concentration of

nonylphenol ethoxylate (NPE) with no statistical difference between exposure
groups.

110

 

 

 

 

 

 

 

E
E.
13
I6
0.
in!
(6
LI.
0)
O)
N
s.
43
>
<
Out 0.3 1 3 10
pgNPEI L
Figure 26. Relationship between the fatpad size and concentration of

nonylphenol ethoxylate (NPE) with no statistical difference between exposure
groups.

111

 

 

 

Average Score (0-4)

 

 

 

pgNPE/L

Figure 27. Relationship between average tubercle diameter and nonylphenol

ethoxylate (NPE) exposure concentration.
between any concentrations.

There was no statistical difference

112

characteristics (the nuptial breeding tubercles and the fatpad) is stimulated by
testosterone produced by the Leydig cells of the testes (Smith, 1978).
Testosterone regulates its own production through a negative feedback of the
hypothalamus and pituitary (Kramer, 1996; Matty, 1985b; Conn, 1995). The
production of testosterone is under the control of FSH and LH, both of which are
regulated by E2 (Junqueira et al. 1986a) in a feedback mechanism. Atrophy of
the breeding tubercles may have resulted from an inhibition of LH, since
nonylphenol and nonylphenol ethoxylates are estrogenic compounds (Soto et al.
1991; Giger et al. 1984; Naylor, 1995; Jobling et al. 1996). The fatpad was not a
good indicator of exposure in any of the experiments with NP, however, the
diameter of the male breeding tubercles were predictive in the first NP
experiment The fatpad of the fathead minnow is composed of mucous cells in a
mucinous matrix bounded by a thickened epidermis (Smith, 1978). The value of
the fatpad as an indicator of exposure may have been lessen due to the use of
ethanol during tissue preservation. The trend observed in the diameter of the
tubules in response to the exposure concentration was nearly identical in both
NP experiments, although there was no statistical difference in any of the
concentrations of NP in the second experiment.

The histologic response of the testes to NP in both experiments included
Sertoli cell changes much like those observed in previous E2 studies. While
Sertoli cells appeared more numerous, it is was not possible to ascertain with
certainty if lesions observed were an absolute or a relative proliferation. More

quantitative measures, such as autoradiography, would be needed to make such

113

a determination. It is clear, however, that Sertoli cells were distended with
residual bodies and myelin figures to a greater degree than what would normally
be expected. Under normal circumstances, Sertoli cells are involved in the
phagocytosis of excess cytoplasm (residual body) that is discarded during the
process of spermiation (Russell et al. 1990) as well as clearing up germ cells
that may under apoptosis (Lofts, 1987; Russell et al. 1990). During these
processes, myelin figures often form as a result of the breakdown of the cell
membranes of the degenerative germ cells (Jones and Hunt, 1983). Therefore,
the Sertoli cell response observed in this study may have been simply an
extension of their normal phagocytic role. Interestingly, the phagocytic response
of Sertoli cells in NP-exposed males was coupled with the presence of
macrophages in the lumina of seminiferous tubules. This was not evident in the
previous (E2) studies. Phagocytic cells present in the lumina appeared to aid the
Sertoli cells in clearing up degenerate spermatozoa. Infiltration of seminiferous
tubules by macrophages has been observed in mammals exposed to foreign
agents (Russell et al. 1990). Perhaps such a response was noted in this study
because NP is a xeno-estrogen unlike the endogenous estrogen, E2.

The female fathead minnow was not a good indicator of exposure to the
estrogen agonists in this study. In females, FSH and LH are necessary for the
production of ovarian follicles, particularly primary and secondary follicles
(Junqueira et al. 1986b). Although these gonadotropins are regulated by a
similar estrogen control mechanism as in males, there was no evidence of an

alteration in the stages of follicular development after exposure to NP or NPE.

114

Previous studies demonstrated a greater number of primary follicles in response
to E2 exposure. Perhaps a similar response was not observed in this study
because NP and NPE are several orders of magnitude less potent than E2
(Naylor et al. 1992). Since E2 is normally present in females at a more elevated
level than in males, the levels of NP and NPE would probably need to have been
much higher in order to cause an alteration in the ratio of the stages of follicular
development.

Exposure to NPE did not induce gross or histologic changes in male or
female fathead minnows. Since commercially produced NPE is composed of a
mixture of isomers with various patterns of branching in the nonyl moiety
(Naylor, 1992), it is not likely to be as problematic as its degradations products,
which include NP. Furthermore, NPE is a more hydrophilic compound than NP
and is therefore more likely to remain in solution (Naylor, 1992) unlike NP which
is more lipophilic and likely to bioaccumulate. Such differences in the chemical
nature of NP and NPE may explain the differences in the gross and histologic

response of exposed males in this study.

CHAPTER 4:
APPLICATION OF THE FATHEAD MINNOW AS A BIOINDICATOR OF
SUSPECT ENVIRONMENTAL ESTROGENS IN WASTEWATER TREATMENT

EFFLUENT: A FIELD STUDY

ABSTRACT

The purpose of this study was to determine if the gross and histologic
responses of the fathead minnow are useful bioindicators of exposure to
suspect environmental estrogens in wastewater effluent. A total of 35 male and
35 female caged fathead minnows were placed in the effluent of mid-Michigan
area wastewater treatment sites for a 3-week exposure period. While it was
unknown whether or not specific estrogenic compounds were released at a given
site, wastewater treatment sites were selected based on the type of sewage
treatment used, the daily volume, the type of users (i.e. industry ,comrnercial, or
residential), and the type of disinfection used at the site. Six wastewater
treatment sites from the mid-Michigan area were selected along with 2 reference
sites. Sites selected were as follows: Owosso, Portland, Williamston, Eaton
Rapids, Delta Township and Bellevue. One reference site was located upstream
from the 6 wastewater treatment plants and the other reference site was a

Michigan State University limnology pond. The breeding tubercles and fatpads

115

116

were significantly larger at the limnology pond as compared with all other sites.
Males had significant testicular lesions consisting of Sertoli cell proliferation at
the Portland site as compared with all other sites. Females had varying trends
in the number of ovarian primary, Graafian, and atretic follicles between sites,
but there was no difference in the average number of secondary follicles at any
site. These results suggest that the breeding tubercles and gonads of sexually
mature male fathead minnows may be a good field indicator of suspect

environmental estrogens.

INTRODUCTION

Concern has been expressed recently in both the scientific and popular
press about the implications of environmental estrogens, particularly with recent
evidence of estrogens in sewage effluent. Many of these estrogenic
compounds are man-made, however natural compounds from plants have been
shown to act similarly. Laboratory studies have shown that when the
phytoestrogen B-sitosterol, present in high concentrations in bleached kraft pulp
mill effluent, is injected into goldfish, synthesis of testosterone, 11-keto
testosterone, and estradiol are decreased in males and females. The
estrogenic activity of wastewater constituents has been evaluated in laboratory
studies. Male rainbow trout (Oncorhynchus mykiss) hepatocytes were used in an
in vitro study to demonstrate that a number of detergent components in sewage

effluent are estrogenic (Russell and Peterson, 1984). Biodegradation products of

117

detergents, such as nonylphenol (NP) and 4-nonylphenoldiethoxylate were
shown to be estrogenic to fish cells in micromolar concentrations.

Several field studies have been conducted on the reproductive effects of
the components of papermill effluent and on sewage wastewater. Years ago,
researchers observed that masculinization occurred in mosquitofish (Gambusia
affinis) exposed to steroidogenic plant sterols in pulp and papermill effluent.
Masculinization was indicated by the induction of a gonopodium in females, a
structure used by males to transfer sperm packets into the female genitalium
during copulation. While exposed females developed this male organ, there
were no abnormalities noted in the ovaries and many of these viviparous fish
were actually pregnant (Howell and Denton, 1989; Denton et al. 1985).

As a result of the observation of masculinization in feral mosquitofish,
many field studies have focused specifically on effects of the constituents of
papermill effluent. More recently, however, field studies have focused on
suspect estrogens in sewage wastewater. A field study which compared ovarian
development in English sole from urban and non-urban sites in Puget Sound,
Washington demonstrated that females with elevated levels of fluorescent
aromatic compounds in bile were less likely to enter vitellogenesis and had lower
plasma concentrations of estradiol than female sole with low levels of
contaminant exposure (Johnson et al. 1993). These results were supported by
laboratory findings in which pretreatment of gravid female English sole with
contaminated sediment decreased levels of endogenous estradiol (Johnson et

al. 1993). In northern England, it was shown that wild populations of flounder,

118

Platichthys flesus, exposed to sewage effluent from a major sewage treatment
works and a number of industrial discharges had induction of the egg-protein
vitellogenin, increased hepatosomatic index and an increased level of testicular
abnormalities (Lye et al. 1997). Male carp collected from the Minnesota River,
which receives agricultural runoff, and from the St. Paul metropolitan sewage
treatment plant had reduced testosterone concentrations as well as vitellogenin
induction in males in the sewage effluent (Folmar et al. 1996). White suckers
(Catostomus commersonr) exposed to papermill effluent exhibited delayed age
to sexual maturation, decreased gonad size, and a reduction of secondary sex
characteristics (Munkittrick et al. 1991; McMaster et al. 1991).

As a result of a 1994 observation of herrnaphroditic fish in wastewater,
researchers have focused attention on compounds which may be estrogenic. It
has been suggested that likely estrogenic substances are alkylphenol
ethoxylates (APEs) which originate from the biodegradation of surfactants and
detergents during sewage treatment, or perhaps from the pharmaceutical use of
ethynylestradiol in the contraceptive pill (Purdom et al. 1994). Other sources
may include up to 10 pmollday of estradiol and estrone excreted by pregnant
women (Fostis, 1987). Agricultural by-products may also provide sources of
estrogen. For example, chicken manure can contain over 1 p mol of estrogen
(Shore et al. 1988). Cows, pigs, horses, and goats may also excrete large
amounts of estrogen into the environment (Knights, 1980).

Previous studies demonstrated that sexually mature fathead minnows

exposed to natural and synthetic estrogens had gross evidence of exposure

119

consisting of atrophy of breeding tubercles and histologic evidence consisting of
apparent relative or absolute Sertoli cell proliferation. There were also germ cell
syncytia, consisting of aggregates of various stages of germ cells in the
spermatogenic sequence. The purpose of this study was to evaluate the
usefulness of the gross morphologic and histologic response of the secondary
sex characteristics and gonads of sexually mature male and female fathead
minnows caged in wastewater treatment effluent as bioindicators of exposure to

environmental estrogenic compounds.

MATERIALS AND METHODS

Study design. Thirty-five sexually mature male and 35
sexually mature female fathead minnows were placed in cages in the effluent of
wastewater treatment plants at 6 wastewater treatment sites in mid-Michigan.
The sites were as follows: Owosso (OW), Portland (PT), Williamston (WM),
Eaton Rapids (EA), Delta Township (DT) and Bellevue (BV). Two reference sites
were selected. One was upstream from all other study sites (RF) and another
was a limnology pond at Michigan State University (LI). The fish were placed at
the sites for 3 weeks and left undisturbed.

Gross examination and_spgcimen collectjon. At the end of the
exposure period, cages were removed from the wastewater effluent, blood was
collected (Appendix B), and fish were sacrificed with an overdose of Finquel®
(Argent Chemical Laboratories, Redmond, VA). Gross secondary sex

characteristics of males were measured prior to histologic examination of the

120

testes (Appendix D). The height of male fatpad and the diameter of nuptial
breeding tubercles were measured using a 27 mm diameter 6X
comparator/magnifier and reticle (Edmund Scientific Company, Barrington, NJ).
Males and females were immersed in Bouin’s solution after the abdomen was
injected with 0.5 cc of the fixative. After 48 hours, carcasses were rinsed by
agitating them for 30 seconds in 30 mls of 70% ethanol. They were stored in
this solution until trimming. The fish were trimmed in serial transverse sections,
processed and embedded sequentially in paraffin (SurgiPath, Inc., Richmond,
IL). Tissues were sectioned at 5pm with a microtome, and stained with
hematoxylin and eosin for examination using a light microscope.

Gonad scoring criteria. Testicular lesions were evaluated
according to the severity of relative or absolute Sertoli cell proliferation and the
percentage of seminiferous tubules affected. Scores ranged from 0 to 4 as
follows:

0 = no Sertoli cell proliferation

1: < 25% Sertoli cell proliferation

2= 2 25% < 50% Sertoli cell proliferation

3= 2 50% < 75% Sertoli cell proliferation

4: >75% Sertoli cell proliferation
Degenerative changes including germ cell syncytia, mineralization of

spermatozoa, or necrotic spermatozoa were indicated by (+).

121

In females, ovaries were evaluated by counting fifty follicles in the ovary
of each female and calculating the percentage of each stage of follicular
development observed. Ovaries were staged according to the following criteria:

primary - large nucleus, abundant basophilic cytoplasm and no yolk

vesicles

secondary- presence of numerous eosinophilic yolk vesicles

Graafian- large follicle bordered by an amphiphilic egg membrane

with a central core of eosinophilic yolk protein

atretic- degenerative follicle with shrunken, irregular border
Statistical analysis. Treatment effects were examined by non-

parametric two-way ANOVA (SAS Institute, Carey, NC; PROC GLM) conducted
on the ranks of the responses. Significant treatment effects showed no
significant interaction. Terms were subsequently examined by Tukey’s HSD
multiple range test. Effects were determined to be statistically significant at the

0.05 level of type I error (alpha) and 0.2 of type II error (beta).

RESULTS
Fish sgwival; Since caged fish placed in the effluent were left
undisturbed, it was not possible to determine the number of fish deaths,
particularly since the carcasses of dead fish would have been subject to the
force of the water current. Other factors which could account for the reduction
in the number of fish recovered after the 3 week exposure include predation,

escape, and vandalism.

122

Gross examinatjm Males exposed to wastewater effluent at Ll had
significantly larger tubercles than males at all other sites (Table 12, Figure 28).
Fatpads of males at LI and PT were also significantly different from one another
and from males at all other sites. There was no difference in the size of the
fatpad between males at OW, WM, EA, DL, BV, and RS (Table 12, Figure 29).

Histologic Examination. Several males and females had mild to
moderate branchitis characterized by blunting and fusion of gill lamellae with
occasional intralesional protozoa consistent with lchthyophthin‘us multifiliis
(Figure 30). Males exposed to wastewater effluent at PT had apparent relative
or absolute Sertoli cell proliferation (Table 13, Figure 31) as compared with
males at other sites except ER and RS which were morphologically and
statistically similar (Figures 32, 33). Females at PT often had a preponderance
of primary follicles in the ovarian stroma (Table 14, Figure 34) compared with

other sites (Figure 35), however this was not statistically significant.

plSCUiSSION
Several studies have demonstrated that fish are important indicators of
water pollution (Haaparanta et al. 1996; Dunier and Siwicki, 1993).
Histopathology has been used as a biomarker of environmental stress in male
(Hinton et al. 1992; Spitsbergen et al. 1991; Wester and Canton, 1986; Wester
et al. 1985), and female (Komen et al. 1989; Johnson et al. 1988; Forsberg et al.
1985) and has gained more attention recently. It has also been shown that

secondary sex characteristics are useful indicators of waterborne exposure to

 

Table 12. Measurements of the diameter of tubercles and the
length of fatpads of male fathead minnows exposed to wasterwater

 

 

effluent.
Site AN Tubercles Fatpad
Median (mm) Median (mm)
(range) (range)
Owosso 1 3 0.4 0.5
(0-0.5) (0.1-1.2)
Portland 27 0.25 0.24
(0-0.4) (0.1-0.9)
Williamston 16 0.2 0.3
(0-0.5) (0.2-0.6)
Eaton Rapids 14 0.27 0.17
(0-0.7) (0.1-1.5)
Bellevue 7 0.26 0.26
(0.2-0.6) (0.1-0.4)
Delta Township 15 0.3 0.3
(0.2-0.8) (0.2-0.5)
Limnology pond 23 0.75 1.02
(0.6-0.9) (0.5-1.5)
Reference site 21 0.28 0.3
(0-0.6) (0.1-0.5)

 

AN= sample size

124

 

 

 

 

 

 

0.8
E 0.7
.5.

g 0.6
In
0
'5 0.5
3
'3 0.4
0
8 0.3
E
< 0.2
0.1
0

 

 

 

PT WM OW DL BV EA RS Ll
Field Sites

Figure 28. Relationship between average tubercle diameter and wastewater
treatment plant effluent at field sites. There was a statistical difference between
the Limnology pond and all other sites which were not different from one another.
(PT= Portland; WM= Vlfilliamston; OW= Owosso; DL= Delta Township; BV=
Bellevue; EA= Eaton Rapids; RS= Reference site; Ll= Limnology pond)

125

 

 

 

 

 

 

 

Average Fatpad size (mm)

 

 

 

PTVIMOND.BVEARSU
FreldSites

Figure 29. Relationship between average fatpad size and wastewater treatment
plant effluent at field sites. Statistical differences are denoted by letters A, B, and C
where the same letter indicates no difference. (PT=Portland; WM=Williamston;
OW=Owosso; DL=Delta Township; BV= Bellevue; EA= Eaton Rapids; RS=
Reference site; Ll= Limnology pond)

126

‘_
"- t)‘ “J”?

:wa::‘\

 

Figure 30. Photomicrograph of gill from a fathead minnow. Note branchitis
characterized by blunting and fusion of gill lamellae and intralesional protozoa
consistant with Ichthyophthin'us multifiliis . Hematoxylin and eosin. x 624.

127

Table 13. Testicular severity scores of males exposed to wastewater
treatment effluent.

 

 

Site "N Severity Score (0-4)
mean median range
Owosso 1 3 1 .5 3 0-4
Portland 27 3.6 4 0-4
Williamston 15 1.86 0 0-4
Eaton Rapids 17 2.7 1 0-4
Bellevue 8 1 .87 1 1-4
Delta Township 15 2 3 0-4
Limnology pond 23 0.2 0 0-2
Reference site 21 2.57 4 0-4

 

AN: sample size

128

 

Figure 31. Photomicrograph of testis from a fathead minnow from the Portland
site. Note numerous Sertoli cells lining seminiferous tubules. Hematoxylin and
eosin. x 499.

 

 

 

129

 

 

 

 

w

 

N
01

 

Score (0-4)

H
AU'IN

p
a:

 

 

 

O

PTVIMOND.BVEAIEU
FreldSilles

Figure 32. Relationship of testicular severity score and wastewater treatment
plant effluent at field sites. Statistical differences are denoted by letters A, B, and C
where the same letter indicates no difference. (PT=Portland; WM= Vlfilliamston;
OW=Owosso; DL=Delta Township; BV= Bellevue; EA= Eaton Rapids; RS=
Reference site; Ll= Limnology pond)

130

 

Figure 33. Photomicrograph of testis from a male fathead minnow at the
Eaton Rapids site. Note the normal appearance of seminferous tubules.
Hematoxylin and eosin. x 62.

131

Table 14. Percentage of ovarian follicular stages in females exposed
to wastewater treatment plant effluent.

 

 

Site AN % Follicular Stage*
P S G A
mean mean mean mean
(range) (rang) (range) (ranggL
Owosso 1 42 8 5O 0
BN/A N/A NIA NIA
Portland 9 65 12 5 7
(70-90) (2-20) (0-38) (0-16)
Williamston 7 73 10 5 6
(50-92) (2-30) (0-26) (0-14)
Eaton Rapids 18 64 12 16 3
(30-96) (2-20) (0-56) (0-14)
Bellevue 8 85 1 1 0 , 4
(76-96) (4-16) (0-0) (0-16)
Delta Township 4 57 19 12 13
(42-79) (16-22) (2-34) (10-22)
Limnology pond 28 54 16 29 0.7
(36-78) (4-28) (14-40) (0-4)
Reference site 32 79 13 3 4
(62-48) (2-28) (0-12) (0-20)

 

-p= primary; S= secondary; G= Graafian; A= atretic

AN= sample size
BNIA= not applicable

132

 

Figure 34. Photomicrograph of ovary from a fathead minnow at the Portland
site. Note the preponderance of primary follicles. Hematoxylin and eosin. x 121.

133

 

Figure 35. Photomicrograph of ovary from a female at the Eaton Rapids site.
Note a relatively equal ratio of primary (P), secondary (S) and Graafian (G)
follicles. Hematoxylin and eosin. x 62.

134

environmental hormone mimics (Denton et al. 1985; Howell and Denton, 1989;
Howell et al. 1980).

Although it was not known if any specific estrogenic compounds were
present in the wastewater treatment sites selected, fish in the Ll site, which
served as a source of all other fish in the study, had significantlylarger fatpads
and tubercles than fish from any other site. The development and maintenance
of these secondary sex characterisitics in male fathead minnows is due to
testosterone, which is regulated by luteinizing hormone (LH). Compounds which
mimic E2 have been identified in sewage wastewaters (Naylor et al. 1992),
therefore it is possible that such compounds were present in the wastewater
treatment effluent in this study. Exposure to environmental estrogen mimics
could lead to an inhibition of luteining hormone (LH) (Junqueira et al. 1986a),
and subsequent atrophy of secondary sex characterstics. Since all of the fish
originated from Ll, it is highly likely that the reduction observed in the tubercles.
and fatpads was real.

There was also a striking difference between sites in the histologic
response of the testes, particularly in fish from the PT site. In previous studies
with E2 and nonylphenol, a similar histologic response was observed with an
increase in Sertoli cell numbers. Sertoli cell proliferation is not known to occur
in mammals, since the number of Sertoli cells do not increase after puberty
(Fawcett, 1975; Fawcett, 1977; Russell et al. 1990; Russell et al. 1981). Further
work is needed to determine if this is true in fish. It is possible that Sertoli cells

appeared more numerous because of a reduction in number and stages of germ

135

cells. A monomorphic population of germ cells observed in PT and EA suggests
an arrest in the spermatogenic maturation sequence. Again, this could be due to
an alteration in the hypothalamic-pituitary axis. It is also possible that Sertoli
cells increased in number because of the need to phagocytize degenerate
spermatozoa (Russell et al. 1990; Fawcett, 1977; Fawcett, 1975) which were
unable to continue along the maturation sequence. In this case, degeneration
would have occurred do to an arrest in the maturation sequence resulting from
the negative feedback of E2 (or E2 agonists) on LH and follicle stimulating
hormone, both of which are necessary for spermatogenesis to proceed normally
(Junqueira et al. 1986a; Eddy and O'Brien, 1994)

Because of the similarity of lesions observed in previous studies with E2,
it is important to identify if estrogenic compounds are actually present in the
wastewater effluent. Water samples have subsequently been collected and
tests are currently undenlvay to determine what, if any, estrogenic compounds
are present in the wastewater effluent. Meanwhile, ancillary tests have
demonstrated that water samples from four sites (PT, WM, BV, and EA) caused
proliferation of estrogen-responsive MCF-7 cells, suggesting the presence of
estrogenic compounds at those sites. There were also some differences in
water quality parameters at two of the four sites containing fish with testicular
lesions. In addition to the presence of apparent Sertoli cell proliferation, the PT
site was the only one of those chosen which had an elevated average and
maximum chlorine content in the effluent. At PT, the chlorine level of the effluent

averaged 0.3 mg/L with a maximum level of 0.45 mglL , whereas all other sites

136

had levels which did not exceed 0-0.1mg/L. Chlorine is toxic to fish at levels of
0.2-0.2 mg/L but primarily affects the gills (Andrews et al. 1988). Another site
(EA) with similar histologic lesions had the highest level of fecal coliform,
measuring 537 lb/100 mls, compared to all other sites which ranged from 8 to
291 lb/100mls. It is unknown, however, if these differences in water quality
parameters could have played a role in the difference in the histologic response
observed.

This study demonstrates that the fathead minnow is a hearty and robust
fish amenable to caged field studies and that the histologic response of the
sexually mature male is a sensitive indicator of suspect environmental
estrogens. Work is currently underway to determine the constituents of the

wastewater effluent to confirm the presence of estrogenic compounds.

W

The overall goal of this research was to determine if the fathead minnow
is a good fish species to use as a bioindicator of environmental estrogens.
Estrogenic compounds and other “endocrine disrupting chemicals” have received
much attention because of the vast number and common use of many of the
compounds implicated and the potential magnitude of adverse effects across
species. Since many of these compounds ultimately appear in wastewaters and
other aquatic basins, a fish would seem a likely species to serve as a sentinel for
not only the health of the ecosystem in question, but also as an indicator of
potential human health effects. The prominent secondary sex characteristics of
the fathead minnow make it an ideal bioindicator species since these features
could serve as a rapid, visual screen of estrogen exposure. The fish is also easy
to maintain in both laboratory and field studies. Furthermore, the robust nature
of the fathead minnow makes it tolerant of extreme environmental conditions and
tolerant of exposure to less that ideal water quality conditions.

There are many uncertainties regarding environmental estrogens,
including, but certainly not limited to, questions of additivity or synergy among
estrogen agonists. The complexities that surround the whole issue of

environmental estrogens suggest that a simple animal model may be difficult to

137

138

develop. Furthermore, we do not yet know how to account for subsequent
exposures to estrogen agonists and antagonists. Also, it is not yet clear what
effects such estrogenic mimics would have on a system that is simultaneously hit
with other hormone mimics. There are may be differences in the number,
location, and binding specificity of the estrogen receptor between various aquatic
and mammalian species. The nature of the endocrine system, with all of its
checks and balances, further muddles our understanding of effects of these
types of compounds on this intricately complex system.

Nonetheless, the obvious course of action appears to be to identify a
sentinel species which can alert humans of the presence of environmental
estrogens and to which other more rapid and inexpensive in vitro screening
assays may be calibrated. Towards that end, this research has provided
valuable information. Although the prominent secondary sex characteristics
were not as useful as originally hypothesized, based on the histologic response
of the testis, and to a lesser extent, atrophy of the breeding tubercles, the
fathead minnow does appear to be a good fish species to pursue as an indicator
of estrogen exposure. The implications of this research are that reproductive
efficiency may be impaired which could affect not only individuals, but also
communities and ultimately populations. Preliminary data collected on the
number of eggs produced by fathead minnows exposed to 17 B-estradiol (E2)
indicate a dose-dependent decrease in eggs spawned. This reduction in eggs

correlated inversely to induction of the yolk precursor protein, vitellogenin. Eggs

139

collected during nonylphenol studies did not follow a typical dose-response
curve, rather, an “inverted U” was noted with decreases in eggs numbers
observed mid concentrations. Highest concentrations, however, had identical
eggs numbers as the controls. This information, though, not included in this
study, demonstrates further the range of responses that could be observed in
real-life situations with environmental estrogens and further underscore the need
for a sentinel species.

Questions regarding whether or not humans would respond in a manner
similar to that demonstrated in this study with fathead minnows remain

unanswered.

W

While this research has provided meaningful information on the use of
fish and the use of pathologic lesions as bioindicators of exposure to
environmental estrogens, there are several areas of research that should be
pursued to further characterize this model. The current study took initial steps in
characterization through the use of basic pathologic techniques of gross,
histologic and ultrastructural morphologic examination. Areas of further study
are focused primarily on quantitative techniques to further characterize and
confirm the morphologic changes observed in this research:

1. The histologic response of the Sertoli cells should be confirmed with
histochemical techniques or autoradiography. A bromodeoxyuridine (BRDU)
study was attempted to determine if cell replication were occurring, however,
there was interference between the mitotic action of spermatogonia which also
lie on the basement membrane of seminiferous tubules along with the Sertoli
cells, thus rendering the results inconclusive. Autoradiographs, prepared from
tissue sections and observed at the electron microscope level, can be used to
address this problem. Alternatively, histochemical or morphometric techniques
can be developed and/or optimized to demonstrate absolute increases in Sertoli
cell number. The usefulness of morphometry was investigated. This type of

analysis requires that samples be collected from identical areas in each fish.

140

141

The variable nature of the size of the fish and location of the testis precluded the
use of morphometrics in this study.

2. Flow cytometry could be used to differentiate testicular germ cells
which degenerated due to apoptosis from those which are necrotic. Since
apoptosis, or programmed cell death normally occurs in reproductive tract, it is
important to differentiate this normal process from the degenerative process of
necrosis.

3. Cytokinetic studies can be undertaken to determine if the Sertoli cell
response was the result of altered cell-cell communication. The Sertoli cell
maintains a tight junction, the blood-testis barrier, which prevents the free
passive of materials from the blood to the germ cells of the seminiferous tubules.
It is believed that these tight junctions also serve to keep cell from uncontrolled
proliferation. Alteration in the tight junctions of Sertoli cells may, therefore be
responsible for the apparent Sertoli cell proliferation observed.

Other studies should be undertaken to determine whether or not the adult
fathead minnow is the most sensitive age to use as a bioindicator and to
document effects in other age groups, particularly immature fathead minnows
and those exposed as embryos. Also, one could further assess effects on
reproductive efficiency by doing backcross studies, such as breeding exposed
males with unexposed females and exposed females with unexposed males, and
so on. This would help to identify potential reductions in reproductive efficiency

and isolate the sex most impaired.

APPENDICES

APPENDIX A

142

APPENDIX A

FATHEAD MINNOW NECROPSY FORM

DATE: TIME:
MANNER OF DEATH: IDENTIFICATION:
SEX: TREATMENT:

 

 

 

GROSS DESCRIPTION OF LESIONS:

 

 

 

CONCLUSION:

 

PROSECTOR:

 

 

 

 

SAMPLES TAKEN FOR HISTOPATHOLOGY:

 

 

 

PROSECTOR:

 

HISTOPATHOLOGIC DESCRIPTION OFLESIONS:

 

 

 

 

CONCLUSION:

 

EXAMINER:

 

 

 

APPENDIX B

143

APPENDIX B

Standard Procedure
Clinical Pathology of Fathead Minnows during Exposure to Xeno-estrogens
Version 1.1
Aquatic Toxicology Laboratory/ Department of Pathology
Michigan State University
August 24, 1994
Author: Stephanie Miles-Richardson

Investigators: Stephanie Miles-Richardson, Vincent J. Kramer, Natalie White
Principle Investigator: John P. Giesy

1. At the end of the exposure sudy, anesthetize fish using MS-222 (FINQUEL) at
a dosage of 37 mglL in a beaker containing culture water.
2. Collect blood immediately.

a. Use a scapel blade to remove tail.

b. Collect blood via tail vein in heparinized microhematocrit tubes.

3. Return fish to a beaker of culture water containing lethal concentration of MS-
222 for euthanasia.

4. Place fish in a labeled 50 ml screwtop vial containing Bouin’s solution. After
24 hours, replace Bouin’s solution with 70% ethanol until fish are trimmed.

5. Centrifuge microhematocrit tubes at 10,000 rpms for 5 minutes.

6. Record packed cell volume (PCV) using a microcapillary reader.

APPENDIX C

144

APPENDIX C

Recipie for Resin Used in Tissue Preparation for Electron Microscopy

1. 20 mls Araldite 502
2. 25 mls of Polybed 812
3. 60 mls of DDSA (Dodecenylsuccinicanhydride)

Mix ingredients for 15 minutes.

Freeze mixture ( -11 °C) and thaw as needed.

Once thawed, add UMP-30 (tri dimethylaminomethyl phenol).

Stir mixture.

Pour into appropriate containers and bake ( 75 °C) until hardened.

(source: Department of Pathology, Electron Microscopy Laboratory, Michigan
State University.)

APPENDIX D

145

APPENDIX D

Standard Procedure

Histologic Examination of Fathead Minnows during Exposure
to Xeno—estrogens
Version 1.1
Aquatic Toxicology Laboratory/ Department of Pathology
Michigan State University

August 23, 1994

Author: Stephanie Miles-Richardson
Investigators: Stephanie Miles-Richardson, Vincent J. Kramer, Natalie White
Principle Investigator: John P. Giesy

1. Make observations of fish health and record any abnormalities in “Fish
Pathology” log.

If fish are observed in distress, i.e. circling, etc., euthanize by administering an
overdose of MS-222 (FINQUEL) in a beaker filled with culture water.

2. Remove any dead fish and preserve in a 50 ml screw-top centrifuge vial filled
with 30—35 mls of Bouin’s solution * for 48 hours. Complete an individual
“Fathead Minnow Necropsy Form” for each fish (see Pathology log). After a 48
hour time period, agitate the fish carcass in 30 mls of 70% ethanol for
approximately 30 seconds and store the fish in this solution until fish are
trimmed. (Place the fish in the vials such that the head is toward the wide part of
the vial and the tail is toward the narrow end. Screw the tops on securely and
store the vials upside-down.)

3. Select a random subsample of fish in each treatment group for photography of
gross features.

5. Prior to trimming fish, using a comparatorl reticle, take measurements of the
nuptial tubercles on the males by averaging the diameters of three randomly
selected tubercles. Measure the length of the fatpad in males and the length of
the female ovipositor using the linear scale on the same instrument.

6. Collect testis for electron microscopy. Cut multiple 10mm transverse sections
across the abdomen of fish, cranial to the anal fin but caudal to the pectoral fin.
Store these sections in 10 ml glass vials containing 2% glutaraldehyde
(approximately 8 mls) for 24 hours. At that point, dissect the testis from the
section and store in a separate vial of glutaraldehyde. After another 24 hour
period, transfer sample to a vial containing 0.1 M phosphate buffer. Deliver the

146

sample, stored in this solution, to the Electron Microscopy Laboratory (Michigan
State University, Department of Pathology, East Fee Hall) for post—fixation and
processing. ‘

7. Trim fish according to the diagram below beginning just cranial to the caudal
musculature taking care to collect 1/4 inch transverse sections of (1) caudal
musculature and gonads; (2) gonads and liver; (3) heart and liver; and a
longitudinal section of the cranium including (4) brain and gills. After making the
longitudinal section and prior to collecting brain and gill samples, measure the
fatpad using the comparator/reticle.

4 ................... H —_//-
5:3 \\

3 2 1

 

 

8. Place trimmed tissues into labeled cassettes and transport in 10% neutral
buffered formalin to the Animal Health Diagnostic Laboratory for paraffin
embedding and staining.

9. Complete “Fathead Minnow Trim Log” located in the trim log notebook.

*BOUIN’S SOLUTION
Picric acid, saturate aqueous solution 7500c
37-40% formaldehyde 2500c
Glacial acetic acid 50cc

(source: Animal Health Diagnostic Laboratory, East Lansing, MI)

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VITAE

The author was born in Nashville, Tennessee on May 17, 1966 to Dr.
Benny M. Miles and Mrs. Bobbie Watson Miles. The author's family relocated to
Grambling, LA in her early childhood years where they remain in residence. The
author received a BS. degree in Biology from Grambling State University in
1988. She recieved a Dector of Veterinary Medicine (D.V.M.) degree in May of
1992 from Tuskegee University in Tuskegee. AL. She began graduate study at
Michigan State University in the fall of 1992. While a graduate student in the
Department of Pathology. she received a National Institutes of Health (NIH)

postdoctoral fellowship.

 

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