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This is to certify that the
dissertation entitled

THE ROLE OF ESTROGEN IN THE RETENTION OF COPULATORY
BEHAVIOR AFTER CASTRATION IN BGDZFl MALE HOUSE
[VDUSE (MUS MUSCULUS)

 

presented by

Kevin Sinchak

has been accepted towards fulﬁllment
of the requirements for

Phi. D . degree in Zool Neu oscience

    

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MSU Ie An mum Mon/Equel Oppottunltv Imam
WWI

 

THE ROLE OF ESTROGEN IN THE RETENTION OF COPULATORY BEHAVIOR
AFTER CASTRATION IN B6D2F1 MALE HOUSE MOUSE (MUS MUSQULUS)

By

Kevin Sinchak

A DISSERTATION

Submitted to
Michigan State University
in partial fulﬁlling of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Zoology and Neuroscience Program

1996

ABSTRACT

THE ROLE OF ESTROGEN IN THE RETENTION OF COPULATORY BEHAVIOR
AFTER CASTRATION IN B6D2F] MALE HOUSE MOUSE (MUS MUSCULUS)

By

Kevin Sinchak

In the B6D2F] hybrid strain of house mouse, (Mu_s musculus), most males continue
to achieve an ejaculatory reﬂex for six to twelve months after castration (continuers), while
others stop copulating within a few weeks after castration (noncontinuersXClemens et al.,
1988; McGill & Manning, 1976). The present study investigated: 1) if continued copulation
after castration in B6D2F 1 males is dependent on steroid hormone stimulation; 2) if continuer
and noncontinuer males differ in steroid hormone physiology.

Serum levels of testosterone (T), and estradiol (E2) were measured in intact and
castrated continuer and noncontinuer males. Castration reduced, but did not eliminate T from
the circulation of B6D2F] males. In contrast, castration did not aﬁ‘ect serum E2 levels.
However, continuer and noncontinuer males did not differ in serum T or E2 levels. The
adrenal gland is not the source of these nongonadal androgens and estrogens, since removal
of the adrenals had no effect on serum T and E2 levels in castrated males. The importance of
nongonadal estrogens in the maintenance of copulation in continuer males was determined
by blocking the aromatization of androgens to estrogens with an aromatase inhibitor, 1,4,6-
androstatriene-Z-l7-dione (ATD) in continuer males. Inhibition of estrogen synthesis by ATD
reduced the percentage of continuer males that achieved ejaculation and intromission, but had
no eﬂ‘ect on percentage of males that mounted. Continuer and noncontinuer males appear to

differ in their responsiveness to estrogens, since they do not differ in circulating levels E2 and

continued copulation is dependent on synthesis of nongonadal estrogens.

Since copulation in continuers appears to be estrogen dependent, and continuers and
noncontinuer appear to differ in responsiveness to estrogens, aromatase activity (AA), and
estrogen receptor (ER) levels were measured in the preoptic area (POA), hypothalamus
(HYP) and amygdala (AM) to determine if continuer and noncontinuer males differ in
estrogen physiology. In general continuers and noncontinuers did not diﬂ‘er in AA or ER
levels. Castration reduced but did not eliminate AA in POA, HYP and AM. Castration did
not aﬂ‘ect nuclear ER levels in the POA and HYP but reduced nuclear ER levels in the AM.
The data support the hypothesis that sexual behavior of castrated B6D2F2 male mice
continues to be inﬂuenced by nongonadal E2. Although continuer and noncontinuer males
appear to differ in responsiveness to estrogens, they could not distinguished by serum '1‘, or

E2 levels, or AA or ER levels in the POA, HYP and AM.

Copyright by
Kevin Lee Sinchak
1996

To

Mom and Dad

ACKNOWLEDGMENTS

I would like to thank my mentor, Dr. Lynwood Clemens for his support, wisdom and
tolerance during the many years of my graduate training. Additionally, I would like to thank
my committee members, Drs. Antonio Nunez, Donald Straney, John 1. Johnson, Martin
Balaban, and Fred Dyer for their participation in my graduate training. I must thank the TG
behavioral-endocrine group in Psychology Research, especially Tony Nunez and Cheryl Sisk.
Their support, advice and friendship were invaluable. Dr. Charles E. Roselli deserves my
gratitude for his generosity, help, and patience for running and teaching me the estrogen
receptor and aromatase activity assays and guidance in interpreting our data. Thanks, Chuck!
I’ve had great technical support over the years, and I would like to thank David Brigham,
Kevin Grant, and Brad Rakerd for their expertise. Further, I would like to thank Dr. William
Raum for running the steroid assays. I wouldn’t be where I am now if it weren’t for Beth
Wee and David Weaver. It is these two that I have to thank for inducting me and getting me
started in reproductive behavior (the scientiﬁc aspects of it that is).

I need to thank the army of undergraduates that collected behavioral data, castrated,
perfused, and injected hormones on the ”mouse project" over the years. I really enjoyed
working with and teaching them. Their contributions were invaluable. I need to recognize
a few special undergraduates that worked above and beyond duty. Ilaben Patel, Linda Kenke

and Mike Dilaberto were great assistants and good friends to have around.

vi

This research was supported in part by PHS Award HD0676O to Dr. Clemens, and
NIH Grant 23293 to Dr. Chuck Roselli of Oregan Health Sciences University. Further, my
graduate studies were funded by teaching and research assistantships from the Zoology
Department and College of Natural Science. Travel funds were received from the
Department of Zoology and the Neuroscience Program in the College of Osteophathic
Medicine. The "Crew” in the Zoology Oﬂice deserves my thanks also for their help over the
years (Tracy, Chris, Jan, et al.). Although it was out of the need to pay rent and keep myself
fed, 1 had two jobs that were excellent learning experiences. They taught me a lot about
teaching and science, and kept the acedemic ﬁres burning. First, I must thank the folks of
Lansing Community College, Carol Hurleburt, Sue Anderson, Chris Marshall, Roscoe Root,
Lou, Pat, and Sandy. It was a great learning experience, and fun people to work with, which
helped compensate for the wages. Second, I thank Drs. Kenneth Moore and Keith
Lookingland for hiring me on as a gloriﬁed technician in their "Neurophannatoxicology Lab".
They gave me the opportunity to get back into research and develop and learn new
techniques. Thanks Keith for your support and ﬁiendship. I doubt that I’ll ever be hired again
on the condition that I play inﬁeld.

I have been blessed with having great group of people that I call my friends. They
have been essential for my sanity, keeping me in line and keeping me in and out of trouble.
Andy, Brent, Francis, Jeﬁ', and Reid were there before, during, and will be there long after.

There have been many that came on board during the ride, and will be there for the years to

vii

come. For starters, these include Chris and Mark Wagner, Dr. "Bob" Cole, Becky and Marc
Bailie, Mike ”Hickmaster” Kashon, Kris Krajnak, Yu-Wen Chung, Liang-Yo and Fu-Mei
Yang. Further, I must mention a number of others, Yu-Ping, Alan, Tony, Gary, Colleen,
Torn, Margo, Mary, Mary, Kristen, John, Rob D, Misty, Sun, Ken, Heather, Suzanne, Bubba
(T om), Dr. Fang, Jay, Hlubek, Rob (Sunday Boy), Carrie, Yvonne, et al.... I am truly lucky!

I can’t say enough for the support my family has given me. I must thank my sister
Karen for having a similar circadian rhythm, so that I could call her when I needed a break
after midnight and my brother-in-law Jim for tolerating ”Sinchak” telephone habits, as well
as all the entertainment and moving services they provided. I have to thank my brother Mike
for keeping the pages "in focus" throughout my schooling, and my nieces Erika and Nikki for
their interest and someone to pick on when I go home. I wish my grandparents, Gladys and
Stanley Baldwin were able to share in my accomplishment, there would have been one helluva
a barbeque to celebrate! Last, but not least, are my Mom and Dad, Yvonne and Sam Sinchak.
I can’t sufﬁciently express the appreciation I have for all the love and support they have
provided me throughout my life. All I can say is that I hope I can do the same for my

children. Thanks!

viii

TABLE OF CONTENTS

LIST OF TABLES

............................................................ xii
LIST OF FIGURES ................................................. xiii
LIST OF ABBREVIATIONS .......................................... xv
INTRODUCTION .................................................... l

Eﬂ’ects of Orchidectomy and Steroid Hormones
on Sexual Behaviors .............................................. 2
Effects of Castration on Copulation .................................. 2
Castration and the Effects of Androgen and Estrogen Replacement on Male
Copulatory Behavior ....................................... 4
Testosterone .............................................. 4
Metabolism of Testosterone .................................. 5
Effects of Estrogen and Reduced Androgen on Copulatory Behavior in Castrated
Males ................................................... 6
Aromatization of Androgens to Estrogen and Behavior ................... 8
Effects of castration on steroid hormone levels .................... 9
Sources of Nongonadal Steroids ................................... 10
Neural Circuits Important for Male Copulatory Behavior: Their Metabolism of, and
Responsiveness to Steroid Hormones .......................... 13
Chemosensory Pathways ......................................... l4
Olfactory Bulbs and Copulatory Behavior ................. l4
Projections of the Main and Accessory Olfactory Bulbs ....... I6
Amygdala (AM) .......................................... l7
Projections of the Amygdala ................................. 18
Stria Terminalis and Bed Nucleus of the Stria Terminalis (BNST) ..... l9
Medial Preoptic Area and Hypothalamus ....................... 20
Regions of the Brain that Concentrate Steroid Hormones and the Effects of
Intracerebral Implants of Steroid Hormones ............... 23
Brain regions that concentrate androgens ................. 24
Brain Regions that Concentrate Estrogens ................. 24
Effects of T microimplants ............................ 25
Neural aromatase activity and copulatory behavior ................ 26
Neural aromatase activity and the effects of castration ........ 26
Estrogen Receptors ....................................... 29

ix

Location of estrogen receptor in copulatory behavior neural circuits . . . 29

Measuring ER ........................................... 30

Effects of castration and hormonal regulation of ER ......... 31

Summary and Purpose ..................................... 32
EXPERIMENT 1: LEVELS OF SERUM STEROID HORMONES IN INTACT AND
CASTRATED CONTINUER AND NONCONTINUER MALES ........ 34
METHODS .................................................. 35
RESULTS .................................................... 41
SUMMARY .................................................. 42

EXPERINIENT 2: DETERMINATION IF MAINTENANCE OF COPULATORY
BEHAVIOR AFTER CASTRATION IN B6D2F] MALES IS DEPENDENT ON
THE AROMATIZATION OF N ONGONADAL ANDROGENS TO

ESTROGENS ................................................. 44
METHODS .................................................. 44
RESULTS .................................................... 47
SUMMARY .................................................. 49

EXPERIMENT 3: DETERMINATION OF AROMATASE ACTIVITY IN POA, HYP
AND AM OF INTACT AND CASTRATED CONTINUER AND

NONCONTINUER B6D2Fl MALES .............................. 55
METHODS .................................................. 57
RESULTS .................................................... 58
SUMMARY .................................................. 59

EXPERIMENT 4: ESTROGEN RECEPTOR LEVELS IN THE POA, HYP AND AM
OF INTACT AND CASTRATED CONTINUER AND NONCONTINUER
B6D2F] MALES .............................................. 62

EXPERINIENT 4A: THE EFFECTS OF CASTRATION AND BEHAVIORAL
STATUS ON ESTROGEN RECEPTOR LEVELS IN THE POA, HYP
AND AM OF INTACT AND CASTRATED CONTINUER AND

NONCONTINUER B6D2F] MALES ........................ 62
METHODS ............................................. 63
RESULTS .............................................. 65
SUMMARY ............................................ 66

EXPERIMENT 4B: COMPARISON OF ER MEASURED IN THE POA, HYP,
AND AM BETWEEN NUCLEAR PELLETS PURIFIED WITH
SUCROSE AND NUCLEAR PELLETS NOT PURIFIED IN INT ACT

AND CASTRATED B6D2Fl MALES ....................... 74
METHODS ............................................. 74
RESULTS .............................................. 76

SUMMARY ............................................ 77

EXPERINIENT 5: DETERMINATION OF AROMATASE ACTIVITY LEVELS IN

POA, HYP AND AM OF INTACT AND CASTRATED C57BL/6J, DBA/ZJ

AND B6D2Fl MALES .......................................... 82
METHODS .................................................. 84
RESULTS .................................................... 86
SUMMARY .................................................. 86

EXPERIMENT 6: SERUM CONCENTRATIONS OF TESTOSTERONE,

ESTRADIOL AND DIHYDROTESTOSTERONE IN B6D2F] INTACT,
CASTRATE, AND CASTRATE-ADRENALECTOMIZED MALES . . . . 90

INTRODUCTION ............................................. 90
METHODS .................................................. 91
RESULTS .................................................... 93
SUMMARY .................................................. 94
GENERAL DISCUSSION ........................................... 101
BIBLIOGRAPHY .................................................. 1 12

xi

Table 1

Table 2

Table 3

LIST OF TABLES

Mean (+/- s.e.m.) circulating levels of testosterone (T) (ng/ml) in intact and
castrated males of several mammalian species, and the levels circulating T in
the castrate as a percent of intact T levels .................................................. l 1

Mean (+/- s.e.m.) circulating levels of estradiol (52) (pg/ml) in intact and
castrated males of several mammalian species, and the levels circulating E2 in
the castrate as a percent of intact E2 levels ................................................. 12

Mean (+/- s.e.m.) concentration of testosterone (T), dihydrotestosterone

(DH'I‘), and WIS-estradiol (E2) in serum of intact and castrated continuer and
noncontinuer B6D2F 1 males ...................................................................... 43

xii

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

LIST OF FIGURES

The percent of the last six copulatory behavior tests with behavior (mount,
intromission, and ejaculation) for intact (Int) and castrated B6D2F]
continuers (Cont) and noncontinuers (None). Each bar represents the total
number of tests with behavior for all males within a behavioral group divided
by the total number of tests for all males (refer to text for deﬁnitions of
behavioral groups) ..................................................................................... 39

Percent of continuer B6D2F] males that expressed behavior A) ejaculatory
reﬂex, B) Intromission, C) Mount per test 24 hours (hr) prior to and 2, 4, and
6 weeks (wk) aﬁer ATD or blank silastic capsule implant. * = signiﬁcantly
less than blank silastic implant group on that test (p < 0.05). + = signiﬁcantly
less than preirnplant (-24 hour) test within treatment group (p < 0.05) ........ 51

Mount latency (sec) +/- (s.e.m.) for blank silastic capsule and ATD treated
continuer B6D2F] males 24 hours (hr) prior to and 2, 4, and 6 weeks (wk)
after ATD or blank silastic capsule implant ............................................... 53

Mean (+/- s.e.m.) aromatase activity levels (fmol/h/mg protein) in the POA,
HYP, and AM for intact (Int), and castrated B6D2F] continuers (Cont) and
noncontinuers (None). * = signiﬁcantly less than intact treatment group (N-K
p < 0.05). + = signiﬁcantly greater than other brain regions within behavioral
group (N-K p < 0.05) ................................................................................ 60

Mean (+/- s.e.m.) nuclear estrogen receptor (ERn) levels (fmol/mg DNA) in
the POA, HYP, and AM for intact (Int) and castrated B6D2Fl continuers
(Cont) and noncontinuers (N one). * = signiﬁcantly less than intact treatment
group, (N-K p < 0.05). + = signiﬁcantly greater than other brain regions
within behavioral group (N-K p < 0.05) ..................................................... 68

Mean (+/- s.e.m.) cytosolic estrogen receptor (ERc) levels (fmol/mg DNA)
in the POA, HYP, and AM for intact (Int) and castrated B6D2Fl continuers
(Cont) and noncontinuers (None). * = signiﬁcantly greater than intact
treatment group (N-K p < 0.05). + = signiﬁcantly greater than other brain
regions within behavioral group (N-K p < 0.05). A = signiﬁcantly greater than
HYP within behavioral group (N-K p < 0.05) ............................................. 70

xiii

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Mean (+/- s.e. m.) total estrogen receptor (ERt) levels (ERn + ERc) (ﬁnal/mg
DNA) in the POA, HYP, and AM for intact (Int) and castrated B6D2Fl
continuers (Cont) and noncontinuers (None). * = signiﬁcantly greater than
intact treatment group (N-K p < 0.05). + = signiﬁcantly greater than other
brain regions within behavioral group (N-K p < 0.05) ................................ 72

Mean (+/- s.e.m.) levels of DNA (pg) in the nonpuriﬁed (N) and sucrose
puriﬁed (S-P) nuclear pellet of brain tissue ﬁ'om the POA, HYP, and AM of
intact (Int) and castrated (Cast) B6D2F] males .......................................... 78

Mean (+/- s.e.m.) nuclear estrogen receptors (ERn) levels (ﬁnol/mg DNA) of
nonpuriﬁed (N) and sucrose puriﬁed (S-P) nuclear pellet of brain tissue from
the POA, HYP, and AM of intact (Int) and castrated (Cast) B6D2F 1 males.
................................................................................................................. 80
Mean (+/- s.e.m.) aromatase activity levels (ﬁnol/h/mg protein) in the preoptic
area (POA), hypothalamus (HYP), and amygdala (AM) for intact (Int), and
castrated C57BU6J, DBA/ZJ and B6D2F 1 male house mice. * = signiﬁcantly
greater than other strains within surgical treatment group (SNK, 2 < 0.05).

.................................................................................................................. 88

Mean (+/- s.e.m.) concentration of testosterone (T)in serum of sham
castrate/sham adrenalectomized (S/S), sham castrate! sham adrenalectomized
(Cas/ S), castrate/3 day adrenalectomized (Cas/Adx3), castrate/14 day
adrenalectomized (Cas/Adxl4) B6D2F] males. * = signiﬁcantly less than 8/8
group (N-K p < 0.05). + = signiﬁcantly less that S/Cas group (N-K p < 0.05).
................................................................................................................. 95

Mean (+/- s.e.m.) concentration of WIS-estradiol (E2) in serum of sham
castrate/sham adrenalectomized (S/ S), sham castrate/ sham adrenalectomized
(Cas/S), castrate/3 day adrenalectomized (Cas/Adx3), castrate/14 day
adrenalectomized (Cas/Adx14) B6D2F] males ........................................... 97

Mean (+/- s.e.m.) concentration of dihydrotestosterone (DHT) in serum of
sham castrate/sham adrenalectomized (S/S), sham castrate/ sham
adrenalectomized (Cas/ S), castrate/3 day adrenalectomized (Cas/Adx3),
castrate/l4 day adrenalectorrrized (Cas/Adxl4) B6D2F] males .................. 99

xiv

LIST OF ABBREVIATIONS

AA = aromatase activity

ADX = adrenalectomy

AB = androstenedione

AH = anterior hypothalamus

AM = amygdala

ANOVA = analysis of variance
AOB = accessory olfactory bulb
ATD = 1,4,6-androstatriene-2-17-dione
BC = bulbocavemosus

cmAM = corticomedial amygdala
cont = continuer

DHT = dihydrotestosterone

E2 = 17B-estradiol

ER = estrogen receptor

ERc = cytosolic estrogen receptor
ERn = nuclear estrogen receptor
ERt = total estrogen receptor
HYP = hypothalamus

int = intact

ir = irnmunoreactivity

LOT = lateral olfactory tract

XV

mAM = medial amygdala
MOB = main olfactory bulb
mPON = medial preoptic

MVPC = medioventral pars compacta
nucleus

N-K = Newman-Keuls' multiple range test
none = noncontinuer

POA = preoptic area

RIA = radioimmunoassay

SDN = sexually dimorphic nucleus

SNB = spinal nucleus of the
bulbocavemosus

S-R = steroid hormone-receptor complex
T = testosterone
TP = testosterone propionate

VNO = vomeronasal organ

INTRODUCTION

Expression of copulatory behavior in males of most mammalian species is dependent
upon the presence of gonadal hormones (reviews: Meisel & Sachs, 1994; Sachs & Meisel,
1988). However, the effect of castration on copulatory behavior differs between species as
well as between individuals within a species or strain. For example, some males continue to
achieve an ejaculatory reﬂex for up to a year in dogs, cats, goats, rats, rhesus monkeys and
the B6D2F] hybrid strain of house mouse (Beach, 1970; Clemens, Wee, Weaver, Roy,
Goldman, & Rakerd, 1988; Hart, 1968; Hart, 1975; McGill & Manning, 1976; Phoenix, Slob,
& Goy, 1973; Rosenblatt & Aronson, 1958; Stone, 1927). In contrast, other castrated
individuals within these same species stop copulating within days or weeks after castration.
Why some males continue to copulate after castration and other do not is not understood.
In the case of the B6D2F] house mouse (Mus musculus), most males continue to achieve an
ejaculatory reﬂex for six to twelve months after castration, while others stop ejaculating
within a few weeks after castration (Clemens et al., 1988; McGill & Manning, 1976). In the
present study, genetically homogenous hybrid B6D2F] male mice that continue to copulate
after castration and those that do not were studied to determine if they differ in their steroid
hormone physiology. Therefore, a review of the effects of steroid hormones on copulatory
behavior as well as the effects of steroids on steroid hormone sensitive neural circuits that

regulate copulatory behavior in male mammals will be provided as a background for this set

of experiments.

Effects of Orchidectomy and Steroid Hormones

on Sexual Behaviors

EM gt Qagmion on Copulation

Removal of the testes eliminates the major source of the steroid hormone testosterone
(T). Castration generally reduces or abolishes the expression of male sexual behavior. The
loss of copulatory behaviors aﬁer castration follows a typical pattern, in which ejaculatory
behavior is ﬁrst to cease, followed by intromission, then mount behavior, and ﬁnally
precopulatory behaviors such as ultrasound production (Clemens & Pomerantz, 1981;
Dizinno & Whitney, 1977; Nunez, Nyby, & Whitney, 1978; Sachs & Barﬁeld, 1976).
However, the length of time that copulatory behavior persists after castration as well as the
levels of copulatory behavior expressed after castration vary among species as well as among
individuals of a given species or speciﬁc strain. For example, in the C57BV6J strain of house
mouse, nearly all males stopped exhibiting an ejaculatory reﬂex 7 weeks aﬂer castration, while
up to 80% of the castrated males in the B6DZF 1 hybrid strain of house mouse (derived from
the cross of a DBA/2J male and a C57BV6J female) continued to exhibit an ejaculatory reﬂex
for several months after castration and some continued for over a year after castration
(Clemens et al., 1988; McGill, 1965; McGill & Haynes, 1973; McGill & Manning, 1976;
McGill & Tucker, 1964). DBA/ZJ males, the paternal strain of B6D2F] hybrids, exhibited
a behavioral response to castration that is between C57BV6J and B6D2F] males. DBA/2J

males showed a rapid decline in ejaculatory behavior seven weeks after castration, like C57

3

males, however, a few individuals continued to achieve an ejaculatory reﬂex very sporadically
up to 25 weeks after castration (Clemens et al., 1988).

In these strains of house mouse, the retention of copulatory behavior is under genetic
inﬂuence. The persistance of copulation alter castration in B6D2F 1 males is due to
heterozygosity between loci (genes), and not due to heterozygosity of alleles (genes)(hybrid
vigor). A hybrid of two inbred strains has a new combination of paired alleles. Some of these
alleles remain homozygous, while many are now heterozygous. This new combination of
alleles ultimately affects phenotypes that are the product of multiple gene expression. It has
been demonstrated that the exact recombination the autosomal alleles is not sufﬁcient to
produce persistance of copulation after castration. Males resulting from the reciprocal cross
of the B6D2F 1 parental strains, D2B6F 1, exhibit a loss of copulatory behavior aﬁer castration
that resembled the parental strains (McGill & Manning, 1976). These data indicate that
genetic information on the sex chromosomes interacts with the autosomes to maintain
copulatory behavior aﬁer castration. The importance of the interaction of the autosomal loci
was demonstrated by the production of recombinant inbred strains of B6D2F] house mice,
BXD strains (Coquelin, 1991). Aﬁer systematically inbreeding B6D2F] males over twenty
generations, several new inbred strains of BXD strains were produced, each with a new
combination of ﬁxed homozygous alleles at all loci. When tested for retention of copulatory
behavior after castration, males from two of the six recombinant BXD strains continued to
copulate 5 months after castration (Coquelin, 1991). Thus, the interaction of autosomal and
sex loci determine the maintenance of copulatory behavior after castration in the house
mouse.

Variability in the retention of the ejaculatory reﬂex is seen in a number of other species

4

as well. For example, in dogs (Cm familiiis), some males stop copulating within one to
two months after castration, while other males continue to intrornit and achieve copulatory
lock which is associated with ejaculation ﬁve years after castration (Beach, 1970; Hart, 1968).
This kind of variability has also been observed in goats, rams, and rhesus monkeys (Clegg,

Bearner, & Bermant, 1969; Hart, 1975; Michael & Wilson, 1974; Phoenix et al., 1973).

Castration and the Effects of Androgen and Estrogen Replacement on Male Copulgog
Behavior

 

o ron
In mammals, copulatory behavior may be maintained or restored after castration by
treating males with exogenous testosterone (T): deer mice, (Clemens & Pomerantz, 1981;
Clemens & Pomerantz, 1982), house mouse (Dizinno & Whitney, 1977; Larsson, 1979;
Nunez et al., 1978; Wee, Weaver, & Clemens, 1988), rabbits (Macmillan, Desjardins, Kirton,
& Hafs, 1969), rats (Beach & Holz-Tucker, 1949; Bloch & Davidson, 1968; Whalen &
Luttge, 1971), guinea pigs (Grunt & Young, 1952), gerbils (Yahr, Newman, & Stephens,
1979), hamsters (Whalen & DeBold, 1971), dogs (Beach, 1970), sheep (Clegg et al., 1969),
and rhesus monkeys (Michael & Wilson, 1974; Phoenix et al., 1973). Administration of
smaller doses of testosterone propionate (TP) is required right after castration to maintain
sexual behavior at pre-castration levels compared to the higher doses of TP required to
restore sexual behavior if lost after castration (Davidson, 1972; Yahr et al., 1979).
Individual differences in sexual capacity and responsiveness to T have been
demonstrated. Mthin guinea pigs and rats, individual intact males exhibit varying levels (high

vs low) of sexual behavior (e. g. number of ejaculations per test, latencies or duration of

5

particular behavior etc...)(Grunt & Young, 1952; Larsson, 1966). After castration, high
activity males required fewer T treatments to restore ejaculation than the low activity group
(Larsson, 1966). Further, these differences in intact levels of behavior were not due to
differences in circulating levels of T. Low and high sexually active males given an equivalent
dose of TP after castration exhibited the their respective intact levels of sexual activity after
castration (Grunt & Young, 1952; Larsson, 1966; Whalen, Beach, & Kuehn, 1961).
Additionally, superphysiological doses of TP given to castrated males did not increase their
levels of sexual behavior beyond intact levels (Larsson, 1966; Riss & Young, 1954).
Therefore, these data indicate that intact levels of copulatory behavior are dependent on the
capacity of the individual to exhibit sexual behavior, not on the absolute concentration of T.
However, these individuals demonstrate differences in the threshold levels of hormone
stimulation required to initiation copulatory behavior (responsiveness) to the activation

eﬂ‘ects of hormones.

Mgabglism of Testosterone

Although T restores and maintains copulatory behavior in males, T may not be the
hormone that facilitates behavior. T may be converted into more metabolically active
androgens or estrogens. The metabolism of T to l7B-estradiol (E2) or dihydrotestosterone
(DHT) is regulated by enzymes which are members of the large superfamily of P450
cytochromes of which hundreds of isoforrns are known to occur naturally (Juchau, 1990).
The P450 isoforms that are responsible for the aromatization of C 1 9 steroids to estrogens and
a- and B—reduction of androgens belong to family XI isoforrns. Aromatase is the P450

enzyme that is responsible for converting T and adrostenedione (AE) to E2 and estrone

6

respectively. Aromatase, converts the A-4, 3 ketone-A ring of aromatizable androgens to an
aromatic benzene ring of estrogens. The reductase enzymes are responsible for the
conversion of T and AE to the or- or B-reduced androgens, DHT and androstanedione. These
reduced androgens are formed when T or AE are reduced at the carbon-5 position. The
reduction of T and AB is a nonreversible reaction, and once reduced, these androgens cannot

be aromatized or converted to estrogens.

Eﬂ'ﬂﬁ ef Estregen Ed Reduced Androgen on Copuletory Beh_avior in CastratQ Males

 

Responsiveness to metabolites of T varies from species to species as well as among
strains. For example, E2 treatment restores or maintains copulatory behavior in castrated
males of the following strains of house mice (Mus musculus): CD-1 mice, Swiss-Webster,
B6D2F], but does not restore ejaculatory behavior in deer mice (Peromyscus maniculetus
beigdj) (Clemens & Pomerantz, 1982; Dalterio, Bartke, & Butler, 1979; Edwards & Burge,
1971; Wallis & Luttge, 1975; Wee et al., 1988). E2 also maintains or restores copulatory
behavior in castrated male rats, guinea pigs, and cats (Antliﬂ‘ & Young, 1956; Davidson,
1969; Feder, Naﬁolin, & Ryan, 1974; Green, Clement, & deGroot, 1957; Sodersten, 1973).

DHT is able to restore or maintain copulatory behavior after castration in B6D2F],
and Swiss-Webster strains of house mouse, and the deer mouse (Clemens & Pomerantz,
1981; Clemens & Pomerantz, 1982; Luttge & Hall, 1973; Sinchak & Clemens, 1990; Wallis
& Luttge, 1975). However, DHT treatment is unable to maintain or restore copulatory
behavior in the CD-l strain of house mouse, as well as rats, hamsters, gerbils, pigs or sheep
(Baum, Kingsbury, & Erskine, 1987; Baum & Starr, 1980; Christensen, Coniglio, Paup, &

Clemens, 1973; Feder, 1971; Levis & Ford, 1989; Luttge & Whalen, 1970, Luttge, 1973 #45;

7
McDonald, Beyer, Newton, Brien, Baker, Tan, Sampson, Kitching, Greenhill, & Pritchard,
1970; Parrott, 1986; Sodersten, Eneroth, & Hansson, 1988; Whalen & Luttge, 1971; Yahr
& Stephens, 1987).

Although treatment of castrate males with either androgens or estrogens alone may
maintain or restore behavior, it is likely that both estrogenic and androgenic stimulation
facilitate copulatory behavior in mice as well as other species. For example, E2 and DHT act
in a synergistic manner to activate intact levels of copulatory behavior in castrated males of
the CD] strain of house mouse (Wallis & Luttge, 1975). Further, in castrated deer mice that
respond to DHT alone, blocking the conversion of T to E2 or DHT reduced the effectiveness
of exogenous T to activate copulatory behavior (Clemens & Pomerantz, 1981; Clemens &
Pomerantz, 1982). The synergism of androgens and estrogens in activating copulatory
behavior has also been demonstrated in the rats and hamsters (Baum & Vreeburg, 1973;
DeBold & Clemens, 1978; Larsson, Sodersten, & Beyer, 1973b). Although E2 treatment
restores copulatory behavior in castrated male rats, if castrated males are adrenalectomized,
the same dose of E2 is unable to restore copulatory behavior (Gorzalka, Rezek, & Whalen,
1975). However, if these same male are then treated with TP, ejaculatory behavior is restored
which suggests that adrenal androgens may facilitate copulation (Gorzalka et al., 1975).
Further evidence of synergism was demonstrated in castrated male rats given subthreshold
doses of E2 that did not facilitate copulation. When DHT was implanted discretely in the
lateral septum or medial amygdala, these E2 treated males started copulating (Baurrr, Tobet,

Starr, & Bradshaw, 1982).

8

Ammatieetieg ef Androgens to Estrogen and Behavior

 

The “aromatization hypothesis” for activation of copulatory behavior was formulated
ﬂour a combination of studies that demonstrated that l) aromatizable androgens were more
eﬂ‘ective than nonaromatizable androgens in activating copulatory behavior in castrated males
of several species (Beyer, Larsson, Perez-Palacios, & Morali, 1973; Beyer & Rivaud, 1973;
Luttge & Hall, 1973; McDonald et al., 1970; Whalen & Luttge, 1971), and 2) E2 either
restores copulatory behavior or facilitates the effects of DHT or other nonaromatizable
androgens to activate copulatory behavior in a number of species (see above). The basic
premise was the aromatization of androgens (T) to estrogens (52) is required for the
activation of copulatory behavior. This aromatization hypothesis was supported by numerous
studies in several species that showed the ability of T to restore copulatory behavior in
castrates could be inhibited by either blocking its aromatization to E2 with systemic aromatase
inhibitors or by blocking estrogenic stimulation with an estrogen receptor antagonist
(Alexandre & Balthazart, 1986; Beyer, Morali, Naﬁolin, Larsson, & Perez-Palacios, 1976;
Carroll, Weaver, & Baum, 1988; Christensen & Clemens, 1975; Floody & Petropoulos, 1987;
Luttge, 1975; Morali, Larsson, & Beyer, 1977).

In summary, in most male mammals, castration causes a reduction in sexual activity
by a reduction in gonadal steroid hormones (mainly T). Sexual behaviors may be restored in
these males by replacing gonadal hormones. Although T is the most eﬂ‘ective in restoring
copulatory behavior to castrate males, its metabolites E2, and DHT appear to be the
metabolites that promote copulation. Alone, E2 restores copulatory behavior in castrate
males of numerous species, however, concurrent administration of DHT and E2 works better

to restore copulatory behavior to intact levels. The fact that copulation in some males is

9

dependent on the presence of gonadal hormones while it is not in others suggests two possible
reasons for the diﬁ‘erent eﬂ‘ects of castration on copulatory behavior among individuals. One
possibility is that continued copulation in castrated males is independent of steroid hormone
facilitation. The other possibility is that castrated males that copulate are more responsive to

circulating steroid hormones that are present after castration.

Eﬁ‘eete ef eastration on steroid hormone levels

 

Castration reduces but does not eliminate T from systemic circulation. Although this
general proﬁle occurs in all species studied to date, relative levels of nongonadal steroids vary
among species. For example, T concentrations in castrates vary from 0.45% (rats) to nearly
12.3% (hamster) of intact levels (Table 1). In the B6D2Fl male mice, castration reduced T
levels to approximately 10% of intact levels (Clemens et al., 1988).

In contrast, for some mammalian species, E2 levels are not affected by castration
while in others they are slightly reduced. For example, E2 concentrations after castration vary
from 26.2% (horse) to 156.1% (ferret) of intact levels (Table 2). This differential effect of
castration on T and E2 levels has been observed in rats, rhesus monkeys, and ferrets
indicating that serum E2 is maintained by an unknown nongonadal source (Carroll et al.,
1988; Roselli & Resko, 1984; West, Roselli, Resko, Greene, & Brenner, 1988).

However, levels of nongonadal E2 or T do not correlate with the maintenance of
copulatory behavior aﬁer castration among species. For example, castrated hamsters have
the highest relative level of T compared to intact males, and castrated rats maintain E2
equivalent to intaets, yet both species stop copulating shortly after castration, while males of

other species with less relative T and/or E2 levels continue to copulate.

10

Nonaheless, the fact that steroid hormones are available after castration suggests that
it is possible that continued copulation after castration is dependent on these nongonadal
steroids. Although T levels are reduced, it is still available to provide androgenic stimulation,
as well as being a substrate for aromatization to E2. Furthermore, the fact that endogenous
E2 levels are less affected by castration (Table 2) than T (Table 1), support the idea that
species whose copulatory behavior is dependent upon estrogenic stimulation would be less

likely to show an eﬂ‘ect of castration on copulation than those species that require androgens.

Sperm efNongonedal Steroids

Nongonadal steroids may originate from several sources. One possible source is the
adrenal gland. However, since copulation often continues after castration and adrenalectomy
in a number of species (Cooper & Aronson, 1958; Schwartz & Beach, 1954; Thompson,
McGill, McIntosh, & Manning, 1976), other sources may also exist if this behavior is
hormone dependent.

Another potential source of nongonadal androgens and estrogens is the brain. A
considerable amount of steroid metabolism takes place in the brain. Local tissue metabolism

(reviewed later) as well as the possibility of _d_e novo synthesis of steroid from cholesterol are

 

potential mechanisms by which the brain could synthesize metabolically active steroids that
affect behavior. Steroids produced by d_e me synthesis or by i_n_ site metabolism of
circulating steroid are referred to as neurosteroids (Baulieu, 1981). Three of the four
enzymes needed for the metabolism of cholesterol to estrogen have been demonstrated in the
adult rat brain (Corpechot, Rebel, Axelson, Sjovall, & Baulieu, 1981; Le Goascogne, Rebel,

Gouezou, Sananes, Baulieu, & Waterman, 1987; Rebel & Baulieu, 1995). While the fourth,

Table 1

Mean (+/- s.e.m.) circulating levels of testosterone (T) (ng/ml) in intact and castrated males
of several mammalian species, and the levels circulating T in the castrate as a percent of intact
T levels.

 

 

Species Intact Castrate % of Intact
B6D2F1 Mouse 1.63 (0_20)2 0.18 (0.9) 11.0
Rat 3.80 (1.10)7 0.070 (0.030) 1.8
2.67 (0.19)5 0.012 (.0018) 0.45
3.80 (036)21 0.030 (0.020) 0.79
Ferret 7.01 (2.67)1 0.030 (0.010) 0.43
2.07 (1.58)6 0.042 (0.023) 2.03
Rhesus Monkey 5.00 (0.80)4 0.30 (0.10) 6.0
17.00 (1.50)4 0.10 (0.10) 0.58
4.32 (1.20)10 0.31 (0.04) 7.18
Horse 2.10 (0.10)3 0.20 (0.03) 9.5
Hamster”) 4.30 (0.90)9 0.53 (0.20) 12.3

 

N_e_te. LDHamsters housed in long day light cycle (14 hour light/10 hour dark).
1Carroll, et al., 1988; 2Clemens, et al., 1988; 3Ganjam, et al., 1975; 4Goodman, Hotchkiss,
Karsch & Knobil, 1974; 5Handa, Reid & Resko, 1986i;S Kastener & Apfelbach, 1987;

7Roselli, et al., 1984; 8Roselli, et al., 1993; 9Sisk & Turek, 1983; 10West, et al., 1988.

12

Table 2

Mean (+/- s.e.m.) circulating levels of estradiol (E2) (pg/ml) in intact and castrated males of
several mammalian species, and the levels circulating E2 in the castrate as a percent of intact
E2 levels.

 

 

Species Intact Castrate % of Intact
Rat 36.7 (2.3)7 39.0 (2.5) 106.1
3.9 (1.4)8 2.6 (1.1) 66.7
Ferret 8.2 (2.6)1 12.1 (7.2) 156.1
Rhesus Monkey 19.0 (6.0)10 14.0 (4.0) 73.7
Horse 43.9 (2.3)3 11.5 (2.0) 26.2

 

N_Qte. lCarrell, et al., 1988; 3Ganjam & Kenney, 1975; 7Roselli, et al., 1984; 8Roselli, et al.,
1993; 10West, et al., 1988.

13
17a-hydroxylase (P450170) with 17,20-desmolase activity, which converts pregnenelone to
dehydroepiandrosterone (DHEA) has not been demonstrated in mammalian brain, there is
circumstantial evidence for its presence: DHEA levels maintain a circadian rhythm and are
higher in the brain of castrated, adrenalectomized rats than in the plasma, suggesting DHEA
may be synthesized in the brain (Corpechot, et al., 1981; Rebel, et al., 1987).

Because steroid hormones interact on so many levels within the body to organize and
regulate important metabolic and behavioral functions, it is not surprising that steroid
synthesis takes place at multiple sites. While gonadal synthesis of steroid hormones and
copulatory behavior are associated in mammals, it is possible that multiple sources of steroid

hormone may inﬂuence copulatory behavior.

MCircuits Importent for Male Copulatory Behevior: Their Meteboliem of. m
Responeiveness to Steroid Hormones

In the central nervous system (CNS) several neural pathways are involved in the
regulation of reproductive behavior. The best understood mechanism by which steroid
hormones exert their inﬂuence on copulatory behavior is through regulating genomic activity
of these neurons that contain steroid receptors in these pathways. As previously mentioned,
the brain is able to synthesize and metabolize steroid hormones. Much of this activity is
located in brain regions within the neural circuits that regulate copulation. The following
section will focus on and review the steroid sensitive neural pathways of the limbic system
that are involved in the regulation of male copulatory behavior, and how steroid hormones
regulate copulatory behavior and steroid physiology (receptor mechanisms and metabolism

of steroids) within the CNS. Since the studies performed in the dissertation investigate the

l4

role of estrogen in maintenance of copulatory behavior in male B6D2F] males after
castration, emphasis will be place on the role of estrogen.

Both external cues (e.g. female odor, or behavioral cues) as well as internal cues (e. g.
hormonal or dietary status) that modulate the sexual motivation appear to converge in the
medial preoptic area (mPOA). In turn, the mPOA modulates three types of behavioral
responses: 1) Appetitive or sexual motivation, 2) Somatomotor regulating copulation, and
3) genital reﬂexes. In general, each of these behavioral responses is regulated by a ”series”
of brain regions that comprise a neural circuit that receives, integrates, and relays information
11m eventually results in behavioral output. The activity of nearly every brain region involved
in these circuits is either directly or indirectly inﬂuenced by steroid hormones. The following
section will ﬁrst focus on the behavioral neural circuits in the limbic system that regulate male
sexual behavior, and then review the steroid hormone physiology of brain regions that

comprise these circuits.

Chmemgry Pathways

Olfgtog Bulbsend Copulatory Beh_avior

In rodents, he olfactory bulbs are the most rostral portions of the brain. There are two
anatomically distinct regions to the olfactory bulb: the main olfactory bulb (MOB) and the
accessory olfactory bulb (AOB). Each region receives and integrates different types of
olfactory information. The MOB receives airborne chemical information that stimulate
olfactory neurons located in the nasal mucosa of the nasal cavity. The AOB receives and
integrates cherrrical stimuli that are taken into the oral cavity and reach the vomeronasal organ

(VNO) through the nasopalatine ducts in the roof of the mouth. During precopulatory ano-

15

genital investigation, the male picks up chemical cues ﬁom the female that stimulate both
olfactory systems. These chemical cues reveal the reproductive status of the female and aﬁ‘ect
the sexual arousal state and motivation of the male. Sensations from olfactory and
vomeronasal organ (VNO) receptors send chemosensory information to the MOB and AOB
respectively via axons that project through the cribiforrn plate of the ethmoid bone and into
the olfactory bulbs. These olfactory and VNO projections synapse with dendrites of mitral
and tuﬂed cells in the glomerular layer of the MOB and AOB respectively (Barber &
Raisman, 1974). There is evidence that some integrative function occurs in the olfactory
bulbs, since chemosensory information is supplemented by trigeminal afferent information and
possibly by projections of the nervus temrinalis (Silver, 1987; Wirsig & Leonard, 1986).
Bilateral removal of olfactory bulbs abolished expression of male copulatory behavior
in mice and hamsters (Devor, 1973; Doty, Carter, & Clemens, 1971; Edwards & Burge,
1973; Lisk, Zeiss, & Ciaceio, 1972; Murphy, 1980; Murphy & Schneider, 1970; Rowe &
Edwards, 1972; Winans & Powers, 1974). However, vomeronasal information from AOB
appears to be more important for the expression of copulatory behavior than olfactory stimuli
of the MOB. For example, peripherally induced anosmia that eliminates olfactory stimuli to
the MOB, but not to AOB, does not affect expression of male copulatory behavior in Swiss-
Webster mice (Edwards & Burge, 1973; Rowe & Edwards, 1972). Whereas, removal of the
VNO causes a decrease in the percent of males that achieve intronrission and ejaculate
(Clancy, Coquelin, Macrides, Gorski, & Noble, 1984). Eliminating chemosensory input to
the MOB in the hamster by irrigating the nasal cavity with zinc sulfate, reduced copulatory
behavior in some studies but not in others (Devor, 1973; Lisk et al., 1972; O'Connell &

Meredith, 1984; Powers, Fields, & Winans, 1979; Powers & Winans, 1975; Winans &

16

Powers, 197 7). Likewise, removal of chemosensory input to the AOB by cutting the
vomeronasal nerve or removing the VNO affects copulation in less than 50% of the animals
(Meredith, 1986;0'Conne11 & Meredith, 1984; Powers et al., 1979; Powers & Winans, 1975;
Winans & Powers, 1977). In the male rat, olfactory bulbectomy mainly produces a reduction
in the percent of males that achieve ejaculation (Larsson, 1969; Larsson, 1975; Lumia,
Zebrowski, & McGinnis, 1987; Meisel, Lumia, & Sachs, 1982; Meisel, Lumia, & Sachs,
1986). Evidence in rats suggests that these olfactory cues are important in identifying the
female’s reproductive state and stimulating sexual arousal of the male. For example, male rats
that continue to copulate after olfactory bulbectomy, no longer exhibit a preference of an
estrous female over a nonestrus female. Moreover, these males take longer to achieve

intromission (intromission latency (11.)), which is suggestive that sexual arousal may be

reduced (Edwards, Grifﬁs, & Tardival, 1990).

Projegions of the Main and Accessory Olfectory Bulbs

In the male hamster, MOB and AOB efferents project to corticomedial nuclei of the
amygdaloid complex via separate ﬁber bundles within the lateral olfactory tract (LOT) (Scalia
& Winans, 1975). Efferent ﬁbers of the MOB carry olfactory information to the "olfactory
amygdala" and the AOB etferents carry VNO information to the medial amygdaloid group
or "vomeronasal amygdala" (Scalia & Winans, 1975). In tunr, each of these nuclei, projects
to the bed nucleus of the stria terminalis (BNST) and to the medial preoptic area/anterior

hypothalamus (POA-AH) via the stria terminalis (Winans, Lehman, & Powers, 1982).

Amygdele (M)

The anatomy, projections, and behavioral properties of amygdala have not been
worked out in the mouse. Therefore, most of the data presented will be from other closely
related rodents, the rat and hamster, which demonstrate the roles of diﬁ‘erent regions of the
amygdala in chemosensory regulation of motivation (arousal) and facilitation of copulatory
behavior.

The amygdala can be divided into ﬁve major anatomical nuclei: cortical amygdala
(olfactory), medial amygdala, basemedial amygdala, lateral amygdala and a central amygdala
(De Olrnos, Alheid, & Beltramino, 1985). Two regions of the amygdala have been
investigated as to their roles in expression of male copulatory behavior: basolateral
amygdaloid nuclei (basemedial and lateral nuclei) and corticomedial amygdaloid nuclei
(cortical and medial amygdala nuclei).

The basolateral amygdala appears to be part of a neural circuit that is involved in
regulation of chemoinvestigative behavior which appears to affect sexual motivation but not
the ability to copulate. For example, male rats and hamsters with lesions of the basolateral
amygdala cepulate if presented with a female (Harris & Sachs, 1975; Kevetter & Winans,
1981; Lehman, Winans, & Powers, 1980). However, male rats that have been trained to
associate the secondary stimulus of bar pressing to gain access to a receptive female, will not
bar press to gain access to an estrous female if a lesion has been placed in the basolateral
amygdala (Everitt, Cador, & Robbins, 1987). Thus, sexual motivation that is stimulated by
primary cues (female odor, vocalizations, or behavior) is not disrupted by these lesion, but
association of secondary stimuli for facilitating sexual motivation is impaired (Everitt et al.,

1987). It appears that the amygdala plays a role in regulating the motivational states of a

18

number of other behaviors including aggressive, fear and ingestive behaviors (reviewed
Everitt, 1989; Everitt et al., 1987).

In contrast, lesions of the corticomedial amygdala cause varying amounts of deﬁcits
in expression of copulatory behavior or motor output, and sexual motivation or arousal in rats
and hamsters. For example, in hamsters, lesions in the rostral region of the corticomedial
amygdala elimimte both chemoinvestigatory and copulatory behavior, whereas lesions in the
caudal corticomedial amygdala produce variable and more short-lived effects on expression
of copulatory behavior (Lehman & Winans, 1982; Lehman et al., 1980). Caudal lesions
increased mount latency and ejaculatory latency shortly after surgery, however, these latencies
rearrned to control levels three weeks after surgery. In rats, corticomedial amygdala lesions
increased the ejaculatory latency and decreased the number of ejaculations achieved by rats
(Giotonio, Lund, & Gerall, 1970). While another study suggested that the effects of these
lesions were actually more subtle in that copulatory deﬁcits only arose in male rats if the
stimulus female was only primed with estrogen versus a female primed with estrogen and

progesterone (Perkins, Perkins, & Hitt, 1973).

Pr jgions of the Amygdala

In the hamster, the caudal corticomedial amygdala, which receives information ﬁom
the AOB, sends projections via the stria terminalis to the medial preoptic area of the
hypothalamus (mPOA) (Kevetter & Winans, 1981). The rostral corticomedial amygdala,
which receives alferents ﬁom the MOB, projects efferents via the ventral ﬁber pathway (VP)
to the caudal medial bed nucleus of the stria terminalis (BNST) which then sends efferents to

the mPOA (Lehman, Powers, & Winans, 1983).

19

Strie Terminelis egd Bed Nucleus of the Stria Terrninalis (BNST)

Lesions in either the BNST or stria terminalis in the male rat produce increased
ejaculatory latencies as seen with corticomedial amygdala (cmAM) lesions, as well as
increased number of intronrissions to ejaculation, and interintromission intervals (Emery &
Sachs, 1976; Giotonio et al., 1970; Paxinos, 1976; Valcourt & Sachs, 1979).

In the hamster, the caudal portion of the cmAM projects directly to the medial
preoptic area/anterior hypothalamus (mPOA-AH) via the stria terminalis (Kevetter & Winans,
1981). Cutting the stria terminalis caused temporary deﬁcits as seen with caudal cmAM
lesions (Lehman et al., 1983). In contrast, the rostral portion of the cmAM projects via the
VP to the caudal medial BNST, which in turn projects to the mPOA-AH (Lehman et al.,
1983). Cutting the ventral ﬁber pathway caused severe deﬁcits in copulatory behavior similar
to rostral cmAM lesions, while cutting both stria terminalis and ventral ﬁber pathway
eliminated copulatory behavior in hamsters (Lehman et al., 1983). Further, these pathways
have been shown to be selectively activated by sexually relevant olfactory stimuli. For
example, expression of the immediate-early gene eﬁs (which increases in some cells when
stimulated) was increased in the POA and mAM when male rats were exposed to stimuli
associated with reproduction (Baum & Everitt, 1992). By unilaterally cutting pathways of
olfactory and vomeronasal information received by mAM, induction e—Qe by sexually relevant
information in the ipsilateral POA was attenuated (Baum & Everitt, 1992; Krettek & Price,
1978)

Thus, it appears that the BNST and stria terminalis do very little processing of sexual
relevant olfactory information. These regions appear to act mainly as a relays of information,

since lesions and knife cuts in these regions produce basically the same effects that lesions of

20

their respective nuclei in the amygdala produce in the rat and hamster (Emery & Sachs, 1976;

Lehman et al., 1983; Lehman & Winans, 1983; Paxinos, 1976; Valcourt & Sachs, 1979).

Maia] Prmptie Area md Hypothalamus

Lesions of the mPOA-AH cause major deﬁcits in male copulatory behavior in all
mammalian species studied thus far which includes mice (Meisel & Sachs, 1994). The roles
of the mPOA are: 1) integrate aﬁ‘erent information; 2) affect the transition from
precopulatory behavior (e. g. sexual arousal behaviors, female investigation, ultrasound
production) to initiation of copulatory behavior (mount, intromission, and ejaculatory
behaviors); 3) modulate arousal state of the male 4) regulate penile reﬂexes

The mPOA receives information indirectly ﬁ'om almost all sensory systems of the
animal via limbic and brainstem sites: 1) olfactory input via the medial amygdala and bed
nucleus of the stria terminalis, 2) auditory, visual and somatosensory input from the neocortex
via the ventral subiculum and lateral septum, 3) visceral and genital input from the central
tegrnental ﬁeld, nucleus of the solitary tract, and the A1 noradrenergic region of the brain
stem (Sirnerly & Swanson, 1986). The mPOA also has reciprocal connections with all these
regions, and therefore may modulate sexually relevant information coming ﬁ’om these regions.

A major role of the mPOA is to initiate copulatory behavior to sexually relevant
stimuli. For example, in the Swiss-Webster strain of house mouse, lesions of the mPOA
eliminated intromissions, and ejaculations in all males tested, and mounting in all but one male
was eliminated (Bean, Nunez, & Conner, 1980). In contrast, arousal and motivation was far
less aﬁ‘ected. For example, the proportion of males displaying ano-genital investigation was

not affected by mPOA lesions, but the latency to start ano-genital investigation was increased

21

(Bean et al., 1980). Furthermore, ultrasound production by the male to a stimulus female or
bedding soiled by female urine was not disrupted by mPOA lesions. These same effects on
sexual behaviors were seen in B6D2F] male mice by blocking protein synthesis in the POA
with cyclohexamine (Quadagno, Albelda, McGill, & Kaplan, 1976). Males that received
implants of cycloheximide in the POA still investigated the genital region of the female, but
signiﬁcantly fewer of these males mounted, gained introrrrission and ejaculated.

In other species, more discrete lesions reveal that POA-AH may regulate several
measures of copulatory behavior. In the rat, dorsal parastriatal lesions of the POA decrease
the percent of male that ejaculated (Arendash & Gorski, 1983). Further, dorsal and ventral
lesions each eliminated ejaculation, while ventral lesions disrupted initiation of copulation
(Kondo, Shineds, Yamanouchi, & Arai, 1990). In some species, small lesions in the POA
eliminate copulatory behavior. For example, in the hamster, lesions of the region of the POA
that receives caudal medial amygdala efferents eliminate copulation (Powers, Newman, &
Bergondy, 1987). Further, in the gerbil small lesions that eliminate the sexually dimorphic
nucleus (SDN) (a nucleus of cells that is larger and more darkly staining in males than it is in
females) also eliminates copulatory behavior (Commins & Yahr, 1985b). In contrast, lesions
of the SDN in the rat do not affect expression of copulatory behavior unless the rat was
inexperienced (de Jonge, Louwerse, Ooms, Evers, Endert, & van de Poll, 1989).

Evidence that the POA plays a role in initiating and directing sexual behavior to female
comes from studies with rats and monkeys where POA lesions eliminated copulation. Males
with these POA lesions still engage in precopulatory behaviors (e. g. investigate, pursue and
climb over females) and sometimes mount females (Hansen & Drake af Hagelsrunr, 1984;

Heinrer & Larsson, 1966/67; Meisel, 1983). Further, both male rats and rhesus monkeys with

22

POA lesions continue to bar press to gain access to an estrous female, but exhibit very little
female directed sexual contact. Further, rhesus monkeys with POA lesions continue to
masturbate which means sexual motivation, motor output and reward pathways are still
ﬁrnctional, but no longer directed towards the female. Thus, the mPOA appears to regulate
initiation of female directed copulatory behavior.

There is also evidence to suggest the mPOA is part of neural circuits that regulate
penile reﬂexes and seminal emission, and that dopaminergic innervation of the mPOA
regulates these responses (Bazzett, Eaton, Thompson, Markowski, Lunrley, & Hull, 1991;
Hull, 1995; Hull, Eaton, Markowski, Moses, Lumley, & Joucks, 1992). Projections ﬁ'om the
mPOA appear to facilitate penile reﬂexes, since stimulation of the POA increased penile
reﬂexes (Hughes, Everitt, Lightman, & Todd, 1987). Furthermore, lesions in regions that
mPOA project to also inﬂuence copulatory behavior and penile reﬂexes (paraventricular
nucleus of the hypothalamus (PVN), median raphe, and nucleus paragigantocellularis) (Chiba
& Murata, 1985; Marson & McKenna, 1990; Marson, Platt, & McKenna, 1993; Monaghan,
Arjomand, & Breedlove, 1993; Monaghan & Breedlove, 1991; Yells, Hendricks, &
Prendergast, 1992).

The projections of the mPOA have not been delineated in the mouse, therefore, most
of the infemratien presented will be from the rat. The mPOA projects to numerous regions,
and appears to be able to modulate or regulate motivational, arousal, consummatory
behaviors and penile reﬂexes.

Most regions that project to mPOA receive reciprocal projections ﬁ'om the mPOA.
Therefore, olfactory information important for motivation and arousal coming from these

rostral brain regions may be modulated by these reciprocal efferents of the mPOA.

23

A main projection from the mPOA appears to regulate the initiation of copulatory
motor patterns as discussed above. This efferent projection of the mPOA is to the midbrain
region via the medial forebrain bundle (MFB) (Swanson, 1976). This efferent pathway
appears to project laterally from the mPOA to join the MFB and then project caudally to the
midbrain, since only knife cuts lateral to the anOA and lesions of the MFB caudal to the
mPOA disrupt copulatory behavior (Caggiula, Antelman, & Zigrnond, 1974; Hendricks &
Scheetz, 1973; Scouten, Burrell, Palmer, & Cegavske, 1980; Szechtman, Caggiula, &
Wulkan, 1978).

Another projection of the mPOA regulates penile reﬂexes and possibly seminal
emission This mPOA efferent projects to the PVN. In turn, the PVN projects to a group of
motoneurons in the lumbesacral region of the spinal cord, the spinal nucleus of the
bulbocavemosus (SNB) (Wagner & Clemens, 1991). This circuit from the mPOA to PVN
to SNB appears to regulate penile reﬂexes (Argiolas, Melis, & Gessa, 1987; Bitran, Hull,
Holmes, & Lookingland, 1988; Bjorklund, Lindvall, & Nevin, 1975; Hull, Bitran, Pehek,

Warner, Band, & Holmes, 1986).

Regiens ef the Brain that Concentrate Steroid Hormones end the Effects of Intrecerebrej
Mime ef Steroid Hormones

Lesions studies in conjunction with tract tracing studies have aided in elucidating
distinct neural circuits that regulate copulatory behavior. However, these studies do not

reveal which horrnenes are acting at which particular sites to facilitate copulatory behavior.

24

Brm'n regions thet concentrate androgens

In the rat, neurons which concentrate T or DHT were determined by autoradiography
(Sar & Stumpf, 1975). Both T and DHT produced similar patterns of accumulation.
Androgens were accumulated in most of the brain regions known to be involved in copulatory
behavior. In the telencephalon, T is accumulated in the olfactory bulbs, amygdala, with
heaviest accumulation in the medial amygdala. In the diencephalon, heavy accumulations of
androgens were seen in the mPOA, BNST, PVN, and lateral POA, and lighter accumulation
in the anterior hypothalamus. In the mouse, although the olfactory bulbs were not were not
sampled, the pattern of androgen accumulating cells in the telencephalon and diencephalon
was similar to the rat (Luttge, 1975; Sheridan, 1978; Sheridan, Howard, & Gandelman,
1982). Additionally, there are numerous other regions that are not associated with copulatory
behavior that also concentrate androgens in the telencephalon and diencephalon in both the
mice and rats.

In the rat, androgen concentrating cells were also found throughout the midbrain and
brainstem of the rat. These neurons were located in the central gray nucleus, tegrnental
nuclei, rnagnocellular reticular nuclei, and pontine nuclei (Sar & Stumpf, 1975; Sar & Stumpf,
1977). In addition, motor nuclei of the spinal cord (SNB and DLN) that are important for

copulation concentrate androgens (Breedlove & Arnold, 1983).

Brgjn Regions that Concentrate Estrogens
In rrrice and rats, estrogen concentrating cells are located from the most rostral regions
of the telencephalon to the brainstem (Sheridan, 1978; Stumpf & Sar, 1974/1975; Stumpf,

Sar, & Keefer, 1974/1975). The regions that concentrate the most estrogen are also areas

25
that are important for regulating copulatory behavior. For example, the olfactory bulb, medial
and cortical AM, BNST, lateral septum, mPOA, periventricular POA, anterior hypothalamus,
diagonal band of Broea, VMH, dorsal raphe, and dorsomedial nucleus gigantocellularis

contain cells that concentrate estrogen.

Eﬂege ef T microimplants

Microimplants of crystalline T in the mPOA (which likely stimulate BNST also) are
the most effective stimulating copulatory behavior in rats, hamsters, and ferrets (Davidson,
1966; Tang & Sisk, 1991; Wood & Newman, 1993b). Wood and Newman (1993) were able
to pinpoint with greater accuracy to regions of the brain that were being stimulated by their
T microimplants by observing androgen receptor (AR) irnmunoreactivity (ir) in the area
around the implants. AR-ir in castrate males is very weak compared to intact males, however,
where the T had diffused from the implant and activated cells, AR-ir was as robust as intact
males. By this method, they were able to determine that implants of T in the AM that were
most eﬂ‘ective in facilitating copulatory behavior were those that increased irnmunoreactivity
in the dorsal medial nucleus of AM near the optic tract. It should be noted that sexual
behavior may be reinstated by the microimplants, however, it is not restored to the level of
the intact, in part due to the lack of peripheral steroid hormone stimulation (Wood &
Newman, 1993b).

Although T implants elucidate the regions of the brain that are targets for the
behavioral effects of steroids, they do not reveal whether androgenic or estrogenic stimulation
is responsible for the behavioral effects. Implant studies using DHT and E2 demonstrate that

the responsiveness to androgenic and estrogenic facilitation of copulatory behavior is site

26

speciﬁc. E2, but not DHT implants are effective in facilitating copulatory behavior in the
mPOA or rats, and hamsters (Baum et al., 1982; Christensen & Clemens, 1974; Christensen
& Clemens, 1975; Lisk & Greenwald, 1983). In contrast, DHT appears to be effective at
stimulating copulatory behavior when implanted into the AM and lateral septum (Baum et al.,
1982). However, E2 also facilitates copulation when implanted in the AM (Rasia-Filho,

Peres, Cubilla-Gutierres, & Lucien, 1991).

Nthet etematase ectivig end copulatory behevior

Since T or E2 implants in the POA facilitate copulatory in male rats, and the ability

 

of the anterior hypothalarnic region of the brain to aromatize androgens to estrogens was
established, it was suggested that the local aromatization of the T to E2 was responsible for
the activation of copulatory behavior (Naftolin, Ryan, & Petro, 1972).

Christensen and Clemens (1975) demonstrated that local aromatization of T to E2 in
the POA was necessary to activate mounting behavior in castrated male rats. Either T or E2
implanted directly into the POA-AH restores mount behavior in male rats that have stopped
copulating. However, if 1,4,6-androstatriene-2-l7-dione (ATD) (an aromatase inhibitor) is
implanted 20 minutes prior to implantation of T, the facilitatory effects of T are blocked,
however, ATD does not block the facilitatory effects E2 implants. Thus, it appears that
estrogenic stimulation derived from the local aromatization of T to E2 within the POA-AH

is necessary for expression of copulatory behavior.

Neeﬂ emmeteg ectivig and the effects of castration

The ability to aromatize androgens to estrogens in discrete brain regions suggests that

27

estrogen concentrations within these regions may be independent of circulating levels of
estrogens. Therefore, these local estrogen concentrations would be dependent on the amount
of available arbstrate (aromatizable androgens) and/or the activity or quantity of the enzymes
involved in the reaction.

In mammals, localization of brain regions that aromatized androgens to estrogens has
been determined by dissecting discrete brain regions and measuring the rate of conversion of
AE to estrone by radioassay. Aromatase activity was measured in regions of the brain that
are associated with copulation, however, AA varies among these brain regions in the
hypothalamus (HYP) and limbic systems (Roselli, Horton, & Resko, 1985; Selmonoff,
Brodkin, Weiner, & Liiteri, 1977). Within the limbic system, the BNST had the greatest
levels of AA followed the mAM and cAM. Neither the lateral septum nor the medial septum
had rrreasurable levels of AA In the HYP, periventricular preoptic nucleus and POA had the
greatest levels of AA Intermediate levels of AA were measured in the anterior hypothalamus,
periventricular anterior hypothalamus, VMH, and SCN. Low levels of AA were measured
in lateral hypothalamus, dorsomedial nucleus and arcuate-median eminence region.

Other methods have been employed to localize aromatase and its activity. The use
of antibodies to localize aromatase by immunohistochemistry has worked well in conﬁrming
localization AA in nonmammalian species, however, in mammals,
it has not been effective (Balthazart, Foidart, Surlernont, & Harada, 1991; Balthazart, Foidarr,
Surlernont, Vockel, & Harada, 1990; Sanghera, Simpson, MePhaul, Kozlowski, Conley, &
Lephart, 1991; Shinoda, Kideo, Hisao, Soawa, & Shiotani, 1989). For example, aromatase
irnmrrnoreactivity is absent or extremely sparse in the POA and BNST of mice and rats where

AA has been demonstrated by other methods (Roselli, Ellinwood, & Resko, 1984; Roselli et

28

al., 1985; Roselli & Resko, 1984; Schleicher, Stumpf, Drews, & Sar, 1986a; Schleicher,
Stumpf, Morin, & Drews, 1986b; Sheridan, 1978).

There are two distinct categories of AA. One type is regulated by gonadal hormones,
while the other is not inﬂuenced by gonadal hormones (Callard, Mak, & Solomon, 1986;
Roddy, Naftolin, & Ryan, 1973; Roselli et al., 1984; Roselli et al., 1985; Roselli, Horton, &
Resko, 1987a; Roselli & Resko, 1984; Roselli & Resko, 1986; Roselli & Resko, 1989;
Roselli, Salisbury, & Resko, 1987b). For example, in the rabbit, castration increased AA in
block dissections of the HYP and amygdala and anterior hippocampus (Reddy et al., 1973).
However, in the male rat, when AA was measured by taking more discrete dissections of the
brain, levels of AA are reduced in the BNST, mAM, periventricular preoptic nucleus, medial
preoptic nucleus, VMH, anterior hypothalamus and suprachiasmatic nucleus following
castration (Roselli et al., 1985). In contrast, AA was not inﬂuenced by castration or T
replacement in the cortical amygdala, periventricular nucleus of the anterior hypothalamus,
arcuate nucleus/median eminence region, lateral preoptic nucleus, and dorsal medial nucleus.
Tlars, potential for aromatization of androgens to estrogen in speciﬁc brain nuclei exists after
castration.

There is also evidence that regulation and distribution of neural AA in the adult ferret
is determined by organizational effects of steroid hormones on the fetal brain in em
(Krohmer & Baum, 1989). Organizational effects of steroids are also responsible for
inﬂuencing levels of sexual and aggressive behavior in adult mice which are activated by
estrogenic stimulation (vom Saal, Grant, McMullen, & Laves, 1983). Therefore, since
castration does not eliminate aromatizable androgens, differences among individuals that

continue to copulate after castration and those that do not may be related to levels of AA

29

activity in brain regions important for copulation.

Egregen Receptors

One way in which steroid hormones alter cellular activity (and behavior) is via
activation of receptors classiﬁed as ligand activated transcription factors that regulate
genomic activity (Evans, 1988). For example, E2 binds to an estrogen receptor (ER) forming
a steroid-receptor complex (S-R). Two S-R's dimerize and then attach to speciﬁc gene
regulatory binding sites on the DNA within the nucleus where they alter the gene's activity.
This in turn, leads to a change in protein synthesis and presumably behavior. Since estradiol
restores and maintains sexual behavior in castrated males of a number of species and the
effects of can be blocked in castrated male by ER antagonists, activation of ER’s most likely
play a role in male copulatory behavior (Callard et al., 1986; Carroll et al., 1988; Christensen

& Clemens, 1974; Christensen & Clemens, 1975; Gorzalka et al., 1975; We et al., 1988).

Lmation ef estrogen receptor in copulatogt behavior neural circuite

ER’s have been identiﬁed throughout the neural circuits that regulate copulatory
behavior of males in numerous mammals (Commins & Yahr, 1985a; Fox, Ross, Handa, &
Jacobson, 1991; Koch & Ehret, 1989; Lieberburg, MacLusky, & McEwen, 1980; Nordeen
& Yahr, 1983; Shughrue, Bushnell, & Dorsa, 1992; Vito, DeBold, & Fox, 1983; Whalen &
Olsen, 1978: Wood, 1992; Wood & Newman, 1993b). The location of ER’s has been
determined by biochemical assays (receptor binding) and histechemieal techniques (uptake
of radiolabeled ligand (reviewed earlier); immunocytochemistry and I_n aim hybridization for

mRNA).

30

By immunocytochemistry ER-ir cells have been visualized in the mPOA, ventral
region of the lateral septum, medial division of the BNST, lateral region of VMH, arcuate
nuclws, and ventral prernammillary nucleus. In the amygdala ER-ir was seen in the anterior
cAM, mAM, central AM, basal AM magnocellular division (Fox et al., 1991; Koch & Ehret,

1989; Wood, Brabec, Swann, & Newman, 1992; Wood & Newman, 1993b).

Memring ER

ER levels within a region may be quantiﬁed by two basic methods, each with it own
set of limitations. ER levels may be detemrined within a region by an estrogen receptor
exchange assay (Machrsky, Roy, Shanabrough, & Eisenfeld, 1986; Roy & McEwen, 1977).
This assay measures the total amount of ER, however, since the tissue is homogenized,
precision of cell location is lost.

ER levels are measured in two populations: nuclear ER (ERn) and cytosolic ER
(ERc). These populations of ER’s are the product of differential centriﬁrgation. ERn are
located in the nuclear pellet of the centriﬁrgation and are presumed to be ER’s that are
dirnerized S-R and bound to DNA. Since these are thought to be bound to the DNA, ERn
levels may indicate the amount of estrogenic stimulation the target tissue is receiving. In
contrast, ERc’s represent the population of receptors that is detected in the cytosolic fraction
of the centrifugatien. These receptors may be bound to estrogen, but are not bound to the
DNA and are not regulating gene expression at that time (Gorski, Welshons, Sakai, Hansen,
Walent, Kasis, Shull, Stack, & Campen, 1986). Irnmunocytochemical evidence suggests that
there is a signiﬁcant proportion of ER’s in the cytoplasm, and therefore, the ERc ﬁ'action may

not be as artifactual as once thought (Wood & Newman, 1993b). Total ER (ERt) of a region

31

may be calculated by summing the ERn and ERc, if both populations of ER are expressed as
a ratio of DNA in the pellet. Using this method, the dynamics of ER regulation and activity
of a region can be determined. However, determining in which cells ER levels are measured
in is not possible.

In contrast, 113 site hybridization, a method which visualizes the levels of ER mRNA
per cell, allows for the possibility of determining in which cells the transcription of ER
message is being altered and under what conditions. On the other hand, determining the
levels of ER mRNA does not indicate the amount of ER that has been transcribed or the
ﬁrnctional state of the ER population.

Like m em hybridization, immunocytochemistry visualizes the location of ER-ir cells.
Additionally, this method may be used in conjunction with other immune labeling to visualize
other substances (AR, neuropeptides, c-fos etc...) that are colocalized in the cell or with tract
tracing methods to determine aﬂ‘erents and efferents of the cells (Wood et al., 1992; Wood
& Newman, 1993a). Furthermore, the localization of the ER within the cell can be visualized
as well, although it is diﬁcult to determine the activity of ER and the ratios of bound and

unbound receptor.

Effﬁte of eastratien and hormonal regelation of ER

In male rats, gonadal hormones cause a down regulation of ER. Castration does not
affect the number of cells that express ER mRNA in the periventricular POA, MPN or BNST.
In ﬁrct there is a trend towards increased number of cells that express ER mRN A following
castration (Liscietto & Morrell, 1993; Morrell, Wagner, Malik, & Lisciotto, 1995). Further,

the relative amount of mRNA per cell increased in castrated males (Liscietto & Morrell,

32

1993; Sirnerly & Young, 1991). Whm castrated males were treated with T, ER mRNA levels
were restored to the level of intact males. Although jg in; hybridization demonstrates that
ER mRNA is increased by castration, it tells nothing about the actual levels or state of the ER.
ER assays demonstrate that castration causes a reduction in ERn in POA, HYP (HYP) and
AM, brrt an increase in ERc (Roselli, Thornton, & Chambas, 1993). These data suggest ERt
may increase, but less estrogenic stimulation is produced due to the reduction in ERn. This
repartitioning of ER is supported by immunocytochemistry. In intact males, ER-ir is
practically entirely conﬁned to the nucleus, however in castrate males, ER-ir is found in both

cytoplasmic and nuclear regions of the cell (Wood & Newman, 1995; Wood & Newman,

1993b).
Summag and Putpese

In the genetically homogeneous B6D2F1 hybrid strain of house mouse, some males
continue to copulate after castration while others do not. Although it appears that continued
copulation after castration in B6D2F] males is independent of gonadal hormones, there is
evidence that suggests that steroid hormones, in particular estrogens, may be necessary for
the continued copulation aﬂer castration. First, estrogenic stimulation appears important
since E2 restores copulatory behavior to B6D2F] males that have stopped cepulating.
Second, castration does not elinrinate T ﬁ'om the circulation of B6D2F1 males, and in other
species E2 levels are not affected by castration. Third, since B6D2F] males are genetically
homogenous, these diﬂ‘erences in behavioral responsiveness to E2 are likely due to differences
in steroid physiology of steroid-regulated behavioral neural circuits in the adult. These

physiological differences among individuals, could be brought by pre- or neonatal steroid

33

hormones which organize neuronal function and morphology of steroid responsive regions
of the brain. Both distribution and levels of ER and aromatase have been shown to been
under the inﬂuence of the pre- or neonatal organizational effects of steroid hormones.
Therefore, levels of aromatase and/or ER may differ in regions of the brain that regulate
copulatory behavior among B6D2F] males that differ in expression of copulatory behavior
after castration. The following experiments investigate the role of estrogen related physiology
in the maintenance of copulatory behavior after castration. The results indicate that retention
of behavior after castration is dependent on aromatization of nongonadal androgens to

estrogens, and that the source of these nongonadal hormones is not the adrenal gland.

EXPERIMENT 1: LEVELS OF SERUM STEROID HORMON ES IN INTACT AND

CASTRATED CONTINUER AND NONCONTINUER MALES

In castrated B6D2F 1 continuer and noncontinuer males, serum T levels do not differ
(Clemens et al., 1988). Although castration reduced T to less than 10% of intact levels, it
was not eliminated entirely. In rats and other species, castration has little or no affect on E2
levels (Table 2). Therefore, it is possible that serum steroids of nongonadal origin maintain
copulatory behavior after castration in B6D2F] males. Since T is the precursor molecule for
the synthesis of both DHT and E2, and administration of DHT or E2 to noncontinuers
restores copulatory behavior (Sinchak & Clemens, 1990; Wee et al., 1988), the possibility
exists that circulating levels of DHT or E2 may account for behavior differences between
continuer and noncontinuer males. Thus, continuers and noncontinuers may diﬂ‘er in the
conversion of T to DHT and/or E2 which would result in a difference in the circulating levels
of one or both of the neurally active metabolites of T.

A diﬂ'erence in circulating levels of DHT or E2 between continuer and noncontinuer
males would affect the levels of steroid hormone stimulation that would facilitate copulation.
Diﬁ‘erences in DHT levels could have effects by two avenues. First, differences in DHT could
directly affect the amount of androgenic stimulation received that facilitates copulatory
behavior. Second, since DHT regulates aromatase activity in some regions of the central
nervous system, differences in DHT may result in changes in the rate at which estrogen is

synthesized (Roselli et al., 1985; Roselli & Resko, 1984). Furthermore, E2 levels may differ

34

35

between males that continue to copulate and those that do not in the absence of differences
in DHT levels.

The purpose of this study was to detemrine if nongonadal T, DHT, and/or E2 are
present after castration to stimulate copulatory behavior, and if so do levels of these

hormones differ between continuers and noncontinuer B6D2F] males.

METHODS
Meats:
Sixty-day old male B6D2F] hybrid house mice (bits We) (the cross of a
C5 7BIJ6J female and a DBA/2J male), were purchased from Jackson Laboratory, Bar
Harbor, ME. All males were individually housed in 7 x 11 x 4h (inches) plexiglas cages while
in the colony and maintained on a 14:10 light-dark cycle with lights out at 11:30AM. Food

and water were available ad libitum except during behavioral testing.

Sexual Behavior Tests

Behavioral testing was performed during the dark phase of the light cycle under red
illumination between 12:30-5:3OPM. Behavioral scoring and testing procedures were as
described previously (Clemens, et al., 1988) except that the experimental males were tested
in their home cages (Wee & Clemens, 1989). The home cage of the male was placed in the
testing room 30 minutes prior to the start of testing. The cage top was removed and replaced
with an inverted cage top to prevent animals ﬁom escaping. Testing started by placing a
hormone-primed stimulus female into the male’s cage. If the male did not achieve

intromission or display intromissive-like behavior within 10 nrinutes after introduction of the

36

female, the ﬁrst female was replaced by another stimulus female. Again if the male failed to
intromit within 10 minutes, the female was replaced by a third and ﬁnal stimulus female. If
intronrission was not achieved within 30 minutes, the male’s test was terminated.

If intromission did occur, the test continued until 1) the male ejaculated, 2) 40 minutes
elapsed between succesive intromissions, or 3) 4 hr passed from the time of ﬁrst intromission,
at which time the test was stopped even if the male was still sexually active. The stimulus
female was replaced with another stimulus female, if she became unreceptive anytime during
the course of testing. The pine chip bedding (Alpha Chips) in the home cage was changed
after the completion of each behavioral test so that the male had two-week old bedding at the
time of behavioral testing. However, during extended periods without behavioral testing, the
bedding was changed on a weekly basis. The data were recorded with a TRS-8O Model 4P
computer using software described by Rakerd et al., (1985).

Adult intact C57BI/6J female mice were used as stimulus females. They were group
housed in the same colony room as the experimental males. Sexual receptivity was induced
in the females by sequential subcutaneous injections of l7B-estradiol benzoate (60ug) 48 and
24 hours before testing and progesterone (0.6mg) 4 hours before testing. All hormones were
delivered in 0.031111 sesame oil.

Six biweekly tests for copulatory behavior with a sexually receptive female were given
to the males prior to castration or sham surgery. Surgeries were performed under
methoxyﬂurane anesthesia (Metofane; Pittman-Moore, Inc.) one week after the sixth
behavioral test. Since continuous testing is not necessary for the maintenance of copulatory
behavior in castrated B6D2F] males (Wee & Clemens, 1989), behavior testing resumed

twelve weeks after surgery and continued on a biweekly schedule until 38 weeks after

37

castration to assure that maintenance of copulatory behavior in castrated males was robust.

Prior to sacriﬁce, males were grouped according to their surgical treatment and

post-castration behavior exhibited in the last six behavioral tests after surgery. The criteria

used for determining behavioral groups were:

Irrtacts - Intact males that received sham castration surgery and exhibited copulatory
behavior in all of the last six behavior tests (int).

Continuers - Castrated males that exhibited the ejaculatory reﬂex on the last behavior test and
on 4 of 5 preceding tests (cont).

Noncontinuers - Castrated males that did not exhibit copulatory behavior in any of the last
6 behavior tests (mount, intromissive and ejaculatory) (none) (Figure 1).

Those males that did not meet the criteria for the above experimental groups were eliminated

from the study. All assays had eight animals per experimental group and were performed

without knowledge of experimental group and were decoded after statistical analyses were

performed.

Steroid Hormone Radioimmunoassay (RIA)

Within one week after their last behavioral test, the mice were sacriﬁced by
decapitation. Trunk blood was collected ﬁom each animal individually and allowed to
coagulate in an ice bath. The blood was centrifuged and serum was collected and quickly
frozen. The coded serum samples were shipped on dry ice to the Hormone Assay Core of the
Population Research Center at University of California, Los Angeles to determine the
concentrations of T, DHT and E2 by RIA The brains of these males were excised and placed

on alunrinum foil on dry ice to quickly freeze the brains and stored at -80°C for ER and AA

38
assays (Exp 3).

To monitor recovery, tracer amounts of [3H]T/E2, DHT/estrone or androstenedione
were added to alternate serum samples and then the serum was extracted with diethyl ether
(10:1 v/v). The organic phase was separated ﬁ'om the aqueous phase and dried under a
stream of dry, ﬁltered air. The dried extract was then solubilized in 0.5m] of nanograde
isooctane. Samples were then applied to celite chromatography columns for fractionation
(Abraham, 1977).

T and E2 were analyzed in an [‘2’I]-RIA with reagents obtained ﬁom ICN
Biomedicals, Inc. (Costa Mesa, CA) and counted in a micromedic 4/600 gamma counter with
automatic data reduction software (RIA AID; Robert Maciel and Associates, Inc., Arlington,
MA 02174). Standard curves were calculated using the four parameter logistic option. DHT
was analyzed in a [3H]-RIA utilizing charcoal separation methods with reagents from ICN
Biomedicals, Inc, and counted in a LS3 55 liquid scintillation counter. Data were calculated

using the software described above.

Figure l. The percent of the last six copulatory behavior tests with behavior (mount,
intromission, and ejaculation) for intact (Int) and castrated B6D2F] continuers (Cont). Each
bar represents the total number of tests with behavior for all males within a behavioral group
divided by the total number of tests for all males. The noncontinuer group is not represented
since sexual behavior was not exhibited by these males (refer to text for deﬁnitions of

behavioral groups).

Percent of Tests with Behavior

100
so]
80.
70.
60.
50.
40.
30.
20.
10.

 

 

Mount

40

I 1m
U Cont

None

I

 

 

——
Intromission Ejaculation

Behavior

Figure l

41

The within assay coeﬂicient of variation at the ED90, ED50, and ED20 were
respectively for T (10.8, 16.4, and 16.8%), for DHT (7.4, 16.1, and 11.5%), and for E2
(12.6, 12.2, and 5.5). Between assay error was not applicable, since all hormones were run
in one assay. The limits of detection for the RIA were as follows: T, 0.07ng/ml; DHT, 0.05
ng/ml; E2, 19.20 pg/ml.

RIA data were analyzed by parametric 1 way analysis of variance (ANOVA) followed
by post hoc Newnran-Keuls‘ multiple range test (N—K) for ANOVA's with F values associated
with p's s .05. For statistical purposes, values below the limit of detection of the assay were

assigned the value of the limit of detection.

RESULTS

Castration signiﬁcantly reduced but did not eliminate T in the serum of continuer and
noncontinuer males compared to intact males (F_(2,21) = 9.88, p < .05; N-K p < .01) (Table
3). T levels did not differ between castrated continuer and noncontinuer behavioral groups
(Table 3). T levels were below the limits of detection in only three of the castrate males (2
noncontinuers and 1 continuer).

Castration did not affect the level of serum E2 in either continuer or noncontinuer
males compared to intact males, nor were there differences between the continuer and
noncontinuer behavioral groups, (F (2,21) = 0.108) (TABLE 3). E2 levels were below the
limits of detection in only 1 noncontinuer male.

Castration did not affect the level of serum DHT in either continuer or noncontinuer
males compared to intact males, nor were there differences between the continuer and

noncontinuer behavioral groups, (F (2,21) = 1.95) (TABLE 3). DHT levels were below the

42

limits of detection in 4 intact, 4 noncontinuer, and 6 continuer males. Therefore the results

of this assay may not reﬂect actual physiological values.

SUMMARY
Results of this experiment indicate that nongonadal T and E2 hormones are present
after castration in B6D2F1 males. Serum T levels were decreased by castration but were not
different between castrated continuer and noncontinuer males as seen previously (Clemens,
et al., 1988). Serum E2 and DHT levels were not affected by castration in either continuer

or noncontinuer B6D2F] males.

Mean
17B-e.

males,

 

a

C0

Non

n=nur
1<0.(

43

Table 3

Mean (+/- s.e.m.) concentration of testosterone (T), dihydrotestosterone (DHT), and
l7B—estradiol (E2) in serum of intact and castrated continuer and noncontinuer B6D2F]
males.

 

 

 

Steroid
Treatment 11 T (ng/ml) DHT (ng/ml) E2 (pg/ml)
Intact 8 3.426 (1.045) 0.095 (0.018) 83.8 (12.6)
Continuer 8 0.140 (0.022)" 0.070 (0.014) 90.0 (16.7)
Noncontinuer 8 0.137 (0.023)“ 0.058 (0.006) 96.4 (25.8)

 

n = number of subjects per group. ** = signiﬁcantly less than intact serum steroid level (N-K
p < 0.01).

EXPERIMENT 2: DETERMINATION IF MAINTENANCE OF COPULATORY

BEHAVIOR AFTER CASTRATION IN B6D2Fl MALES IS DEPENDENT ON

THE AROMATIZATION OF NONGONADAL ANDROGENS TO

ESTROGENS

The fact that castration does not affect serum E2 level or eliminate T ﬁ'om the
circulation of B6D2F] males may indicate that maintenance of copulatory behavior after
castration is facilitated by the continued presence of these hormones. E2 stimulation has been
shown to be critical for the expression of copulatory behavior. F or example, in noncontinuer
B6D2F1 males, E2 treatment restores copulatory behavior (Wee et al., 1988). Further, in
castrated male rats, the aromatization of androgens to estrogens in the POA-AH is necessary
for the facilitation of mounting behavior (Christensen & Clemens, 1975). Therefore, it is
possible that endogenous nongonadal estradiol may facilitate copulation in castrated continuer
B6D2F] male mice.

To test whether non-gonadal estrogens play a role in the retention of the ejaculatory
reﬂex after castration in the male B6D2F] house mouse, the aromatase inhibitor, ATD, that

blocks the enzymatic aromatization of testosterone to 17B-estradiol, was administered to

continuer males.

METHODS
Male mice (Mus musculus) of the B6D2Fl hybrid strain purchased ﬁom Jackson
laboratories (Bar Harbor, ME) were housed individually and maintained on a 14L: 10D light-
dark cycle, with lights on at 2130 hours. Commercial chow (Breeder Blox) and water were

44

45

available at! li_bi_t_m except during testing. The males were allowed to adjust to the colony
room two weeks prior to the beginning of the experiment and were approximately 45 days
of age at the start of behavioral testing. A total of 77 males completed the 47-week
experiment.

Adult Swiss Webster females were used as stimulus females for behavioral testing and
were made sexually receptive by sequentiaL subcutaneous injections of estradiol benzoate (60
pg, 48 hours prior to testing) and progesterone (0.6mg, 4 hours prior to testing). Both
hormones were administered in .O3ce of sesame oil vehicle. The females were grouped house

in the same colony room as the experimental males.

Surgical Procedures

The nrales were anesthetized using methoxyﬂurane (Metofane; Pittman-Moore, Inc.)
for both castration and silastic implant surgeries. Castration was performed via a single
nu'dline scrotal incision. Silastic capsules were implanted subderrnally between the shoulder

blades through a small incision made in the nape of the neck.

Silastic Capsules

Silastic capsules (1.47 id. x 1.96 o.d. mm) 17mm in length were ﬁlled with ATD
(Steraloids, Wilton, NH) or no steroid (Blank). The ends of the capsules were plugged with
Dow silastic silicone cement. Capsules were dried overnight in a 37° C oven and weighed

prior to implantation. The capsules were not incubated before implanting.

46

Behavioral Testing

Behavioral testing was conducted between 1200 and 1700 hours under red light
illumination Behavioral scoring and testing procedures were similar to Clemens et al. (1988),
a briefsummary is presented here. Testing of the experimental males took place in their home
cage rather than an arena (Wee & Clemens, 1989). The pine chip bedding (Alpha Chips) in
the home cage was changed after the completion of each behavioral test so that the male had
two week old bedding at the time of behavioral testing. However, during extended periods
without behavioral testing, the bedding was changed on a weekly basis. The data were
recorded on a TRS-80 Model 4P computer (Rakerd, Brigham, & Clemens, 1985).

Males were given 6 biweekly tests for copulatory behavior, and castrated one week after
the sixth behavioral test. Four biweekly tests commenced one week after surgery, then the
males were not tested for 5 weeks. Behavioral testing was resumed and a ﬁnal series of
eleven biweekly tests were given. Within 24hrs alter the eighth test after surgery, the
castrated males were divided into three behavioral groups based on their performance in the
last four copulatory behavior tests. Assignment to these three groups was based upon three
criteria:

1) Continuers displayed ejaculatory reﬂex in at least 3 of the 4 tests;

2) Intermediates displayed ejaculatory reﬂex in less than 3 of the 4 tests;

3) Noncontinuers displayed no ejaculatory reﬂex in the last 4 tests.
Males were randomly assigned to either the ATD or blank implant treatment group and
subsequently implanted subcutaneously with a 17mm blank or ATD ﬁlled silastic capsule.
Implant treatment continued through the last 3 biweekly behavioral tests. At the time of the

implant, all males were treated for mites with 0.04% rotenone solution (Canex, Pittman-

47

Moore, Inc.) in mineral oil vehicle. A stripe of the solution was painted on the back of each

male with a cotton swab applicator.

Statistical Analysis

Percentage data for the last six behavioral tests were analyzed by nonparametric
statistics (Siegel, 1956). The effects, within treatment group over time, of ATD or sham
treatment on the percent of males that mounted, intromitted or ejaculated for the three weeks
prior to and after treatment were analyzed by Cochran's Q test. For Cochran's Q tests with
p s 0.5, within group comparisons of percent of males that exhibited behavior were made
between the last test prior to silastic capsule implantation (~24hr test) and each of the three
tests after implantation by mst h_ee Fisher exact probability test. Between treatment group
comparisons of blank and ATD silastic implanted within the continuer, intermediate, and
noncontinuer behavioral groups were analyzed by Chi2 analysis for independent samples
between the -24hr test and the three post-implant behavioral tests. Since, the number of
males that continued to show intromissive and ejaculatory behaviors after ATD treatment
were so few, only the mount latency data of the ATD and sham treated continuer groups were

analyzed by parametric two-way ANOVA.

RESULTS
There was a main effect of time from implantation on expression of the ejaculatory
reﬂex within each treatment (Cochran Q ATD - p < 0.001 It; 26.67, df = 5; Shams - p < 0.05
1t; 9.8889, df = 5). ATD signiﬁcantly reduced the percent of continuer males that achieved

an ejaculatory reflex 4 and 6 weeks after initiation of ATD treatment when compared to their

48

-24hr pro-ATD implant test performance (Fisher exact test: p = 0.041; p = 0.0045 1t;
respectively). The percent of sham continuer males achieving an ejaculatory reﬂex on the test
conducted 6 weeks after implant surgery was signiﬁcantly reduced compared to their -24 hour
preimplant test (Fisher exact test: p < 0.049, It). However, the percent of ATD treated
continuer males that achieved an ejaculatory reﬂex was signiﬁcantly less than the blank
implant continuer group 4 and 6 weeks after implantation (Fisher exact test: p = 0.042 and
p = 0.011 respectively) (Figure 2).

The percent of continuers that achieved intromission within each treatment was
affected over time after implantation (Cochran Q: ATD - p < 0.005 It; 19.69, df= 5; Shams -
p < 0.025 It; 12.5, DF = 5). ATD signiﬁcantly reduced the percent of continuer males that
aclu'eved intromission 4 and 6 weeks after ATD treatment when compared to their -24hr pre-
ATD implant test performance (Fisher exact test: p = 0.037; p = 0.012 It; respectively). The
percent of sham continuer males that achieved intronrission on the test conducted 6 weeks
after implant surgery, was signiﬁcantly reduced compared to their -24 hour preimplant test
(Fisher exact test: p = 0.049, 1t). However, the percent of ATD treated continuer males that
achieved an ejaculatory reﬂex was signiﬁcantly less than the blank implant continuer group
4 weeks after implantation (Fisher exact test: p = 0.043) (Figure 2).

The percent of continuers that mounted after implantation was not affected overtime
(Cochran Q: ATD - p < 0.30 It; Q = 5.0, df= 5; Shams - p < 0.30 It; Q = 5.0, df= 5).
Further, ATD and blank continuer implant groups did not differ in the percent of tests with
a mount (X2 = 9.859 df= 11) or the latency to mount (F = 0.073; df= 1,88) over all six tests
prior to and after implantation. However, there was a signiﬁcant effect of the repeated testing

on the latency to mount for both ATD and blank continuer implant groups post implantation

49
(F = 4.041; df= 3,88; p < 0.025) (Figures 2 & 3).

There was a main effect of repeated testing over the last six tests (three prior to
implantation and three post implantation) on the percent of intermediate males that achieved
an ejaculatory reﬂex, achieved intromission or mounted (Cochran Q: ejaculatory reﬂex: p <
0.001 It; Q = 21.67, df. = 5; intromission: p < 0.001 It; Q = 21.67, df= 5; mount: p < 0.02
It; Q = 14.32, df = 5) (data not presented). However, compared to last test prior to
implantation (—24hr test), ATD had no effect on the percent of intermediates that achieved an
ejaculatory reﬂex or mounted 2, 4, and 6 weeks after implantation (Fisher exact test:
ejaculatory reﬂex: p = 0.297, p = 0.11, and p = 0.11; mount: p = 0.50, p = 0.12, and p = 0.12
respectively). Compared to -24hr test, ATD reduced the percent of intermediates that
achieved intromission 4 and 6 weeks after implantation (Fisher exact test: 2 weeks p = 0.322,
p < 0.05, and p < 0.05 respectively) (data not presented).

Repeated testing over the last six tests did not affect the percent of ATD
noncontinuer males that achieved an ejaculatory reﬂex, achieved intromission or mounted
(Cochran Q: ejaculatory reﬂex, p > 0.30 it; Q = 5.0, df= 5; intronrission: p > 0.10 1t; Q =

8.07, df= 5; mount: p > 0.20 1t; Q = 7.27, df = 5) (data not presented).

SUMMARY
The results of the present study are consistent with the idea that nongonadal estrogens
contribute to the maintenance of copulatory behavior after castration in male B6D2F] hybrid
house mice. When synthesis of E2 was inhibited with ATD, fewer continuer B6D2Fl males
achieved ejaculations and intromissions when compared to their pretreatment test as well as

to continuers that received blank silastic capsules. In contrast, inhibition of estrogen synthesis

50

had no eﬁ‘ect on the percent of continuer males that mounted nor on their latency to mount.

51

Figure 2. Percent of continuer B6D2F] males that expressed behavior A) ejaculatory reﬂex,
B) Intronrission, C) Mount per test 24 hours (hr) prior to and 2, 4, and 6 weeks (wk) after
ATD or blank silastic capsule implant. * = signiﬁcantly less than blank silastic implant group

on that test (p < 0.05). + = signiﬁcantly less than preimplant (-24 hour) test within treatment

group (p < 0.05).

Percent of Tests with Behavior

52

I Blank
[:I ATD

100.

*+

 

111+

 

O
L

 

w Ejaculatory Reﬂex >
01
.°

(0:501me
000000
I I I I I I

     

 

 

Mount

 

 

 

 

 

 

 

 

 

-24hr Implant 2 wk 4 wk 6 wk

Time from Implant
Figure 2

53

Figure 3. Mount latency (sec) +/- (s.e.m.) for blank silastic capsule and ATD treated
continuer B6D2F] males 24 hours (hr) prior to and 2, 4, and 6 weeks (wk) after ATD or

blank silastic capsule implant.

.h
0 U" 0
C O O
1 r r

O
O
l

Latency to Mount (sec)
er
.0

A-‘NNODOD
U"
0
r

01 O
O O
r r

 

O
l

 

 

 

54.

 

 

 

 

 

-24hr Implant 2 4 6
Weeks from Implant

Figure 3

110

Cir

8011

C215

than
as 11.
dlﬁ‘ej

QSlra

EXPERIMENT 3: DETERMINATION OF AROMATASE ACTIVITY IN POA, HYP
AND AM OF INTACT AND CASTRATED CONTINUER AND
NONCONTINUER B6D2F] MALES
Since inhibition of aromatase activity reduced copulatory behavior in continuer males,

and continuers and noncontinuers do not differ in serum steroid levels, it is possible that

continuers and noncontinuers differ in the levels of neural aromatase in regions of the brain
that are important for copulatory behavior which could result in difference in E2
concentration within those regions among continuers and noncontinuers. Since aromatization
of T to E2 implanted directly into the POA-anterior hypothalamus (AI-l) is all that is necessary
to restore mounting to castrated male rats that have stopped copulating, it is possible that
noncontinuers lack the, or have a reduced ability, to convert aromatizable androgens to
estrogens in the POA or other regions of the brain important for copulation (Christensen &

Clemens, 1974; Christensen & Clemens, 1975).

In rats, levels of aromatase activity (AA) within the POA-AH are dependent on
gonadal androgens (Roselli et al., 1984; Roselli et al., 1985; Roselli & Resko, 1984).
Castration reduces AA in the POA of the rat and AA is restored with androgen treatment.
Therefore, even though serum E2 concentrations are maintained after castration, it is possible
that estrogenic stimulation is reduced in POA-AH because castration lowers local AA as well
as the availability of aromatizable androgens (T). Thus, it is possible that behavioral

differences between continuer and noncontinuer B6D2F] male house mice produced by

castration may be due to differences in the ability to aromatize androgens to estrogens within

55

56

the POA.

The medial amygdala (mAM) has also been shown to concentrate estrogens, contain
ER, aromatize androgens, express aromatase irnmunoreactivity, and project efferents to the
POA that are part of a neural regulatory pathway of male copulatory behavior (Akesson,
Simerly, & Micevych, 1988; Callard et al., 1986; Koch & Ehret, 1989; Roselli et al., 1985;
Sanghera et al., 1991; Sheridan, 1978; Simerly, Chang, Murarnatsu, & Swanson, 1990;
Stumpf & Sar, 1974/1975; Winans et al., 1982). In rodents, the amygdala (AM) receives
olfactory and vomeronasal information from the main and accessory olfactory bulbs
respectively (Scalia & Winans, 197 5). Reproductively relevant olfactory bulb sensory
information increases expression of the immediate-early gene e—f_os_ in mAM in male rats
(Baum & Everitt, 1992). Further, this olfactory and vomeronasal information received by
mAM is in part responsible for mating induced increases in & levels of the POA and for
the regulation of male copulatory behavior (Baum & Everitt, 1992; Krettek & Price, 1978).
Thus, it is likely that estrogen acting in the mAM facilitates male copulatory behavior, and
that in B6D2F] males castration may induce differences in estrogen synthesis in the AM
between continuers and noncontinuers.

The following experiment investigates the possibility that continuers and
noncontinuers differ in the ability to aromatize available androgens to estrogen in regions of
the brain that are important for copulatory behavior (POA, hypothalamus (HYP) and AM)

by measuring aromatase activity (AA) in these brain regions.

57

METHODS
Subjects

The males for this study were the same males from experiment one.

Brain Tissue Preparation

The brain was quickly excised ﬁ'om the cranium, frozen on dry ice, and shipped
overnight to Dr. Roselli in Oregon to measure AA, cytosolic ER (ERc), nuclear ER (ERn)
and total ER (ERt). On the day of the assays (2 days aﬂer sacriﬁce) the brains were placed
ventral side up on a cold platform and three sequential coronal cuts were made: 1)
approximately lmrn anterior of the optic chiasm, 2) immediately posterior to the optic chiasm
and 3) immediately posterior to the mammillary bodies. The POA dissection extended from
the medial septum to the caudal border of the optic chiasm, bilaterally to the lateral border
of the supraoptic nucleus and dorsally to the superior border of the third ventricle. The HYP
dissection extended ﬁom the optic chiasm to the mammillary bodies, bilaterally to the optic
tract and dorsally to the superior border of the third ventricle. The AM dissection consisted
of the temporal cortex lying immediately adjacent to the HYP and having the same anterior

to posterior boundaries.

Aromatase Activity Assay
AA was quantiﬁed with a radiometric assay that measures the stereospeciﬁc loss of
tritium ﬁ'om the C-113 position of [3H-lB]androstenedione and its incorporation into3 H20

which is produced in proportion to the amount of estrogen formed (Roselli et al., 1984). This

assay has been previously validated for mouse brain (Wozniak, Hutchinson, & Hutchison,

58
1992).

Brieﬂy, brains tissues were homogenized in 250 111 ice-cold TEGD buffer (lOmM Tris,
1.5mM EDTA, 10% glycerol, 1 mM dithiothreitol, pH 7.4). The homogenates were
centriﬁrged at 1,000x g for 10 min. The pellets from this ﬁrst spin (1,000x g pellets) were
reserved for the ERn assay. The low-speed supematants were harvested and centriﬁrged for
10 min. at 106,000x g to generate cytosols (106,000x g supernatants) and mixed
mitochondrial-microsemal pellets (106,000x g pellets). These pellets were suspended in 30
volumes of phosphate buffer (10 mM KPO,, 100 mM KCl, 1 mM EDTA; pH 7.4) and
sonicated. Aliquots were then incubated for 1 h at 37°C with 0.3 11M
[’H-Bl]androstenedione. The reactions were stopped with 10% trichloroacetic acid
containing 20 mg/ml charcoal, and the 3[-120 generated was puriﬁed on small Dowex columns.
The reaction exhibited Michaelis-Menton kinetics (Vmax = 89.8 ﬁnth'mg protein; Km = 9.0
nM).

Aromatase activity data were analyzed by parametric 3x3 way ANOVA (brain region
by behavioral group) followed by post hoc one way ANOVA and N-K for ANOVA's with F
values associated with p's s .05. Behavioral group codes were not revealed until statistical

analyses were complete.

RESULTS
There was a main effect of behavioral group on AA levels (F(2,63) = 22.39, p <
0.0001). Further, there was a main effect of brain region on AA (F(2,63) = 122.08, p <
0.0001). However, there was no signiﬁcant interaction of between behavioral group and

brain region (E(4,63) = 2.31, p = 0.068). Between brain regions within each behavioral

59

group, AA levels in the AM were greater than both POA and HYP, and POA AA levels were
greater than HYP (mtact, E(2,63) = 56.35, p < 0.001; continuer, E(2,63) = 38.59, p_ < 0.001;
noncontinuer, E(2,63) = 31.74, p < 0.001; all N-K, p < 0.05).

Within brain region, AA was signiﬁcantly decreased in the POA, HYP, and AM of
castrated continuer and noncontinuer B6D2F] males compared to intact males, (POA,
F(2,63) = 17.59, p < 0.001; HYP, F(2,63) = 17.71 p < 0.001; AM, E(2,63) = 7.98, p < 0.001;
all N-K p < 0.05) (Figure 4). Within each brain region AA was not different between

castrated continuer and noncontinuer males.

SUMMARY
The results of this experiment support the idea that aromatizable androgens of
nongonadal origin may be converted to estrogens in regions of the brain important for
copulation. Following castration, AA in POA, HYP, and AM was reduced but not
eliminated. In none of the three brain regions was the level of AA correlated with the
maintenance of copulatory behavior after castration in continuer and noncontinuer B6D2F1

males.

60

Figure 4. Mean (+/- s.e.m.) aromatase activity levels (fmol/h/mg protein) in the POA, HYP,
and AM for intact (Int), and castrated B6D2F] continuers (Cont) and noncontinuers (None).
* = signiﬁcantly less than intact treatment group (N-K p < 0.05). + = signiﬁcantly greater

than other brain regions within behavioral group (N-K p < 0.05).

3H20 fmol/h- mg protein

200..

150.

100.

 

61

 

Brain Region

Figure 4

111.

for

[05!

gene

Castr

1980

e131.

EXPERIMENT 4: ESTROGEN RECEPTOR LEVELS IN THE POA, HYP AND AM
OF INTACT AND CASTRATED CONTINUER AND NONCONTINUER
B6D2F] MALES

EXPERIMENT 4A: THE EFFECTS OF CASTRATION AND BEHAVIORAL
STATUS ON ESTROGEN RECEPTOR LEVELS IN THE POA, HYP AND
AM OF INTACT AND CASTRATED CONTINUER AND NONCONTINUER
B6D2Fl MALES
Since continuers and noncontinuers do not differ in serum T, E2, or DHT levels, or

in neural aromatase activity, it seems likely that they may differ in their responsiveness to

these hormones. One measure of responsiveness to E2 that may differ between continuers
and noncontinuers is the levels of estrogen receptors (ER) in regions of the brain important
for copulatory behavior.

One way in which steroid hormones alter cellular activity (and behavior) is via
activation of receptors that regulate genomic activity. For example, E2 binds to an estrogen
receptor (ER) forming a steroid-receptor complex (S-R). Two S-R's dimerize and then attach
to speciﬁc gene regulatory binding sites on the DNA within the nucleus where they alter the
gene's activity. This in turn, leads to a change in protein synthesis and presumably behavior.

In earlier rat studies, nuclear estrogen receptor (ERn) levels in POA were reduced by
castration and were associated with circulating androgen levels (Krey, Kemel, & McEwen,
1980). This reduction in ERn in the mPOA was also seen in continuer and noncontinuer

B6D2F 1 males, however, ERn levels did not differ between these behavioral groups (Clemens

et al., 1988). Unfortunately, ERc levels were not measured in these B6D2F1 males, which

62

my have (eve
continuer and
appear to dc
returned to
ilurtmrtsu.
heath-'81
urn! amo
by our 1111

continuers

Est!
ERc

mtlhods (it

63

may have revealed possible diﬂ‘erences in the dynamics of ER regulation in the POA between
continuer and noncontinuer males (Clemens et al., 1988). For example, testicular hormones
appear to down regulate ER. In rats, castration increased ER mRN A levels, which were
returned to intact levels in some brain regions by T and/or E2 treatment (Lauber, Mobbs,
Muramatsu, & Pfaff, 1991; Lisciotto & Morrell, 1993; Simerly & Young, 1991).
Alternatively, castration may elicit a slower turnover or degradation rate of ER protein. The
actual amount of ER synthesized, used and degraded per unit of time cannot be determined
by our method of analysis. However, if there were difference in ER turnover between
continuers and noncontinuers it would be revealed in the ERn to ERc ratios.

In addition, only ERn in the mPOA was measured by Clemens et al., (1988). It is
known that the AM of a number of species contains ER synthesizes estrogen from circulating
androgens and is responsive to estrogen (Lieberburg & McEwen, 1977; Roselli & Resko,
1984; Sanghera et al., 1991; Schleicher et al., 1986b; Sheridan, 1978; Simerly et al., 1990;
Stumpf& Sar, 1974/1975; Winans et al., 1982).

The purpose of this experiment was to extend the ﬁndings of Clemens, et al., (1988)
to determine if there are differences in ER levels and dynamics in POA, HYP and AM
between castrated continuer, noncontinuer, and intact B6D2F 1 male house mice by measuring

ERn, ERc, and ERt levels.

METHODS
Estrogen Receptor Assay
ERc and ERn measurements were made using modiﬁcations of previously described

methods (MacLusky et al., 1986; Roy & McEwen, 1977). Cytosols (106,000x g pellets)

64

were adjusted to 225 pl total volume. To measure ERc, 100111 aliquots were incubated for
2h at 0—4C with 2 nM[2,4,6,7 3H]-E2 with or without a ZOO-fold excess of radioinert E2.

To measure ERn, the crude nuclear pellets were mixed with Cellex (5mg/25 111 TEGD
buffer) and washed twice with 200111 TEGD buffer followed each time by centriﬁrgation at
15,600 x g for 5 min in an IEC Centra-M microcentriﬁrge (International Equipment Co.,
Needham Heights, MA). ERn were salt extracted by suspending the washed pellets in 115
pl TEGDB buffer (TEGD buﬂ‘er + 0.5 mM bacitracin) and an equal amount of TEGDB buffer
containing 0.8 M KCl was added to give a ﬁnal salt concentration of 0.4 M. After a 30 min
extraction period, the tubes were centriﬁrged at 15,600 x g for 5 min. Aliquots (100 pl) of the
supernatant (nuclear extract) were incubated for 5h at 25C with 2nM [3H]E2 with or without
a ZOO-fold excess of radioinert E2. The DNA contents of the washed nuclear pellets were
estimated by the diphenylamine method (Giles & Myers, 1965).

Bound [’HJEZ was separated from free steroid on Sephadex LH-20 columns. Speciﬁc
binding was calculated by subtracting nonspeciﬁc binding (measured in the presence of excess
radioinert E2) ﬁ'om total binding (measured in the absence of excess radioinert E2). Results
for both ERc and ERn are expressed as femtomoles (frnol) of [3H]E2 bound per mg DNA.
Total binding (ERt) was calculated as the sum of ERc + ERn.

Estrogen receptor assay data were analyzed by parametric 3x3 way ANOVA (brain
region by behavioral group) followed by post hoc one way ANOVA and N-K for ANOVA's
with F values associated with p's s .05. Behavioral group codes were not revealed until

statistical analyses were complete.

65

RESULTS

Nuclear Estrogen Receptors

There was a main effect of brain region on ERn levels, (F(2,59) = 3.361, p < 0.05)
with a signiﬁcant interaction between region and behavioral group (F(4,59) = 2.462, p =
0.05).

Between brain regions within intact males, ERn levels in the AM were greater than
POA and HYP (E(2,59) = 7.286, p < 0.01; N-K p < 0.01). However, there were no
differences in ERn levels between brain regions of the continuer (E(2,59) = 0.312) and
noncontinuer (F(2,59) = 0.767) males.

ERn levels in the AM were signiﬁcantly decreased in the castrated continuer and
noncontinuer behavioral groups compared to intact males (F(2,59) = 7.19, p < 0.01; all N-K
p < 0.05) however, ERn levels were not signiﬁcantly different between intacts and castrated
continuers and noncontinuers in the POA and HYP (POA, E(2,59) = 0.310; HYP, E(2,59) =
0.264) (Figure 7). Within each brain region ERn levels were not signiﬁcantly different

between castrated continuer and noncontinuer males.

Cytosolic Estrogen Receptors

There was a main effect of behavior group and brain region on ERc (behavior, F(2,59)
= 23.074, p < 0.001; region, F(2,59) = 14.221, p < 0.001). However, there was not an
interaction between behavioral group and brain region (F (4,59) = 1.100).

Between brain regions, ERc levels were signiﬁcantly greater in the AM compared to
the POA and HYP of continuer and noncontinuer males (continuer, E(2,59) = 13.91; p <

0.005; noncontinuer, F(2,59) = 6.201; p < 0.005; all N-K p < 0.05). In intacts, ERc levels

66
in the AM were greater than the POA (intacts, F(2,59) = 5.524; p < 0.05; N-K p < 0.05)
(Figure 6).

Within brain region, ERc levels of castrated continuer and noncontinuer males were
signiﬁcantly greater in POA and AM compared to intact males (POA, E(2,59) = 4.56, p <
0.05; AM, E(2,59) = 8.00, p < 0.005, all N-K p < 0.05). In HYP there was a main effect of
behavioral group on mean ERc levels (112,59) = 4.06; p < 0.05). ERc were signiﬁcantly
increased in HYP of noncontinuers compared to intacts (N-K p < .05), while ERc levels in

the continuers were not signiﬁcantly different from either noncontinuers or intacts (Figure 6).

Total Estrogen Receptors

There were main effects of behavioral group on ERt (F(2,59) = 10.46, p <0.001) and
brain region (_F_'(2,59) = 23.45, p_ <0.001), but no signiﬁcant interaction between behavioral
group and brain region (F (4,59) = 0.570).

Between brain regions, AM ERt levels within each behavioral group were signiﬁcantly
greater than in both POA and HYP (N-K p < 0.05), however there were no diﬁ‘erences in ERt
levels between POA and HYP.

Within brain region, both continuer and noncontinuer males had greater ERt levels in
the POA, HYP, and AM than intact males (POA, F(2,59) = 3.568, p < 0.05, HYP, E(2,59)

= 3.934, p < 0.05; AM, E(2,59) = 4.514, p < 005; all N-K p < .05) (Figure 7).

SUMMARY
Castration did not affect the level of ERn in POA and HYP, while in the AM ERn

were signiﬁcantly reduced after castration. Overall, castration increased ERt in POA, HYP,

67

and AM. Although castration altered steroid levels, AA, and ER dynamics, none of these
changes were associated with maintenance of copulatory behavior after castration in continuer

males compared to noncontinuer males.

68

Figure 5. Mean (+/- s.e.m.) nuclear estrogen receptor (ERn) levels (ﬁnong DNA) in the
POA, HYP, and AM for intact (Int) and castrated B6D2F1 continuers (Cont) and
noncontinuers (None). * = signiﬁcantly less than intact treatment group, (N-K p < 0.05). +

= signiﬁcantly greater than other brain regions within behavioral group (N -K p < 0.05).

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Brain Region

Figure 5

70

Figure 6. Mean (+/- s.e.m.) cytosolic estrogen receptor (ERc) levels (fmol/mg DNA) in the
POA, HYP, and AM for intact (Int) and castrated B6D2F1 continuers (Cont) and
noncontinuers (None). * = signiﬁcantly greater than intact treatment group (N -K p < 0.05).
+ = signiﬁcantly greater than other brain regions within behavioral group (N -K p < 0.05). A

= signiﬁcantly greater than HYP within behavioral group (N-K p < 0.05).

 

 

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Brain Region

Figure 6

72

Figure 7. Mean (+/- s.e.m.) total estrogen receptor (ERt) levels (ERn + ERc) (ﬁnol/mg
DNA) in the POA, HYP, and AM for intact (Int) and castrated B6D2F] continuers (Cont)
and noncontinuers (N one). * = signiﬁcantly greater than intact treatment group (N -K p <
0.05). + = signiﬁcantly greater than other brain regions within behavioral group (N-K p <

0.05).

ERt (fmol/mg DNA)

500.
450.
400.
350.
300.
250.
200.
150.
100.

 

0

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73

 

 

Brain Region

Figure 7

EXPERIMENT 4B: COMPARISON OF ER MEASURED IN THE POA, HYP, AND
AM BETWEEN NUCLEAR PELLETS PURIFIED WITH SUCROSE AND
NUCLEAR PELLETS NOT PURIFIED IN INTACT AND CASTRATED
B6D2F] MALES
In EXP 4A castration did not affect the level of ERn in the region of the POA of

continuer or noncontinuer B6D2F 1 males. These data are in contrast to earlier work, where

mPOA ERn levels of castrated continuers and noncontinuers were signiﬁcantly decreased
compared to intact males (Clemens et al., 1988). Because of this discrepancy, EXP 4B was
designed to verify the effects of castration on ERn levels obtained in EXP 4A, and to
determine if ER measures are equivalent between procedures where the nuclear pellet is

sucrose puriﬁed (Clemens et al., 1988) compared to not purifying the nuclear pellet (EXP

4A).

METHODS

Male B6D2F1 hybrid house mice, 60 days old, were castrated or sham castrated and
housed individually. Five weeks after surgery the male mice were sacriﬁced by decapitation.
Their brains were excised from the cranium, frozen on dry ice and shipped to Dr. Roselli.
Each experimental group consisted of six animals.

In order to compare Clemens et al., (1988) preparation of the nuclear pellet by sucrose
puriﬁcation (sucrose puriﬁed) to our washed pellet procedure in EXP 4A (non-sucrose
puriﬁed), each brain tissue sample was homogenized in 250 111 ice-cold TEGD buﬁ‘er (10mM
Tris, 1.5 mM EDTA, 10% glycerol, 1 mM dithiothreitol, pH 7.4) and centrifuged at 1,000

74

75

x g for 10 min. The 1,000 x g pellet (cnrde nuclear pellet) was either washed twice with
TEGD buffer as in EXP 4A (non-sucrose puriﬁed) or puriﬁed through sucrose before
extraction as in Clemens et al., (1988). For sucrose puriﬁcation, the pellets were ﬁrst
resuspended in 25 ul of low sucrose buffer (lmM KHZPO“ 0.32 M sucrose, 3 mM MgC12,
lmM DTT, 10% glycerol, pH 6.8) that contained 5mg of Cellex 410 (BioRad Laboratories,
Richmond, CA.). An additional 200 111 high sucrose buffer (lmM KH2P04, 2.1 M sucrose,
3 mM MgCl, lmM DTT, 10% glycerol, pH 6.8) was added to make a 2 M sucrose solution.
This suspension was centriﬁrged at 60,000 x g for 10 min. to obtain a puriﬁed nuclear pellet.
Both the washed and puriﬁed nuclear pellets were extracted with 0.4 M KCl and aliquots
(100 111) used to measure ERn. Receptor measurements were performed using 2 nM
[2,4,6,7-’H]-E2 in the absence (total binding) or presence (nonspeciﬁc binding) of a 200-fold
excess cold E2. Bound [3H]-E2 was separated ﬁ'om free steroid on Sephadex LH-20
minicolumns. The DNA contents of the washed nuclear pellets were estimated by the

diphenylanrine method (Giles & Myers, 1965).

Statistical Analysis

The DNA content of the nuclear pellet was analyzed with a 3x2x2 way ANOVA to
make comparisons between brain region, puriﬁcation procedure, and surgical treatment and
analyzed by post hoc N-K for ANOVA's with p s .05. The ERn levels of the nuclear pellet
were analyzed with a 3x2x2 way ANOVA to make comparisons between brain region,
puriﬁcation procedure, and surgical treatment and analyzed by post hoc N-K for AN OVA's
with p s .05. Signiﬁcant main effects of puriﬁcation were analyzed post hoc by 1 way

ANOVA within brain regions and N-K's for ANOVA's with p < 0.05. Individual

76

measurements that did not produce positive values were omitted from the statistical analyses.

RESULTS

DNA Recovery

There was a main effect of sucrose puriﬁcation of the nuclear pellet with no
interaction with surgical treatment, brain region or surgical treatment and brain region
(ANOVA puriﬁcation - E(1,60) = 59.7, p < .0001; p = 0.78; puriﬁcation-surgical treatment
- E( 1,60) = 1.49, p = .2382; puriﬁcation-brain region - F_(2,60) = 1.798, p = .1745;
puriﬁcation-surgical treatment-brain region - F(2,60) = 1.072, p = .3489)(Figure 8). Sucrose
puriﬁcation signiﬁcantly reduced the mean amount of DNA (u g) recovered from the nuclear
pellet within each brain region (N-K p < 0.05). The mean (+/- s.e.m.) amount of DNA (ug)
recovered from the puriﬁed nuclear pellet was 63% (+/- 4.3) of the total amount extracted
ﬁom the nonpuriﬁed nuclear pellet (Figure 8).

There was a main effect of brain region on DNA recovery levels (ANOVA _F_(1,60)
= 3.253, p = 0.0456) with no interaction with surgical treatment (ANOVA F(2,60) = 1.933,
p = 1.537) (Figure 8). There was no main effect of surgical treatment on the recovery of

DNA ﬁ'om the nuclear pellet (ANOVA surgical treatment - F(1,60) = 0.077) (Figure 8).

ERn

There was a main effect of puriﬁcation of the nuclear pellet on the mean concentration
of ERn (ﬁnol/mg DNA) (ANOVA F(1,42) = 5.907, p = 0.0194). Puriﬁcation had no effect
on ERn levels in POA and HYP, (ANOVA POA, F(1,42) = 1.099; HYP, F( 1,42) = 0.737).

However, in the AM there was a signiﬁcant effect of puriﬁcation (ANOVA AM, E(l,42) =

77
5.351; p < 0.05) (Figure 9).
There was a main effect of brain region on the levels of ERn (ANOVA £(2,42) =
9.318, p = 0.0004). The AM had greater levels of ERn compared to POA and HYP (N-K,
p < 0.05).
There was no main effect of castration (5 weeks) on the mean level of ERn (fmol/mg

DNA) (ANOVA £0.42) = 0.085).

Summary EXP 4B
Sucrose puriﬁcation reduced the amount of DNA recovered from the nuclear pellet,
and reduced ERn levels in the AM but not in the POA and HYP compared to nonpuriﬁed
nuclear pellets. In spite of these reductions in DNA recovery and AM ERn levels by sucrose
puriﬁcation, sucrose puriﬁed and nonpuriﬁed nuclear pellets produced equivalent results in
ERn levels with respect to surgical treatment and brain region. These ﬁndings are consistent

with our results from EXP 4A.

SUMMARY
The results of EXP 4 support the notion that brain regions important for copulatory
behavior in castrated B6D2F] males continue to receive estrogenic stimulation that may
facilitate the maintenance of copulatory behavior. However, changes in ER levels after
castration were not associated with the maintenance of copulatory behavior in castrated
continuer B6D2F1 males. Differences in the effects of castration on ERn levels in POA of
B6D2F] continuer males between Clemens et al. (1988) and EXP 4A were not due to

differences in preparation of the nuclear fraction prior to measurement of ERn.

'78

Figure 8. Mean (+/- s.e.m.) levels of DNA (rig) in the nonpuriﬁed (N) and sucrose puriﬁed
(S-P) nuclear pellet of brain tissue from the POA, HYP, and AM of intact (Int) and castrated

(Cast) B6D2F] males.

79

 

 

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Brain Region

Figure 8

80

Figure 9. Mean (+/- s.e.m.) nuclear estrogen receptors (ERn) levels (ﬁnol/mg DNA) of
nonpuriﬁed (N) and sucrose puriﬁed (S-P) nuclear pellet of brain tissue ﬁ'om the POA, HYP,

and AM of intact (Int) and castrated (Cast) B6D2F] males.

81

 

 

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EXPERIMENT 5: DETERMINATION OF AROMATASE ACTIVITY LEVELS IN

POA, HYP AND AM OF INTACT AND CASTRATED C57BL/6J, DBA/21

AND B6D2Fl MALES

Continuer and noncontinuers cannot be distinguished by levels of serum androgens
or 17B—estradiol nor by levels of AA or ER levels in the POA, HYP, and AM (EXP 1-4).
However, in mice, reproductive behavior, physiology, neuronal anatomy and the effects of
castration on these phenotypes are known to be inﬂuenced by genotype. For example,
castration abolishes copulatory behavior in both C57BV6J and DBA/2J strains of mice.
However, compared to DBA's, C57 males stop achieving an ejaculatory reﬂex sooner after
castration. In contrast, most B6D2F] males continue to copulate for six months to over a
year after castration (Clemens et al., 1988; McGill & Manning, 1976). Other related
behaviors also differ between these strains. DBA, C57 and B6D2F] males diﬂ‘er in
expression and retention of aggressive behavior after castration (Sinchak & Clemens, 1988;
Wee, Weaver, Sinchak, & Clemens, 1985). Additionally, DBA and C57 males differed in
levels of infanticide (Svare, Kingsley, Mann, & Broida, 1984).

DBA, C57 and B6D2F1 strains also differ from one another physiologically and
morphologically in a number of reproductively related measures, as well as their responses
to castration. For example, testicular weight, spermatogenesis, cholesterol concentration,
testosterone levels and glycolipid make-up differ between DBA/2] males and C57Bl/10J

strains (Bartke, 1974; Bartke & Shire, 1972; McCluer, Deutsch, & Gross, 1983; Shire &

Bartke, 1972). The weight of the bulbocavemosus (BC) muscle does not differ among intact

82

C5'

C5

15

of

83

C57BV6J, DBA/2] and B6D2F1 males, however, castration reduces the mass of the BC in
C57 and B6D2F] males compared to DBA males (Wagner, Popper, Ulibarri, Clemens, &
Micevych, 1994). Furthermore, DBA males have fewer motoneurons in the spinal nucleus
of the bulbocavemosus (SNB). Levels of CGRP in cells of the SNB also differ among these
strains after castration (Wagner et al., 1994; Wee & Clemens, 1987).

The POA-anterior hypothalamus is important for regulation of reproductive behavior
and physiology which includes copulatory behavior, scent marking, and secretion of gonadal
hormones (Bean et al., 1980; Quadagno et al., 1976; Yahr, Commins, Jackson, & Newman,
1982). A major role of the mPOA is to facilitate copulatory behavior in response to sexually
relevant stimuli. For example, in the Swiss-Webster strain of house mouse, lesions in the
mPOA eliminated intronrissions, and ejaculations (Bean et al., 1980). These same effects on
copulatory behavior occur in B6D2F] male mice by blocking protein synthesis in the POA
with cyclohexamine (Quadagno et al., 1976).

Interestingly, regional POA morphology differs between C57 and DBA males. In the
medial preoptic nucleus (mPON), DBA/2] male mice have a set of densely packed, darkly
staining cells (medioventral pars compacta (MVPC)) that are not seen in C57BL/6J males
(Robinson, Fox, & Sidman, 1985). The ﬁrnction of this set of neurons in not known.
However, these gross morphological differences among strains that differ in expression of
reproductive behavior suggest that other differences in cell physiology may exist between
these strains that regulate behavior. Since retention of copulatory behavior after castration
in B6D2F] males appears estrogen dependent, and C57BL/6J and DBA/2] males stop
copulating shortly after castration, the following experiment investigates if there are

diﬂ‘erenccs in estrogen synthesis (AA) in the POA, HYP, and AM of intact and castrated

C5

60

of

in

bi;

84

C57BL/6J, DBA/2], and B6D2F] mice.

METHODS

Subjects

 

Adult male inbred C57BL/6J and DBA/2], and hybrid house mice M musculus),
60 days old, were purchased from Jackson Laboratory, Bar Harbor, ME. All males were
individually housed while in the colony and maintained on a 14:10 light-dark cycle with lights
out at 11:30AM. Food and water were available at mm; One week after arrival, each
male was either castrated or sham castrated 18 weeks prior to removal and microdissection

of the brain for AA assay (6 per treatment group).

Procedures for Aromatase Activity Assay

Procedures of tissue preparation and aromatase assay are detailed in EXP 3.

Brain Tissue Preparation

Brieﬂy, the mice were sacriﬁced by decapitation, and each brain was quickly excised
ﬁom the cranium and blocked by removal of the olfactory bulbs and cerebellum with a razor
blade. The blocked brain was positioned dorsal side down on aluminum foil and placed on dry
ice to rapidly freeze the brain. The brains were stored overnight on dry ice. Trunk blood was
also taken for each animal upon decapitation. On the following day, (3 days after sacriﬁce),

the brains were placed dorsal side down on ice and the preoptic area (POA), hypothalamus

(HYP) and amygdala (AM) dissected.

85

Aromatase Activity Assay

AA was quantiﬁed with a radiometric assay that measures the stereospeciﬁc loss of
tritium from the C-18 position of [3H-18]androstenedione and its incorporation into’ H20
which is produced in proportion to the amount of estrogen formed (Roselli et al., 1984).

Brieﬂy, brains tissues were homogenized in 250 pl ice-cold TEGD buffer (10mM Tris,
1.5mM EDTA, 10% glycerol, 1 mM dithiothreitol, pH 7 .4). The homogenates were
centrifuged at 1,000x g for 10 min. The low-speed supematants were harvested and
centrifuged for 10 min. at 106,000 x g to generate cytosols (106,000 x g supematants) and
mixed mitochondrial-microsemal pellets (106,000 x g pellets). These pellets were suspended
in 30 volumes of phosphate buffer (10 mM KPO4, 100 mM KCl, 1 mM EDTA; pH 7 .4) and
sonicated. Aliquots were then incubated for 1 h at 37°C with 0.3 11M
[3H-Bl]androstenedione. The reactions were stopped with 10% trichloroacetic acid
containing 20 mg/ml charcoal, and the 31-120 generated was puriﬁed on small Dowex columns.
The reaction exhibited Michaelis-Menton kinetics in all three strains of mice (C57BV6J: intact,
Vmax = 78.34 ﬁnol/hmg protein, Km = 10.10 nM; castrate, Vmax = 31.2 ﬁnol/h'mg protein;
Km = 5.92 nM; DBA/2J2 intact, Vmax = 103.1 fmth'mg protein, Km = 4.37 nM; castrate,
Vmax = 52.76 ﬁnol/hmg protein; Km = 24.3 nM; B6D2F]: intact, Vmax = 88.31 fmol/hmg

protein, Km = 7.47 nM; castrate, Vmax = 42.42 ﬁnol/h'mg protein; Km = 4.97 nM).

Statistical Analysis
Aromatase activity data within brain region were analyzed by parametric 2 way
AN OVA (genetic strain by gonadal status) followed by post hoc Student-Newman-Keuls'

multiple range test (SNK) for ANOVA's with F values associated with p's s .05.

86

RESULTS

In the POA, compared to intact DBA males, AA levels were lower in intact C57 and
B6D2F] males (ANOVA E(2,30) = 7.84, p < 0.05; SNK, 9 < 0.05)(Figure 10). Castration
reduced AA in POA all three strains (E(1,30) = 249.90, p < 0.05; SNK, 2 < 0.05), and
eliminated the differences in AA among the strains (E(2,30) = 3.78, p < 0.05; SNK, 2 > 0.05)
(Figure 10).

Similarly, in the HYP, compared to intact DBA males, AA levels were lower in intact
C57 and B6D2F] males (ANOVA E(2,30) = 4.61, p < 0.05; SNK, 9 < 0.05)(Figure 10).
Castration reduced AA in POA all three strains (E(l,30) = 119.90, p < 0.05; SNK, 2 < 0.05),
and eliminated the differences in AA among the strains (F (2,30) = 1.21, p = 0.312) (Figure
10).

In the AM, AA levels did not differ among the three strains (ANOVA F(2,30) =
1.104, p = 0.3446). Castration signiﬁcantly reduced AA in all three strains (150,30) = 24.003,
p < 0.05), and as in the other brain regions, there was no difference in AA among the three

strains of mice (E(2,30) = 0.412, p = 0.6662)(Figure 10).

SUMMARY
These data demonstrate that AA levels in some brain regions of intact male mice are
inﬂuenced by genotype, while in another brain region AA did not vary among genotypes
analyzed here. DBA/2] males had higher levels of AA in the POA and HYP compared to
C57BL/6J and B6D2F1 males, however, in the AM, AA did not differ among the strains.

Castration reduced AA levels in all three regions of the brain in all three strains and eliminated

87

differences among the strains in AA levels in the POA and HYP. Although the behavioral and
physiological signiﬁcance of different AA levels among strains of intact male mice is unclear,
these differences in AA do suggest that there are physiological differences among these strains
that may account for diﬂ‘erences in behavioral and physiological responses among strains of

mice.

88

Figure 10. Mean (+/- s.e.m.) aromatase activity levels (ﬁnol/h/mg protein) in the preoptic
area (POA), hypothalamus (HYP), and amygdala (AM) for intact (Int), and castrated
C57BL/6J, DBA/2] and B6D2Fl male house mice. * = signiﬁcantly greater than other strains

within surgical treatment group (SNK, p < 0.05).

Aromatase Activity 3H20 fmollh-mg protein

89

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EXPERIMENT 6: SERUM CONCENTRATIONS OF TESTOSTERONE,
ESTRADIOL AND DIHYDROTESTOSTERONE IN B6D2Fl INTACT, CASTRATE,
AND CASTRATE-ADRENALECTOMIZED MALES
INTRODUCTION

A possible source of gonadal-like steroids in castrated B6D2F 1 males is the adrenal
gland. However, some castrated B6D2Fl males, as well as cats and dogs, continue to
cepulate after adrenalectomy (Beach, 1970; Cooper & Aronson, 1958; Schwartz & Beach,
1954; Thompson et al., 1976). Thus, other sources of steroid hormones may exist, since
maintenance of copulatory behavior in B6D2Fl males appears estrogen dependent.

As reviewed earlier, the brain is a potential source of steroid hormone synthesis and
E2 production (review, (Corpechot et al., 1981; Le Goascogne et al., 1987; Rebel, Bourreau,
Cerpechot, Dang, Halberg, Clarke, Haug, Schlegel, Synguelakis, Vourch, & Baulieu, 1987;
Rebel, Synguelakis, Halberg, & Baulieu, 1986). The ability of the brain to produce
measurable circulating levels of E2 is not unprecedented, since the brain of the zebra ﬁnch has
been shown to be the birds major source of estrogen synthesis (Schlinger & Arnold, 1991;
Schlinger & Arnold, 1992).

To determine if there are tissues other than the adrenal gland that synthesize T, DHT
or E2, serum concentrations of T, DHT and E2 were measured by RIA in intact, castrate,

and castrate-adrenalectomized B6D2Fl males.

90

91

METHODS
Subjects
Sixty-day old male B6D2F 1 hybrid house mice (Mus mugales) were purchased from
Jackson Laboratory, Bar Harbor, ME. All males were individually housed while in the colony
and maintained on a 14:10 light-dark cycle with lights out at 11:30AM. Food and water were

available 9.4 1161mm except during behavioral testing.

Surgical procedures

At approximately 150 days of age, all males were either castrated or sham castrated.
Castration surgeries were performed under methoxyﬂurane anesthesia (Metofane;
Pittman-Moore, Inc.). Adrenalectomies were performed six months after castration or sham
castration surgery. Castrated and sham castrated males were either bilaterally
adrenalectomized or sham adrenalectomized to produce the following groups:

1) sham castrated/sham adrenalectomized

2) sham castrated/adrenalectomized

3) castrated/sham adrenalectomized

4) castrated/adrenalectomized.
Males in the castrated/adrenalectomized group were divided into two recovery groups.
Blood samples were taken from one set of males 3 days after adrenalectomy, and from the
second set 14 days after adrenalectomy.

Each male was given a 0.05cc IP injection of 30mg/ml of pentobarbital. If this dose
did not produce an adequate anesthetic state, methoxyﬂurane (Metophane; Pittman-Moore)

inhalant was administered. Adrenalectomies were performed under a dissection microscope

92

at 10X. An incision was made on the lateral dorsal side of the abdomen just caudal to the
ribs. The tissue ventral to the adrenal gland was clamped with toothed forceps and ligated
ventral to the forceps with 0-4 silk. The musculature of the abdomen was sutured with
catgut, and the skin was closed with wound clips. In a rare case where bleeding occurred that
could be controlled, a small piece of Gel Foam was inserted in the abdominal cavity rostral

to the kidney and the abdominal cavity was sutured closed.

Steroid Hormone Radioimmunoassay (RIA)

Males were anesthetized and approximately lml of blood was collected from the right
ventricle of each animal individually and allowed to coagulate in an ice bath. The blood was
centrifuged and serum was collected and quickly frozen. The coded serum samples were
shipped on dry ice to the Hormone Assay Core of the Population Research Center at
University of California, Los Angeles to determine the concentrations of T, DHT and E2 by
RIA.

To monitor recovery, tracer amounts of [3H]T/EZ, DHT/estrone or androstenedione
were added to alternate serum samples and then the serum was extracted with diethyl ether
(10:1 v/v). The organic phase was separated from the aqueous phase and dried under a
stream of dry, ﬁltered air. The dried extract was then solubilized in 0.5ml of nanograde
isooctane. Samples were then applied to celite chromatography columns for fractionation
(Abraham, 1977).

T and E2 were analyzed in an [mu-RIA with reagents obtained from ICN
Biomedicals, Inc. (Costa Mesa, CA) and counted in a micromedic 4/600 gamma counter with

automatic data reduction software (RIA AID; Robert Maciel and Associates, Inc., Arlington,

MA 1
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93

MA 02174). Standard curves were calculated using the four parameter logistic option. DHT
was analyzed in a [3H]-RIA utilizing charcoal separation methods with reagents from ICN
Biomedicals, Inc, and counted in a LS355 liquid scintillation counter. Data were calculated
using the software described above.

The within assay coefficient of variation were less than 6% for each assay. Between
assay error was not applicable, since all hormones were run in one assay. The limits of
detection for the RIA were as follows: T, 0.07ng/ml; DHT, 0.05 ng/ml; E2, 19.20 pg/ml.

RIA data were analyzed by parametric 1 way ANOVA followed by post hoc
Newman-Keuls' multiple range test (N-K) for AN OVA's with F values associated with p's s
.05. For statistical purposes, values below the limit of detection of the assay were assigned
the value of the limit of detection.

RESULTS

Serum T levels were reduced by adrenalectomy alone, castration alone and the
combination of castration and adrenalectomy (F(4,35) = 19.3, p < .0001; N-K p < .05)
(Figure 11). It appeared that castration alone reduced T levels further than adrenalectomy
(N-K p < 0.05), however, T levels were not signiﬁcantly different between adrenalectomized
and the castrated/adrenalectomized groups. T levels were below the limits of detection in
four of the castrated/sham males, one castrated/adrenalectomized 3 day males and four
castrated/adrenalectomized 14 day males.

In contrast, castration, adrenalectomy, and the combination of castration and
adrenalectomy had no effect on serum E2 levels compared to intact males (F(4,36) = 0.463,
p = 0.7627) (Figure 12). E2 levels were below the limits of detection in only 1 intact male.

Castration, adrenalectomy and the combination of castration and adrenalectomy did

94

not aﬂ‘ect the level of serum DHT compared to intact males (F(4,38) = 0.883, p = 0.4833)
(Figure 13). DHT levels were below the limits of detection in 2 intact males, one
castrated/sham male, six castrated/adrenalectomized 3 day males and seven
castrated/adrenalectomized 14 day males. Therefore the results of this assay may not reﬂect

actual physiological values.

SUMMARY
Results of this experiment support the idea that steroid hormones (T, E2, and DHT)
are present after removal of the testes and adrenal glands. Serum T levels were decreased by
adrenalectomy alone and even further by castration, however, the combination of castration
and adrenalectomy was net additive in their effects on reduction of serum T levels. In
contrast, the serum concentrations of E2 and DHT, the metabolites of T, were not affected

by adrenalectomy, castration, or the combination of castration and adrenalectomy.

95

Figure 11. Mean (+/- s.e.m.) concentration of testosterone (T) in serum of sham
castrate/sham adrenalectomized (S/S), sham castrate/ sham adrenalectomized (Cas/S),
castrate/3 day adrenalectomized (Cas/Adx3), castrate/ 14 day adrenalectomized (Cas/Adx14)
B6D2Fl males. * = signiﬁcantly less than S/S group (N-K p < 0.05). + = signiﬁcantly less that

S/Cas group (N—K p < 0.05).

Scrum Testosterone (ng/ml)

1.5 -

1.0 -

0.5 -

 

0.0 -

96

i

     

'5 x“
0* 31»
S/S S/Adx Cas/S c g1» mgr»
Surgical Treatment

Figure 11

97

Figure 12. Mean (+/- s.e.m.) concentration of l7B-estradiol (E2) in serum of sham
castrate/sham adrenalectomized (S/S), sham castrate/ sham adrenalectomized (Cas/S),
castrate/3 day adrenalectomized (Cas/Adx3), castrate/14 day adrenalectomized (Cas/Adxl4)

B6D2Fl males.

98

400 F

é’

N
8

100

Serum Estradiol Levels (pg/ml)

 

 

SIS SIAdx CuIS CuIAde CuIAdxM

Surgical Treatment

Figure 12

99

Figure 13. Mean (+/- s.e.m.) concentration of dihydrotestosterone (DHT) in serum of sham
castrate/sham adrenalectomized (S/S), sham castrate/ sham adrenalectomized (Cas/S),

castrate/3 day adrenalectomized (Cas/Adx3), castrate! 14 day adrenalectomized (Cas/Adx14)

B6D2Fl males.

Serum DHT Levels (nglml)

100

0.15 -

P
A
O

l
I

 

 

0.05 -

 

Serum DHT Levels (ng/ml)

 

    

0.00 J
'5 x“
6* a.
S/S S/Adx Cas/S 05w 0 3}.

Surgical Treatment

Figure 13

[[11

C1

GENERAL DISCUSSION

The results of the present studies are consistent with the idea that nongonadal steroids,
in particular estrogens, contribute to the maintenance of copulatory behavior aﬁer castration
in male B6D2Fl hybrid house mice.

The ability to achieve an ejaculatory reﬂex and intromission after castration in
B6D2Fl male house mice is dependent on the aromatization of nongonadal androgens to
estrogens that are present after castration. Although circulating T levels are reduced by
castration, or castration and adrenalectomy, E2 levels remain unaffected. The ability to
convert androgens to estrogens in regions of the brain important for copulatory behavior
(POA, HYP, and AM) was reduced but not eliminated aﬁer castration. Likely, it is in these
regions where the aromatase inhibitor ATD is having its behavioral effects. Furthermore,
estrogenic stimulation appears to be maintained in POA and HYP aﬁer castration, and is
present in the AM as well, but at reduced levels. However, continuer and noncontinuer males

did not differ in serum T, E2 and DHT levels, AA or ER levels in the POA, HYP and AM.

Effect; of Qastration on Steroid Hormone Levels in gontinuer and Noncontinggr Males

Serum T levels were decreased by castration but were not different between castrated
continuer and noncontinuer males as seen previously (Clemens, et al., 1988). However,

serum E2 levels were not affected by castration in either continuer or noncontinuer B6D2Fl

101

males.
rhesus
nongo:
Resko
castrai
aroma
suppo
stimu'
with h
males
consi
reSpc

Youn

and it
adren

may ]

0sz

data

102

males. This differential effect of castration on T and E2 levels has been observed in rats,
rhesus monkeys, and ferrets indicating that serum E2 is maintained by an unknown
nongonadal source (Carroll, Weaver & Baum, 1988; Roselli & Resko, 1984; West, Roselli,
Resko, Greene & Brenner, 1988). Since T is not eliminated from the circulation after
castration, it may facilitate behavior via androgenic stimulation and act as a substrate to be
aromatized to E2. More interestingly though, castration does not affect E2 levels which
supports the notion that copulation aﬁer castration may be maintained by estrogenic
stimulation. Although retention of copulatory behavior after castration was not associated
with higher serum levels of T, E2 or DHT in continuers, it is possible that continuer B6D2Fl
males are more responsive to hormone stimulation than noncontinuers. This notion is also
consistent with ﬁndings on other individual diﬂ‘erences, e. g. males with "high sex drives”
respond diﬂ‘erently to the same level of hormones as males with "low sex drive" (Grunt &

Young, 1952; Larsson, 1966; Whalen et al., 1961).

Eﬁgtg of Adrenalectomy on Testosterone and Estradiol Levels

Adrenalectomy reduces serum T levels, but not to the same extent that castration
does. However, adrenalectomy and castration were not additive: removal of both adrenals
and testes did not decrease T levels any further than castration alone. Therefore, although the
adrenals appear to contribute to circulating levels of T, their effect on circulating levels of T
may be via indirect regulation of steroidogenesis in the testes.

As seen with the testes, the adrenal glands do not appear to regulate circulating levels
of E2. Removal of both adrenals and testes also did not aﬂ‘ect E2 levels. Thus, based on these

data the adrenal gland does not contribute signiﬁcantly to circulating E2 levels either by

pror
We
not

Hor

B62
res

hor

Scl

ma

be:
wir

me

ac]

103

providing aromatizable androgens or as a site for aromatization of androgens to estrogens.
This lack of adrenal contribution to E2 levels has been seen in the zebra ﬁnch where AA was
not detectable in the adrenals (Schlinger & Arnold, 1991; Schlinger & Arnold, 1992).
However, substrates that can be aromatized to estrogens are synthesized in the zebra ﬁnch
adrenal which may be aromatized elsewhere (the brain) to contribute to circulating E2 levels
(Schlinger & Arnold, 1991).

That circulating levels of T and E2 remain after adrenalectomy and castration in
B6D2Fl males demonstrates that continued copulation aﬁer removal of both adrenals and
testes as seen in B6D2Fl mice, cats and dogs may still be under the inﬂuence of steroid
hormones and independent of adrenal hormones (Beach, 1970; Cooper & Aronson, 1958;
Schwartz & Beach, 1954; Thompson et al., 1976). However, in the rat, adrenals appear to
secrete androgens necessary for the ability of E2 to activate copulatory behavior in castrated
males (Gorzalka et al., 1975).

As reviewed earlier, a potential source of nongonadal-nonadrenal androgens and
estrogens is the brain. Local metabolism of circulating steroid hormones in neural tissue has
been well established. However, the dc M9 synthesis of neurosteroids ﬁom cholesterol
within neural tissue is a potential mechanism by which the brain could be inﬂuenced by
metabolically active steroids that aﬁ‘ect behavior (Baulieu, 1981; Corpechot et al., 1981; Le

Goascogne et al., 1987; Robe] & Baulieu, 1995).

lb! Effgg gt" Inhibiting Aromatase on Copulatog Behavior

When castrated males were treated with ATD, fewer continuer B6D2Fl males

achieved ejaculations and intromissions compared to their pretreatment test as well as

inr

1'65

ha

ab

CO

01'

dir

104

continuers that received blank silastic capsules. The results are consistent with the idea that
nongonadal estrogens contribute to the maintenance of copulatory behavior alter castration
in male B6D2Fl hybrid house mice.

The inhibitory effects of ATD on copulation in continuer B6D2Fl males probably
resulted from the ability of ATD to block neural aromatization of androgens to estrogens
(Brodie, Marsh, Wu, & Brodie, 1979; Lieberburg, Wallach, & McEwen, 1977). The
importance of local neural aromatization of T to E2 for the activation of copulatory behavior
has been demonstrated in castrated rats. ATD implanted directly in the POA inhibits the
ability of T implants in the POA to restore mounting behavior (Christensen & Clemens,
1975). Castrated B6D2Fl males retain aromatase activity in POA as well as the amygdala
and hypothalamus where non-gonadal T may be converted to estrogen (EXP 3). Thus, it is
likely that ATD treatment inhibited aromatase activity in these brain regions of continuer
B6D2Fl males which reduced the amount of estrogen available to bind with estrogen
receptors that are maintained in the POA, HYP and AM of castrated B6D2Fl males even
after castration (EXP 5). However, since ATD was administered systemically, both
peripheral and CNS aromatization of androgens to estrogens were inhibited. Thus, estrogens
that normally originate ﬁom the periphery that may have acted in the CNS to facilitate
copulatory behavior would be reduced as well. Therefore, the possibility that estrogen
originating in the periphery may be in part responsible for maintenance of copulation aﬁer
castration in continuer B6D2Fl male mice cannot be excluded.

In contrast, ATD did not affect the percent of continuer males that mounted, nor their
latency to mount which is in contrast to the rat (Christensen & Clemens, 1975). While ATD

did not block mounting, it is still possible that E2 plays some role in this behavior, since it is

105

unlikely that ATD blocked all estrogen production. Alternatively, mount behavior may be
maintained in ATD treated continuer males via androgenic stimulation, since androgens are
not completely eliminated by castration (EXP’s 1 and 6). Furthermore, since ATD is a steroid
it is possible that ATD may have some action of its own (Christensen & Clemens, 1975;
Kaplan & McGinnis, 1989; Landau, 1980). This is unlikely, however, since ATD treatment

did not facilitate mounting in noncontinuer B6D2Fl males.

1-. . ;I'Illlr‘ nmt;; Acii _r 'n rnin rnann In
Mala

Following castration, AA in POA, HYP, and AM was reduced but not eliminated. In
none of these three brain regions was the level of AA associated with the maintenance of
copulatory behavior aﬁer castration in continuer and noncontinuer B6D2Fl males. Although
reductions in neural AA and aromatizable androgens (T) indicate that local neural
concentrations of estrogens may be reduced after castration, nonetheless, these results in
conjunction with ATD inhibiting copulation support the notion that maintenance of
copulatory behavior alter castration may be due to estrogenic stimulation derived ﬁ'om the
aromatization of nongonadal androgens in regions of the brain important for copulatory
behavior.

As seen in other species, AA in B6D2Fl males appears to be regulated by gonadal
hormones (Connolly, Roselli, & Resko, 1990; Roselli et al., 1984; Roselli et al., 1985; Roselli
et al., 1987a; Roselli & Resko, 1984; Roselli & Resko, 1986; Roselli & Resko, 1989; Roselli
et al., 1987b; Weaver & Baum, 1991). The decline in AA levels in the POA, HYP, and AM

following castration may indicate that AA in B6D2Fl males is regulated by gonadal

androg
Resko,

of T 1e

were e
POA,
Continr

91 al.

Contra
asSOci.
Castrat
"Ongor

B6D2F

106

androgens as seen in other species (Roselli et al., 1984; Roselli & Resko, 1984; Roselli &
Resko, 1986; Roselli & Resko, 1989). In other species, AA in parts of the AM is independent
of T levels, while in other regions of the AM they are not (Roselli et al., 1984). Because the
AM dissection in the present study contained a mixture of these regions, it cannot be

determined if regulation of AA in regions of the AM of B6D2Fl males is independent of

gonadal hormones.
ﬂ’ 1’ i n n Es ro en R e tor Lev Is in Continuer n N ncontinu r
Male;

Nuclear Estrogen Receptors (ERn)

Castration did not aﬁ‘ect ERn levels in the POA and HYP, but did signiﬁcantly reduce
ERn levels in the AM. It should be noted, that ERn levels (ﬁnal/mg DNA) were greater in
the AM than in either the POA or HYP of intact males, and that after castration, ERn levels
were equivalent in all three brain regions. Although present after castration, ERn levels in the
POA, HYP, and AM were not associated with expression of copulatory behavior in
continuers and noncontinuers, but suggest that estrogenic stimulation is still present (Gorski
et al., 1986).

Maintenance of ERn levels after castration in the POA of B6D2Fl males mice is in
contrast with those in rats. In the rat, ERn levels in POA were reduced by castration and
associated with circulating androgen levels (Krey et al., 1980). However, it is unclear in
castrated B6D2Fl mice, whether ERn in POA and HYP are affected by circulating
nongonadal estrogens. In the AM, ERn levels may be associated with T concentrations in the

B6D2Fl male since both were reduced after castration, as reported in rats (Krey et al., 1980;

 

10

in

di

107
Roselli et al., 1993).

The present observation that POA ERn levels are unchanged alter castration in
B6D2Fl males contrasts with our previous report that ERn in POA declined in castrated
B6D2Fl males (Clemens et al., 1988). Diﬁ‘erences in preparation of the nuclear ﬁaction
prior to measurement of ERn do not account for differences between the two studies.
Pruiﬁcation and nonpuriﬁcation of the nuclear pellets prior to KCL extraction produce similar
results in measuring ERn levels between brain regions and with respect to the effects of
castration However, sucrose puriﬁcation reduced the total amount of DNA extracted from
the nuclear pellet by an average of 37 percent compared to the nonpuriﬁed nuclear pellet.
Further, sucrose puriﬁcation may aﬁ‘ect ERn levels in that the concentrations of ERn
(fmol/mg DNA) were reduced in the AM compared to nonpuriﬁed samples; however, sucrose
puriﬁcation did not affect ERn levels in the POA or HYP (EXP 4B). Sucrose puriﬁcation
may also artiﬁcially increase ERc levels (expressed as frnol/mg DNA) since the ratio of ERc
to mg DNA would be greater due to decreased DNA recovery.

It is unlikely that differences in ERn between the two studies were due to the use of
frozen versus fresh tissue, since fresh tissue appears to yield greater ERn levels than ﬁ'ozen
tissue (MacLusky, et al., 1986), and ERn levels in the POA of intact males were comparable
between the studies (between 20-30 ﬁnol/mg DNA). Nonetheless, the differences that
occurred between the two studies may have been due to diﬁ‘erences in the dissections,
heterogeneity of the tissue, and/or the length of time the animals were castrated.

In contrast to EXP 4A, although there appeared to be a slight reduction, castration
did not signiﬁcantly reduce ERn levels in the AM in EXP 4B. It is possible the effects of

castration on ERn levels in the AM are time dependent since the assays were run 5 (EXP 4B)

and 38

been 0t

Castral
mainly
(EXP .
while E
or non
paralk
mRNA
regior
mRNr
(hub
is that
11011121]
by Dr

dyna;

("1
:2"
n

/

.3

/:.?'

108

and 38 (EXP 4A) weeks aﬁer castration. Alternatively, the effects of castration may have

been obscured by a lower number of animals assayed, and/or tissue heterogeneity.

Cytosolic and Total Estrogen Receptors (ERc & ERt)

Our results suggest that ERt levels were down-regulated by testicular hormones.
Castration increased ERt levels in the POA, HYP, and AM. These increased ERt levels
mainly reﬂect changes in ERc levels, since ERc levels are 3 to 7 times greater than ERn levels
(EXP 4A). ERc levels were increased in the HYP of noncontinuers compared to intacts,
while ERc levels in the HYP of continuers were not signiﬁcantly different from either intact
or noncontinuer males. Down regulation of ERt in B6D2Fl males by gonadal hormones may
parallel regulation of ER mRN A levels seen in the male rat where castration increased ER
mRNA in numerous nuclei (Lisciotto & Morrell, 1993; Morrell et al., 1995). In some brain
regions it appears that both androgens and estrogens may play a role in regulation of ER
mRNA levels, while in other brain regions gonadal hormones do not affect ER mRNA

(Lauber et al., 1991; Simerly & Young, 1991). An alternative explanation for increased ERt
is that castration elicits a slower turnover or degradation rate of ER protein. However, the
actual amount of ER synthesized, used and degraded per unit of time cannot be determined
by our method of analysis. Thus, gonadal hormones appear to regulate ER levels and

dynamics, but the mechanism of action has yet to be deterrrrined.

f n n Aromatase Activi in CS7BL/6J DBA/2J nd B6D2Fl M I
Miss

The data demonstrate that AA levels in some brain regions of intact male mice are

rhrmced
greater let
do not dt
signiﬁcam
trim
may accou
Incentrasr
he strains
aeration 2

mi AM.

109

inﬂuenced by genotype, while in others it is not. In the POA and HYP, DBA/2] males have
greater levels of AA than C57Bll2] and B6D2Fl males. However, levels of AA in the AM
do not diﬂ‘er among the three strains of mice. Although the behavioral and physiological
signiﬁcance of different AA levels among strains of intact male mice is unclear, these
diﬂ'erences in AA do suggest that there are physiological diﬂ‘erences among these strains that
may account for diﬂ‘erences in behavioral and physiological responses among strains of mice.
In contrast, AA levels in all three strains were reduced by castration and diﬁ‘erences among
the strains in AA levels were eliminated. Therefore, strains that continue to copulate after
castration and those that do not could not be distinguish by their levels of AA in POA, HYP

andAM.

E17 1’ tion on Aromatase Activi Levels in C57BV6J DBA/2.1 and B6D2Fl
Mala
Following castration, AA in POA, HYP, and AM was reduced in males of all three
Strains. Furthermore, castration eliminated differences in AA levels that were seen between
the three strains of intact males. Thus, we could not distinguish between these three strains
0f males based on AA levels in regions important for copulatory behavior (POA, HYP, and
AM) aﬁer castration.

The decline in AA levels in the POA, HYP, and AM following castration may indicate
that AA in C57BV61, DBA/2] and B6D2F 1 males is regulated by gonadal androgens as seen
in Other species (Connolly et al., 1990; Roselli et al., 1984; Roselli & Resko, 1984; Roselli
& Resko, 1986; Roselli & Resko, 1989; Roselli et al., 1987b; Weaver & Baum, 1991).

However, in rats, AA in parts of the AM is independent of T levels, while other regions of the

110

AM are not (Roselli et al., 1984). Because our AM dissection contained a mixture of these
regions, we cannot determine if regulation of AA in regions of the AM of male mice in these
three strains is independent of gonadal hormones.

That the levels of AA were not different between the strains aﬁer castration may
reﬂect the limited ability of our assay to discriminate diﬁ‘erences at the cellular level. On the
other hand, the failure to see differences in AA levels between strains that continue copulate
after castration and those that do not suggests that other estrogenic processes that may
facilitate reproductive behavior may differ between the strains. These processes would not

be revealed by our analysis.

Qiﬂ'gggnggs Between Continuer and Noncontinggr B6D2Fl Mglg

Since circulating levels of T, DHT and E2 do not differ between continuers and
noncontinuers, continuer B6D2Fl males appear to be more responsiveness to steroids than
noncontinuer males. This difference in responsiveness cannot be accounted for at the level of
the steroid receptor since aromatase activity and estrogen receptor levels in the POA,
amygdala and hypothalamus do not differ between continuers and noncontinuers (EXP 4).
Alternatively, continuers and noncontinuers may differ in their responsiveness to androgenic
stimulation, since androgens appear important for copulation. For example, DHT restores
copulatory behavior to noncontinuer B6D2Fl males (Sinchak & Clemens, 1990).
Furthermore, estradiol and DHT given simultaneously restore copulatory behavior in the CD-
1 strain of house mouse as well as rats better than either hormone individually (Baum,
Sodersten, & Vreeburg, 1974; Baum & Vreeburg, 1973; Larsson, Sodersten, & Beyer,

1973a; Larsson et al., 1973b; Wallis & Luttge, 1975).

(I)

c:
a
"S

m .
Mari.

Ethan

111

n ncl i n

The present studies demonstrate that maintenance of copulatory behavior aﬁer
castration in B6D2Fl male house mice is dependent on the aromatization of nongonadal
androgens to estrogens that are present aﬁer castration. Although a reduction in circulating
T levels occurs aﬁer castration, or castration and adrenalectomy, E2 levels are not aﬁ‘ected.
The ability to convert androgens to estrogens is reduced but not eliminated aﬁer castration
in regions of the brain important for copulatory behavior (POA, HYP, and AM). Likely, it
is in these regions where the aromatase inhibitor ATD is having its behavioral effects.
Furthermore, estrogenic stimulation appears to be maintained after castration in POA and
HYP, and present in the AM, but at reduced levels as reﬂected by ERn levels.

It is not surprising that numerous sites within the body synthesize steroid hormones,
since steroids interact at numerous levels to organize and regulate vital metabolic and
behavioral functions. Therefore, although gonadal steroid hormone synthesis and copulatory
behavior are associated in the B6D2Fl house mouse, our data support the idea that non-
gonadally produced steroid hormones facilitate copulatory behavior in the castrate B6D2Fl
male house mouse.

Although continuers and noncontinuers could not be distinguished by serum T, E2 or
DHT levels, AA or ER levels in the POA, HYP and AM, the ability to study a genetically
homogenous strain that behaves differently after castration is an important model to the

determination of the role of steroid hormones and their mechanisms that regulate behavior.

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Akess

Alexar

has

rirendr

rilgiol;

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