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This is to certify that the
dissertation entitled

THE DEVELOPMENT OF FEMALE- AND MALE-ORIENTED
PARTNER PREFERENCE IN THE MALE LABORATORY RAT:
INDICATION FOR THE ORGANIZATIONAL ROLE OF GONADAL
HORMONES

presented by
Casey Leigh Henley

has been accepted towards fulfillment
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PhD. degree in Zoology

 

 

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5/08 KIProj/Acc8Pres/ClRC/DateDueindd

THE DEVELOPMENT OF FEMALE- AND MALE-ORIENTED PARTNER
PREFERENCE IN THE MALE LABORATORY RAT: INDICATION FOR THE
ORGANIZATIONAL ROLE OF GONADAL HORMONES
By

Casey Leigh Henley

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Zoology

2008

ABSTRACT
THE DEVELOPMENT OF FEMALE- AND MALE-ORIENTED PARTNER
PREFERENCE IN THE MALE LABORATORY RAT: INDICATION FOR THE
ORGANIZATIONAL ROLE OF GONADAL HORMONES
By

Casey Leigh Henley

Sexual differentiation of brain and behavior takes place during a critical period in
development. In males, this is a time of masculinization, the enhancement of brain
systems that mediate male-typical responses, and defeminization, the suppression of brain
systems that mediate female-typical responses. In the rat, adult male partner preference is

organized early in development by testicular steroids.

There is broad acceptance for the idea that the estradiol metabolite of testosterone is
responsible for the organization of the neural systems that mediate reproductive behavior
including partner preference in the rat. The notion that estradiol plays an organizational
role in development of partner preference is drawn from studies where estrogen action
was in some way blocked, either through aromatase or estrogen receptor inhibition during
development in male rats. Lack of estrogens neonatally results in a decrease in the male’s
preference for a female. Therefore, it is likely that estrogens are responsible for the

development of female-oriented partner preference in the male.

Super-physiological doses of testosterone (hyper—androgen) during development may also
affect partner preference. In the rat, an animal that goes through a male-typical

development will show male sexual behavior in adulthood. Administration of

testosterone after neonatal castration also causes masculinization of behavior. However,
if an intact male is treated neonatally with testosterone, male sexual behavior in
adulthood is disrupted. There is a possibility that the hyper-androgen treatment is altering
the male’s partner preference, decreasing the male’s interest in a female partner, and thus
resulting in a suppression of male sexual behavior. Therefore, exposure to super-
physiological levels of androgen during development may play a role in the development

of male-oriented partner preference in the male.

In this dissertation, I examine the role of early postnatal treatment with steroid hormones
in the development of adult partner preference in the laboratory rat. The role of estrogen
in the development of female-oriented adult partner preference and sexual behavior in the
male rat will be evaluated initially by treating females during the early postnatal period
with estradiol and determining if their behavior was masculinized. Then the role of
hyper-androgen exposure during development on the expression of male-oriented partner
preference and sexual behavior in the male will be examined, followed by a study
determining if the estradiol metabolite of testosterone is responsible for the effects seen
after hyper-androgen exposure in the male. Finally, to rule out any possibility that the
hormonal effects seen were due to changes in maternal behavior, the role of maternal
licking and grooming on adult partner preference and sexual behavior in males and

females will be assessed.

I would like to dedicate this dissertation to my parents, John and Gail McGovern, and to
my husband, Kevin Henley. Without their unwavering support, encouragement, belief,

and patience, I would not be the scientist, scholar, or person I am today.

iv

ACKNOWLEDGMENTS

I would like to thank my major professor, Dr. Lynwood G. Clemens, for his guidance,
advice, and support throughout my late undergraduate and graduate career. He taught me
about science and life, gave me confidence when I needed it and direction when I was
lost, led me to build more character than I sometimes wanted, and prepared me to take

my degree into the world to continue the advancement of knowledge.

I would also like to thank my dissertation committee members, Drs. Antonio A. Nunez,
Cynthia L. Jordan, S. Marc Breedlove, and Lori L. Badura for all of their help and
suggestions on my dissertation projects. Along with Lyn, Tony taught me how to
approach research with an open, yet critical mind. Cindy and Marc provided me with
technical assistance and use of their laboratory facilities whenever needed, and Lori
offered me a view into the world of industry and science I would not have received

otherwise.

Further thanks must go to those individuals who spent countless hours with me in the lab,
and without whom many of my studies may have never been completed successfully.
Erin Gruley, Melannie Richmond, Erinn Laimon-Thomson, Scott Taylor, Mike Daniels,
Kyle Keener, Jamie Joswick, and Sarah Hay provided me with unlimited assistance.
Additionally, the Zoology Department and Neuroscience Program office staff helped me

outside of the lab countless times, and I am appreciative of all their assistance.

 

I would also like to thank my friends who have been a network of support over the years,
whether near or far, old or new, scientist or not. Special thanks go to Cathy Peltz, Kristen
Osterlund, Damian Zuloaga, Jennifer Cummings, Jamie J ohansen, Kimberly. D’Anna,

Molly Ryan, and Elizabeth Lang.

Finally, my sincerest gratitude must be expressed to my family. My husband, parents,
brother, and grandparents have always had faith in me, a faith that I hold dear and that

drives me to accomplish my best.

The work in this dissertation was supported by a National Institute of Health grant
(RDKO69991A) to L.G. Clemens. Financial assistance was also provided by National
Science Foundation Graduate Research Fellowship program, National Institute of Mental
Health Training Grant on the Integrative Neurobiology of Social Processes to the
Neuroscience Program, the Graduate School, the College of Natural Science, and the

Department of Zoology.

vi

TABLE OF CONTENTS

LIST OF TABLES ............................................................................................................. ix
LIST OF FIGURES ........................................................................................................... xi
CHAPTER ONE: General Introduction ............................................................................... 1
Partner Preference in Animal Models ........................................................................... 1
Partner Preference in Humans ...................................................................................... 9
Overview of Studies .................................................................................................... 12

CHAPTER TWO: Estrogen is Responsible for the Development of Female-
Oriented Partner Preference in the Male Laboratory Rat as Indicated by its

Masculinizing Effects on Adult Behavior in Females .................................................... 14
Introduction .................................................................................. . ............................... 14
Methods ........................................................................................................................ 15
Results .......................................................................................................................... 25
Tables and Figures ....................................................................................................... 32
Discussion .................................................................................................................... 45

CHAPTER THREE: Hyper-Androgen Exposure During Development Alters
Adult Partner Preference and Reproductive Behavior in Gonadally Intact Male

Rats ................................................................................................................................. 59
Introduction .................................................................................................................. 59
Methods ........................................................................................................................ 60
Results .......................................................................................................................... 66
Tables and Figures ....................................................................................................... 72
Discussion .................................................................................................................... 83

CHAPTER FOUR: Estrogen is Not Responsible for the Effects of Hyper-
Androgen Exposure During Development on Adult Partner Preference and

Reproductive Behavior in Gonadally Intact Male Rats .................................................. 90
Introduction .................................................................................................................. 90
Methods ........................................................................................................................ 9 1
Results .......................................................................................................................... 94
Tables and Figures ....................................................................................................... 98
Discussion .................................................................................................................. 109

CHAPTER FIVE: Natural Variation in Maternal Licking and Grooming Does Not

Alter Adult Partner Preference Expression in either Male or Female Rats .................. 115
Introduction ................................................................................................................ 1 15
Methods ...................................................................................................................... 1 17
Results ........................................................................................................................ 121
Tables and Figures ..................................................................................................... 125

vii

Discussion .................................................................................................................. 129

CHAPTER SIX: General Discussion ............................................................................... 133
Neural Control ........................................................................................................... 136
Future Directions ....................................................................................................... 140
Overall ........................................................................................................................ 145

REFERENCES ................................................................................................................ 146

viii

LIST OF TABLES

Table 2.1. One-sample t-test analyses of preference scores and duration of time
spent in the female chamber during the partner preference tests ................................. 32

Table 2.2. Proportion of experimental females that received sexual behavior from
the stimulus male during the partner preference tests .................................................. 33

Table 2.3. Proportion of experimental females that received sexual behavior from
the stimulus male during the female sexual behavior tests .......................................... 34

Table 3.1. One-sample t-test analyses of preference scores and duration of time
spent in the male chamber during the partner preference tests (Experiments 1
and 2) ........................................................................................................................... 72

Table 3.2. Proportion of experimental males that displayed sexual behavior with
the stimulus female during the partner preference tests (Experiments 1 and 2) .......... 73

Table 3.3. Proportion of experimental males that displayed sexual behavior with
the stimulus female during the social and sexual behavior tests (Experiments 1
and 2) ........................................................................................................................... 74

Table 3.4. One-sample t-test analyses of preference scores and duration of time
spent in the male chamber during the partner preference tests (Experiment 3) ........... 75

Table 3.5. Proportion of experimental males that displayed sexual behavior with
the stimulus female during the final partner preference test in Experiment 2. ............ 73

Table 4.1. One-sample t-test analyses of preference scores and duration of time
spent in the male chamber during the partner preference tests .................................... 98

Table 4.2. Proportion of experimental males that displayed sexual behavior with
the stimulus female during the final partner preference test and during the
social and sexual behavior test ..................................................................................... 99

Table 5.1. One-sample t-test analyses of preference scores and duration of time
spent in the male chamber during the female offspring partner preference tests ...... 125

Table 5.2. One-sample t-test analyses of preference scores and duration of time
spent in the female chamber during the male offspring partner preference tests ...... 126

LIST OF FIGURES

Figure 2.1. Treatment and testing schedule for the experimental females ........................ 35

Figure 2.2. Behavioral testing schedule for the experimental females receiving
early postnatal treatments of cholesterol (C, n=16) or estradiol benzoate [EB
either 5% (n=14) or 10% (n=16)] ................................................................................ 36

Figure 2.3. Photomicrograph of pelvic ganglion tissue processed for Nissl staining ........ 37

Figure 2.4. Time (in seconds) mothers of experimental females spent licking and
grooming during the first postnatal week. ................................................................... 38

Figure 2.5. Duration of time experimental females spent (A) with the stimulus
female, (B) with the stimulus male, and (C) in the middle chamber during
both the initial and final partner preference tests. Preference score (D) is
calculated as time spent with stimulus female minus the time spent with
stimulus male ............................................................................................................... 39

Figure 2.6. Time experimental females spent in the male area of the observation
chamber during the female sexual behavior tests ........................................................ 40

Figure 2.7. Frequencies of mounts, intromissions, and ejaculations displayed by
the stimulus male during the female sexual behavior tests .......................................... 41

Figure 2.8. Frequency of proceptive behaviors shown by experimental females
during the female sexual behavior test after adult treatment with EB + P .................. 42

Figure 2.9 Photomicrograph of cross-sections through ovarian tissue processed for
hematoxylin and eosin staining .................................................................................... 43

Figure 2.10. Number of motoneurons present in the SNB of experimental females ......... 44

Figure 3.1. Treatment and testing schedule for the experimental males
(Experiments 1 and 2) .................................................................................................. 76

Figure 3.2. Treatment and testing schedule for the experimental males
(Experiment 3) ............................................................................................................. 77

Figure 3.3. Behavioral testing schedule for the experimental males (Experiments
1-3) ............................................................................................................................... 78

Figure 3.4. Time spent with in the stimulus male chamber during the partner
preference tests (Experiments 1 and 2) ........................................................................ 79

Figure 3.5. Preference scores of the experimental males during the partner
preference tests (Experiment 1) ................................................................................... 80

Figure 3.6. Frequencies of mounts, intromissions, and ejaculations displayed by
the experimental males during the social and sexual behavior tests when
paired with a stimulus female (Experiments 1 and 2) .................................................. 81

Figure 3.7. Levels of circulating testosterone in a subset of males from

Experiments 1 and 2 ..................................................................................................... 82
Figure 4.1. Treatment and testing schedule for the experimental males ......................... 100
Figure 4.2. Behavioral testing schedule for the experimental males ............................... 101

Figure 4.3. Time (in seconds) mothers of experimental females spent licking and
grooming during the first postnatal week .................................................................. 102

Figure 4.4. (A) Duration of time experimental males spent in the stimulus male
chamber and (B) preference scores ............................................................................ 103

Figure 4.5. Frequencies of mounts, intromissions, and ejaculations displayed by
the experimental males during the final partner preference test ................................ 104

Figure 4.6. Frequencies of mounts, intromissions, and ejaculations displayed by
the experimental males during the social and sexual experience test when

paired with a female ................................................................................................... 105
Figure 4.7. Mount and intromission latencies shown by the experimental males

during the social and sexual experience test when paired with a female ................... 106
Figure 4.8. Number of neurons present in the PG of experimental males ....................... 107
Figure 4.9. Levels of circulating testosterone in the experimental males ........................ 108

Figure 5 .1. Time female offspring spent in the stimulus male chamber during the
partner preference tests .............................................................................................. 127

Figure 5.2. Lordosis quotient of the female offspring during the female sexual
behavior test ............................................................................................................... 128

xi

CHAPTER ONE:

GENERAL INTRODUCTION

Sexual differentiation of brain and behavior takes place during a critical period in
development (Baum, 1979; Baum, 2006). This is a time of masculinization, the
enhancement of brain systems that mediate male-typical responses, and defeminization,
the suppression of brain systems that mediate female-typical responses (Adkins-Regan,
1988; Bakker, 2003). Research on this topic was initiated by studying the organizing
effects of testosterone (Phoenix et al., 1959), and since then many studies have been
conducted examining the effects of steroid hormones secreted perinatally on sexual
differentiation in a number of animal models. In rats, castration at birth eliminates or
greatly reduces the expression of male sexual behavior in adulthood (Beach and H012,
1946) and increases the expression of female receptivity (Beach et al., 1969). Normal
masculine behavior is reinstated if testosterone is replaced soon after castration (Beach et
al., 1969). In addition, female rats treated neonatally with testosterone show a decrease in
receptivity (Barraclough and Gorski, 1962) and, in some cases, an increase in male
typical responses (Mullins and Levine, 1968; Whalen and Edwards, 1967). These
findings suggest that perinatal endogenous testosterone mediates behavioral sexual

differentiation in male rats.

Partner Preference in Animal Models
In addition to male sexual behavior, adult male partner preference is also organized early

in development by gonadal hormones. Partner preference can be assessed by giving an

experimental animal a choice between a sexually active male and a sexually receptive
female and measuring the amount of time spent in the vicinity of each stimulus animal.
There is a broadening literature studying numerous animal models to determine the role

steroid hormones play in the development of adult sex-typical partner preference.

Rat

In rats the normal adult preference usually seen is that for an opposite sex partner (Hetta
and Meyerson, 1978; Meyerson and Lindstrom, 1973). When given the choice between a
stimulus female or a stimulus male, males prefer to spend time with the female, and
females prefer the male. This choice has been shown to be influenced by testicular
steroids early in development. If a male is castrated at birth and given ovarian hormones
in adulthood, he will approach a sexually active male over an estrous female (Hetta and
Meyerson, 1978; Matuszczyk et al., 1988). Neonatal testosterone replacement after
castration, however, will result in the male-typical preference for an estrous female
(Matuszczyk et al., 1988; Meyerson et al., 1980). In addition, if a female is treated early
in life with testosterone, and also again in adulthood, she will prefer an estrous female
over a stimulus male (Dejonge et al., 1988; Meyerson et al., 1980; Meyerson and

Lindstrom, 1973).

Testosterone is a steroid hormone that can have its effects by acting directly on the
androgen receptor itself, or by being metabolized into dihydrotestosterone by 5d-
reductase (J affe, 1969) and into estradiol by aromatase (Ryan et al., 1972). The two

metabolites have different effects on the expression of male behavior because they act

upon different steroid receptors. Studying the effects of the metabolites on adult behavior
can give insights into the mechanisms that underlie sexual differentiation by testosterone
during development. Many accept the idea that estradiol can play an important role in the
organization and activation of male-typical sexual behaviors. Male and female rats have a
greater ability to aromatize testosterone to estradiol in the hypothalamus and amygdala
during perinatal development than at any other time in life (Tobet et al., 1985). Neonatal
treatment with estradiol benzoate defeminizes both female and neonatally castrated male
rats (Baum, 1979). It also appears that estrogens have some masculinizing effects since
neonatal treatment can increase the expression of male-typical sex responses in both

females and neonatally castrated males (Baum, 1979).

The effect of perinatal exposure to testosterone on the development of male-typical
partner preference is thought to be a result of the aromatization of testosterone to
estrogen. Females treated neonatally with dihydrotestosterone (DHT), a nonaromatizable
androgen, continue to show a female-typical preference for a stimulus male (Brand and
Slob, 1991b). It has also been shown that males castrated at birth and treated neonatally
with DHT do not show a preference for an estrous female, an effect similar to non-
treated, neonatally castrated males (Brand and Slob, 1991a). The aromatization
hypothesis for partner preference development is also supported by studies examining the
effect of neonatal exposure to the aromatase inhibitor 1,4,6 androstatriene-3,l7-dione
(ATD) on adult partner preference. Neonatal treatment of males with ATD has been
shown to have effects similar to those of neonatal castration. Whether tested intact or

after adult castration and estrogen treatment, males neonatally treated with ATD either do

not show a preference for either stimulus animal or show a preference for the male
stimulus (Bakker et al., 1993a; Bakker et al., 1993b; Bakker et al., 1996; Brand and Slob,
1991a). Control males, however, after adult castration and estrogen treatment continue to

prefer estrous females (Bakker et al., 1996).

Studies by Matuszczyk and Larrson (1995) give further indication that perinatal exposure
to estrogen plays a substantial role in the development of a male-typical partner
preference. Males treated with an anti-estrogen during the prenatal period show a reduced
preference for females in adulthood. However, males treated with an anti-androgen

prenatally continue to prefer an estrous female over a stimulus male.

Finally, previous work from this laboratory shows that females that receive postnatal
exposure to polychlorinated biphenyl (PCB) 77 on postnatal days (PND) 1-21 exhibit a
lower preference for a sexually active stimulus male than do control females (Cummings,
personal communication). PCB 77 has been reported to have estrogenic effects under
other circumstances (Jansen et al., 1993; Nesaretnam et al., 1996; Seegal et al., 2005).
Similarly, males with the testicular feminizing mutation (Tfm), which is characterized by
a dysfunctional androgen receptor (Zuloaga et al., 2008), also indicate an estrogenic role
in the development of adult partner preference. Tfm males exhibit female phenotypes due
to a lack of androgenization during deveIOpment. However, these males do have high
levels of circulating estrogen and normal estrogen binding in the brain (Roselli et al.,
1987). It has been shown that in adulthood, Tfm males show male—like partner

preference, approaching an estrous female more than a stimulus male (Hamson, personal

communication), supporting the idea that organizational estrogen is responsible for male-

typical preference in adulthood.

The prenatal environment also seems to be important in adult preference behavior. Male
rats castrated neonatally, but treated with testosterone in adulthood, show a normal
preference for the female (Brand and Slob, 199la; Meyerson et al., 1980). Likewise,
males treated neonatally with ATD and given adult testosterone replacement also show a
preference for the female (Bakker et al., 1993b). These effects do indicate that some
masculinization of adult partner preference occurs prior to birth, but testosterone is
necessary as the activational hormone for the expression of this prenatally organized

male-typical behavior.

Ferret

Stockman et al (1985) were the first to study the development of partner preference in the
ferret. Males castrated shortly after birth and treated with estradiol in adulthood show a
higher preference for a stimulus male than do control males (Stockman et al., 1985).
Females ovariectomized in early life and immediately treated with testosterone show a
lower preference for the stimulus male than do control females (Martin and Baum, 1986;
Stockman et al., 1985). Early neonatal manipulations were unable to completely reverse
the partner preference of ferrets, which could mean prenatal hormones are necessary for
the display of sex-typical partner preference. When female ferrets are treated with
testosterone during the prenatal period, immediately at birth, and during the early

postnatal period, they show a preference similar to control males (Baum et al., 1990).

Treatment during only one of these periods leads to a preference that is intermediate to

that of control males and control females (Baum et al., 1990).

Perinatal estrogen appears to have a significant role in the development of adult partner
preference in the ferret. As mentioned above, female ferrets that receive neonatal
testosterone treatment show lower preference for stimulus males than controls. However,
neonatal treatment with DHT does not appear to disrupt female-typical partner preference
(Martin and Baum, 1986), indicating that testosterone is most likely working through its
estrogenic metabolite. Also, males treated with ATD prenatally show decreased latencies
to approach a stud male when tested after adult treatment with estradiol (Baum and

Tobet, 1986).

Mouse

Mice have been an important model for studying the effects of estrogen during
development on adult partner preference due to gene knock out animal availability. Males
with the estrogen receptor a gene knocked out (ERdKO), castrated in adulthood and
given testosterone replacement show no preference between an estrous female and a
stimulus male, whereas wild-type males show a preference for the female (W ersinger et
al., 1997). ERaKO males also do not prefer an estrous female over an unreceptive female
(W ersinger et al., 1997). Finally, wild-type males prefer to approach and sniff odors from
an estrous female, but ERaKO males show no preference (Wersinger and Rissman,

2000).

However, one problem arises with ERa knock out studies. It cannot be determined if the
effects are due to the absence of either the organizational or activational effects of
estrogen. The lowered preference for the stimulus female shown by the male mice may
be due to a lack of estrogen effects during the perinatal critical period but it could also be
due to the fact that the adult testosterone treatment cannot have an estrogenic activational
effect in adulthood. Other studies have attempted to resolve this issue by studying mice in
which the aromatase gene, Cyp-19, has been knocked out (ArKO). Wild-type males
prefer estrous female odors over male odors, but ArKO males show no preference for
female odors over male odors and do not show a preference for a stimulus female over an
empty room (Bakker et al., 2004). The preference for females odors is not increased after
adult estradiol treatment, indicating the effects seen can be attributed to the

organizational effects of estrogen and not activational effects.

The androgen receptor (AR) may also play a role in the development of partner
preference in the mouse, unlike in the rat. Female mice treated with DHT neonatally
display a male-like partner preference for both a stimulus male animal and male bedding
over an estrous female or female bedding (Bodo and Rissman, 2008), whereas neonatal
DHT does not have a masculinizing effect on the partner preference of female rats (Brand
and Slob, 1991b). Also, Tfm male mice, unlike Tfm male rats, show a female-typical
partner preference (Bodo and Rissman, 2007), indicating that perinatal activation of the
AR is necessary for the development of masculine partner preference behavior in the

mouse. More research is needed, however, to explain why the ERaKO and ArKO models

described above show feminine partner preference behaviors despite having normal male

levels of circulating DHT.

Pig

Pigs are another animal model that have been used to evaluate the role of perinatal
exposure to gonadal hormones on adult partner preference. In intact adult pigs, males
usually show no preference for either stimulus animal, whereas estrous females show a
preference for a male over a female stimulus animal (Signoret, 1970). Male pigs castrated
at birth and treated with estradiol in adulthood show a female-typical preference for a
boar (Adkins-Regan et al., 1989; Ford, 1983). Males castrated later in life, show no
preference, similar to the behavior of intact males (Adkins-Regan et al., 1989; Ford,
1983). Both males and females gonadectomized early in life and then treated with
estradiol during the prepubertal period show an adult preference for a stimulus female
after adult estradiol treatment (Adkins—Regan et al., 1989). Prepubertal hormone
treatment has an effect because the pig appears to have a much longer postnatal critical

period than the majority of other animal models studied (Adkins-Regan, 1988).

Hamster

Little work has studied the organizational effects of hormones on the adult expression of
partner preference in the hamster. Johnson and Tiefer (1972) studied partner preference
in the hamster and discovered that neonatal hormones are important for the display of
sex-typical partner preference. A male hamster castrated neonatally and given ovarian

hormones in adulthood prefers a stimulus male over a female (Johnson and Tiefer, 1972).

The role of estrogen in the development of partner preference has not been studied in the

hamster.

Dog

Similar to the hamster, few studies have researched the role of hormones during
development on sexual partner preference in the dog. Beach et al (1977) tested female
dogs after pre- and/or postnatal exposure to testosterone. Adult females were first tested
after estradiol treatment with a stimulus male and then after testosterone treatment with
an estrous female. Even though the paradigm is not a choice test, the females treated with
perinatal testosterone spend more time with the stimulus female than they do with the
stimulus male. Control females, however, spend more time near the stimulus male than
the stimulus female (Beach et al., 1977). The role of estrogen in the development of

partner preference has not been studied in the dog.

Partner Preference in Humans

Although numerous studies have examined the role of steroid hormones on the
development of brain and behavior, many questions remain. Among these is the role
steroid hormones play in the development of human orientation. Understanding hormonal
control of the development of adult partner preference and sexual behavior in an animal
model such as the laboratory rat, can have a wide range of implications. Examining the
endocrine components and neural factors that play a role in sexual motivation and partner
preference in laboratory animals, can give insight into understanding the basis of human

orientation.

The prenatal environment appears to play a role in the development of male sexual
orientation in humans. It has been shown that for each older brother a boy has, his
chances of growing up to be homosexual increases by one-third (Blanchard and Bogaert,
1996), and approximately one homosexual male in seven can attribute his orientation to
this fraternal birth order effect (Cantor et al., 2002). One explanation could be due to
possible hyper—androgen exposure during development. There is the notion that male
preference for another male may not always be due to feminizingeffects since it has been
shown that homosexual men have larger genitalia (Bogaert and Hershberger, .1999) and
more masculine auditory evoked potentials (McFadden and Champlin, 2000) than
heterosexual men, indicating a higher level of androgen during development. Also,
studies indicate that having more older brothers can lead to hyper-masculinization on
some measures such as digit length ratios. The index finger (2D) to ring finger (4D) ratio
is sexually dimorphic in humans and believed to be affected by prenatal androgen levels
(Brown et al., 2002; McFadden et al., 2005; Williams et al., 2000). In females the 2D is
similar in length to 4D, whereas males tend to have shorter 2D (Williams et al., 2000).
The 2D:4D ratio becomes more masculine in men who have more than two older brothers
compared to men with no older brothers, and there is a correlation between number of
older brothers and 2D:4D ratio (Williams et al., 2000). Studies also show that
homosexual men tend to have a lower, or more masculine 2D:4D ratio than heterosexual
men (Rahman, 2005; Rahman and Wilson, 2003; Robinson and Manning, 2000). These
findings suggest that men with more older brothers are more likely to be homosexual and

are exposed to higher androgen levels prenatally than men with no older brothers.

10

Further indication for a role of steroid hormones in the development of human sexual
orientation can be found in studies of human endocrine disorders. Women with
congenital adrenal hyperplasia are exposed to higher than normal levels of androgen
throughout development. These women show an increased incidence of bisexual or
homosexual orientation (Meyer-Bahlburg et al., 1996; Zucker et al., 1996). In addition,
genetic males with complete androgen insensitivity syndrome (CAIS; the human
equivalent of Tfm), look like females at birth, are usually raised as female throughout
life, and show a preference for males in adulthood. Women that were exposed to the
synthetic estrogen diethylstilbestrol (DES) prenatally also have an increased incidence of
bisexual orientation compared to women not exposed to DES (Ehrhardt et al., 1985;
Meyer-Bahlburg et al., 1995). DES is a nonsteroidal synthetic estrogen used through
1970 to decrease the occurrence of miscarriages. It is interesting to note, however, that
although estrogen exposure appears to affect the orientation of women, lack of estrogen
plays little role in the development of female-oriented male sexual orientation. Genetic
males with CAIS are still exposed to male-typical levels of estrogen during development
yet show a preference for men in adulthood. Also, although subjects are limited,
individuals who carry a mutant ERa or aromatase gene self report as being heterosexual

males (for review, Baum, 2006).

Finally, the role of steroid hormones on human orientation can be assessed in gender
reassignment cases. In instances of partial AIS or cloacal exstrophy, genetic males are
born with ambiguous genitalia (Gooren, 2006). In the past, the most common solution

posed by doctors was to assign the newborn a female identity and surgically reconstruct

ll

female genitalia. In a long term study conducted by Reiner and Kropp (2004), eighteen
patients with sexual differentiation disorders were evaluated throughout adolescent life.
Of the patients, three were raised as boys and continued to identify with the male gender
over the evaluation period. Fifteen were assigned as females at birth, and seven of them
reassigned to the male gender before twelve years of age. Of the remaining females, one
refused to declare a sexual identity, one died of unknown causes, and five continue to live
as females. However, the five patients that remained female had mood scores and social
competence evaluations lower than those of the individuals that reassigned to the male
gender. Of the individuals (male or female) that were old enough to declare a sexual
orientation, all expressed a preference for a female partner. These studies all indicate that
prenatal hormones have a profound influence on sexual orientation and gender identity of

humans.

Overview of Studies

In this dissertation, the role of early postnatal treatment with steroid hormones in the
development of adult partner preference in the laboratory rat will be examined. The role
of estrogen in the development of female-oriented adult partner preference and sexual
behavior in the male rat will be evaluated initially by treating females during the early
postnatal period with estradiol and determining if their behavior was masculinized. Then
the role of hyper-androgen exposure during development on the expression of male-
oriented partner preference and sexual behavior in the male will be examined, followed
by a study determining if the estradiol metabolite of testosterone is responsible for the

effects seen after hyper-androgen exposure in the male. Finally, to rule out any possibility

12

that the hormonal effects seen were due to changes in maternal behavior, the role of
maternal licking and grooming on adult partner preference and sexual behavior in males

and females will be assessed.

l3

CHAPTER TWO:
ESTROGEN IS RESPONSIBLE FOR THE DEVELOPMENT OF FEMALE-
ORIENTED PARTNER PREFERENCE IN THE MALE LABORATORY RAT AS
INDICATED BY ITS MASCULINIZING EFFECTS ON ADULT BEHAVIOR IN

FEMALES

INTRODUCTION
As reviewed in Chapter One, adult male partner preference is organized early in
development, and it appears that the estradiol metabolite is responsible for the ontogeny
of partner preference behavior. A normal adult male rat will show a preference for an
estrous female over a sexually active male (Hetta and Meyerson, 1978), neonatal
castration significantly reduces this preference (Brand and Slob, 1991a), and this
reduction in preference is prevented when testosterone is replaced following castration
(Brand and Slob, 1991a). However, a male treated with DHT after neonatal castration
continues to display a reduced preference for the estrous female (Brand and Slob, 1991a).
The success of testosterone administration in overcoming the loss of normal partner
preference after castration, but not DHT, is consistent with the idea that estradiol is

required for the development of this behavior.

Other studies also support the notion that estrogens plays a role in the organization of
adult partner preference behavior. Blocking estrogen action during development, either
through aromatase inhibitors (Bakker et al., 1993a; Bakker et al., 1993b), antiestrogens

(Matuszczyk and Larsson, 1995), estrogen receptor knock outs (Rissman et al., 1997;

14

Wersinger and Rissman, 2000; Wersinger et al., 1997) or aromatase gene knock outs

(Bakker et al., 2002) results in a decrease in the male’s preference for a female.

From these studies, it appears that estradiol plays a significant role in the organization of
partner preference behavior in the rat during postnatal development. However, this idea is
drawn from studies where estrogen action was in some way blocked in male rats. In the
present study, the possible masculinizing effect of estrogens was tested directly by
treating female rats during the early postnatal period with estradiol benzoate and
examining their adult partner preference and sexual behavior. In addition, the effects of
exposure to estradiol on the morphology of the ovaries and cell number in the spinal

nucleus of the bulbocavemosus and pelvic ganglia were also measured.

METHODS

Animals

Experimental Females: Time-mated pregnant Long-Evans rats (Charles River, Raleigh,
NC) were housed individually with ad lib food and water in plastic cages (45.5 x 24 x 21
cm) in a 14:10-hr light dark cycle with lights on at 01:00. For nest building material
thirty, one-inch paper towel strips were given to the dams on gestation day (GD) 20. A
subset of the female offspring of these dams became the experimental females of this
study (see below). On the day of birth, PND 0, the litter was reduced to four male and
four female pups. For litter reductions, the anogenital distance (AGD) for each pup was
measured, and since the AGD is shorter in females than in males, the four shortest and

the four longest were retained (Figure 2.1A).

15

Stimulus Animals: Sexually experienced, gonadally intact, adult Long Evans rats (Charles
River, Raleigh, NC) were used as stimulus animals for the behavioral tests (females at

least 60 days old; males at least 90 days old).

Animals were maintained in a facility accredited by the Association for Assessment and
Accreditation of Laboratory Animal Care, and all experimental procedures were

approved by the Michigan State University Animal Care and Use Committee.

Hormone Treatments

Silastic capsules (Dow Corning; inner diameter 1.47 mm; outer diameter 1.96 mm; length
5 nun) were used to administer estradiol treatments on the day of birth (Figure 2.1A).
Estradiol benzoate (Sigma; EB) was mixed with cholesterol (Sigma; C) to achieve 5%
and 10% EB to C mixtures. On PND 0, pups received Silastic capsules containing either
cholesterol or 5% or 10% EB-C mixtures. These animals are referred to as C, 5%, and
10% females, respectively. While these estradiol treatments are most likely outside of the
physiological range of estrogen exposure, this study was also designed to provide a
positive estradiol control to evaluate the claim from studies in this laboratory that
environmental contaminants, such as polychlorinated biphenyls, that have estrogenic
effects (Jansen et al., 1993; Nesaretnam et al., 1996; Seegal et al., 2005) affect partner

preference in female rats (Cummings et al, in press).

Capsules were implanted subcutaneously (s.c.; one treatment per litter) through a small

incision on the back of the animal, while the pups were under ice anesthesia. The incision

16

was closed using superglue (Loctite). Following surgery, pups were warmed under a heat
lamp until the incision was dry (approximately one hour) and then returned to their
mothers. Capsules were left in place for 3 weeks. On PND 21, at weaning, the pups were
anesthetized with isoflurane (Isoflo, Abbot Laboratories), and the implants were removed
through an incision made near one end of the capsule (Figure 2.1C). The incision was
closed with an Auto Clip (Clay Adams) and covered with First Aid Cream (Johnson and
Johnson). Pups were then housed with same-sex litterrnates. Only female pups were used

in this Experiment.

After animals reached 60 days of age, two females (C, n=16; 5%, n=14; 10%, n=16) from
each litter were ovariectomized and implanted with a Silastic capsule containing either
25% or 12.5% EB-C mixtures (Figure 2.1D). The capsules were implanted so while the
animals were anesthetized with isoflurane; the incision was closed with an Auto Clip and
covered with First Aid Cream. After 4 weeks, the capsules begin to lose efficacy because
of connective tissue growth around the capsule (personal observation). The capsules were
removed and reimplanted via a new incision in the neck. For some tests (see below), the
females were injected so with 0.5 mg progesterone (Sigma; P, in sesame oil) four hours
prior to data collection. Adult behavioral tests were run after treatment with one of two
doses of EB treatment in adulthood and with or without progesterone treatment. This
hormonal regime was used to test the females under two naturally occurring hormonal
conditions: estrogen alone, typical of early proestrus, and estrogen plus progesterone,

typical of late proestrus. Female rats mate during both early proestrus, when estradiol is

17

available but before the surge in progesterone, and during late proestrus, when both

ovarian hormones are present (Blaustein, 2008).

Stimulus females were implanted with a Silastic capsule containing 25% EB-C mixture
prior to testing but were not ovariectomized. It is the practice of this laboratory to not
ovariectomize stimulus females for testing procedures. The hormone treatments used
reliably induce sexual receptivity while avoiding the trauma of an unnecessary surgery.
Stimulus females were injected so with 0.5 mg P four hours prior to partner preference

and sexual behavior testing.

Maternal Licking and Grooming

Studies have shown that neonatal hormone treatments can alter the display of maternal
behavior (Cummings et al., 2005; Moore, 1982), and changes in maternal care can affect
the behavior of the offspring (Cameron et al., 2008; Champagne et al., 2001; Champagne
and Meaney, 2006; Francis et al., 1999). To determine if the early postnatal treatments
caused alterations in maternal care, the maternal licking and grooming behavior of the
mothers of the experimental females was analyzed (Figure 2.1B). Maternal behavior was
videotaped during the last hour of the light phase and the first hour of the dark phase of
the light-dark cycle on PND 1, 2, 4, and 6. These recordings, as well as those for all other
behavioral tests, were analyzed using The Observer 5.0 (Noldus), a behavioral data
acquisition computer program. The amount of time the darn spent licking and grooming

the litter was determined.

18

Behavioral Testing

Partner Preference: Tests for partner preference were conducted in a three-chamber
apparatus (91 x 61 x 41 cm) made of Plexiglas with a transparent front and opaque sides
and inner walls. Each chamber (30 x 61 x 41 cm) had openings in the two inner walls at
the back of the apparatus which allowed the experimental animal to move freely among
the three chambers. One intact male and one sexually receptive female stimulus animal
were tethered to a bar at the front end of the two outer chambers using a 25 cm wire fitted
with a swivel. The stimulus animals wore a harness that was attached to the bar allowing
the experimental animal to make physical contact, but limiting the movement of stimulus
animal to its chamber. The middle chamber had no stimulus animal. Both experimental
and stimulus animals were adapted to the apparatus, and stimulus animals were adapted
to the harness prior to testing. All adult behavioral testing took place under dim red light

illumination in the middle part of the dark phase of the light-dark cycle.

During the 20-minute partner preference test, the experimental animal could freely move
among the three chambers and interact with the stimulus animals. The test was
videotaped, and the following behaviors were quantified: time spent in each chamber,
latency to first enter each chamber, and the occurrence of mounts, intromissions, and
ejaculations between the experimental and stimulus animals. Preference scores were
calculated by subtracting the duration of time spent in the stimulus male chamber from
the duration of time spent in the stimulus female chamber. Therefore, a positive

preference score indicates more time spent with the stimulus female, whereas a negative

19

preference score indicates more time spent with the stimulus male. The experimental

females were sexually naive prior to the initial partner preference test.

Female sexual behavior: When tested for female sexual behavior, a barrier with four
holes (4.5 x 4.5 cm) was placed in a Plexiglas observation chamber (46 x 58 x 51 cm),
which divided it into a female escape area (46 x 22 x 51 cm) and a male area (46 x 36 x
51 cm). The holes in the barrier were too small for the male to get through but gave the
female free access to both chambers. The tests lasted 30 minutes and were videotaped.
The frequency of male mounts, intromissions and ejaculations were scored, as was the
latency to show these behaviors. The latency of the experimental female to approach the
male, the amount of time the female spent in the male area of the observation chamber,
and sexual receptivity were also recorded. Receptivity was measured using lordosis
quotient (LQ), which was calculated for all females who received at least six mounts. For
the first 10 mounts (including intromissions or ejaculations) female responses were
scored as a 0 (no lordosis) or 1 (lordosis), from which a percentage of number of lordoses
per test was calculated. The frequency of exits and latency to return to the male were not
measured because the females treated with estradiol during the early postnatal period did
not receive intromissions or ejaculations from the stimulus males (see Results). Finally,
proceptive behaviors, which include hopping and darting, ear wiggling, and approaching

the male, were scored during the test following progesterone treatment.

Male-like sexual behavior: Tests for male-like sexual behavior displayed by the

experimental females were conducted in a Plexiglas observation chamber (46 x 58 x 51

20

cm). During the test, the experimental female had unrestricted access to the female
stimulus animal. The tests lasted 30 minutes, and video recordings were analyzed to
determine frequency of mounts, intromissions, and ejaculatory patterns shown by the

experimental females and the latency to show these behaviors.

Testing Schedule: Experimental animals were tested twice a week for 6 weeks (Figure
2.2). During the first test each week, the females were tested with only the adult estradiol
treatment (EB alone). For the second test, each female was injected with 0.5 mg
progesterone four hours prior to testing (EB + P). The initial partner preference of the
female was tested in week 1 (Figure 2.2A). Each experimental female received sexual
and social experience with both male and female stimulus animals during weeks 2 and 3,
but data were not collected. During experience weeks, experimental females were
partnered with stimulus animals for 30 minutes during which time sexual behavior could
occur. During week 4, half of the experimental females were tested for sexual behavior
with a male and the other half with a female (Figure 2.2B). The sex of the stimulus
animals was switched for week 5 (Figure 2.2C). Sexual behavior during weeks 4 and 5
was recorded and scored. In week 6, the female’s final partner preference was evaluated

(Figure 2.2D).

Histology
Ovaries: To determine if the early estradiol treatment had an effect on the estrous cycle
of the females, the ovaries were examined for the presence of corpora lutea (Figure

2.1D). The ovaries were removed as described above during the ovariectomy surgery and

21

placed in 0.1 M phosphate-buffered 10% formalin fixative at room temperature for at
least 30 days. One ovary from each female was then washed in phosphate-buffered saline
(PBS) solution (pH 7.4) and transferred to a 50% ethanol solution. The tissue was
embedded in paraffin, sectioned at 4 um, and stained with hematoxylin and eosin.
Stained sections were viewed on a light microscope by an experimenter blind to the
treatment groups, and the presence of corpora lutea was determined. Tissue embedding,
sectioning, and staining were completed by the Investigative HistoPathology Laboratory

at Michigan State University.

Pelvic Ganglia (PG): After behavioral testing was concluded, the experimental females
were anesthetized with an intraperitoneal (i.p.) injection of sodium pentobarbital
(Nembutal, 75 mg/kg). The major pelvic ganglion was fresh dissected from both sides of
each animal (Figure 2.1D). The ganglia were placed in a Cryomold (Tissue-Tek; 15 mm
x 15 mm x 5 mm), covered with O.C.T. Compound (Tissue-Tek), and immediately frozen
in dry ice—cooled isopentane. Ganglia were stored at -80° C until sectioning. Cryostat
sections at a thickness of 14 um were taken in four series of sequential sections and
mounted onto gelatin-subbed slides. Slides were stored at -80° C until staining. One series
of every fourth section was processed for Nissl staining using thionin. Slides were then
dehydrated with ethanol, cleared with xylene (Baker), and coverslipped with Permount
(Fisher Scientific). Stained sections were viewed on a light microscope, and the number
of neurons present were counted by an experimenter blind to the treatment groups.

Neurons were identified as darkly stained, large nucleated cells (Figure 2.3). Only one

22

female from each litter (10% EB and C treatment groups only) was used for analysis of

the pelvic ganglia.

Spinal Nucleus of the Bulbocavernosus (SNB): Directly after pelvic ganglia removal,
females were perfused intracardially with chilled saline and then 10% buffered formalin
fixative. The lumbosacral spinal cord was dissected and placed into buffered formalin
fixative at room temperature for at least 30 days (Figure 2.1D). The spinal cord was then
sectioned at 50 um in three series of sequential sections. Sections were mounted onto
gelatin-subbed slides, and one series of every third section was processed for Nissl
staining and counted by an experimenter blind to the treatment groups. Motoneurons

were identified as darkly stained, large nucleated cells (see Figure 2.10).

Analysis

Maternal licking and grooming data were analyzed using a 3 x 4 (early postnatal
treatment x day) ANOVA with repeated measures on the second factor. The data for the
behavioral measures during the partner preference tests were analyzed using a 3 x 2 x 2 x
2 (early postnatal treatment x adult estradiol treatment x progesterone treatment x initial
or final test) AN OVA with repeated measures on the third and fourth factors. Preference
scores and duration of time spent in the female chamber during the partner preference
tests were also analyzed within each early postnatal treatment group using a one-sample
t-test. This test was used to determine if the animals showed a preference for one stimulus
animal above chance (preference score = 0) or spent more time with the female above

chance (duration = 400 seconds).

23

The data for the behavioral measures during the sexual behavior tests were analyzed
separately for female or male behaviors using a 3 x 2 x 2 (early postnatal treatment x
adult estradiol treatment x progesterone treatment) AN OVA with repeated measures on
the third factor. The presence or absence of corpora lutea in the ovaries was analyzed
using a Chi-Square test. SNB cell counts were analyzed using a one-way ANOVA by
early postnatal treatment. PG cell counts were analyzed using an independent samples I-

test.

For some variables, the data did not meet homogeneity of variance assumptions, even
after the prescribed transformations (i.e. square root). For these measures, nonparametric
statistics were used for analyses. In these situations, data from the 5% and 10% females
were collapsed into an early postnatal-estradiol treated group (EB females) because no
significant differences between the two early postnatal estradiol treatments were found
for any of the variables analyzed using nonparametric statistical tests. Also, no significant
differences were found with the same tests between the two adult EB doses within the
early postnatal treatment groups, so data were collapsed across adult EB doses as well.
The Mann-Whitney U and the Fisher’s exact probability tests were used for these

analyses.

Abbreviations
C females — female pups treated on PND 0-21 with cholesterol
5% females -— female pups treated on PND 0-21 with a 5% EB-C mixture

I 0% females - female pups treated on PND 0-21 with a 10% EB-C mixture

24

EB females — 5% and 10% females collapsed into one treatment group used for non-
parametric analyses

EB alone — adult hormonal testing condition when females were treated with only EB
(either 25% or 12.5% dose)

EB + P — adult hormonal testing condition when females were treated with both EB and

P

RESULTS
Maternal Licking and Grooming
No significant differences in maternal licking and grooming were seen among treatment

groups (p=.292) or days (p=0.078), with no significant interaction (p=0. 147; Figure 2.4).

Partner Preference

Eflects of Early Postnatal Treatments: Early postnatal treatment with EB altered the
partner preference of the female experimental animals. The 10% and 5% females spent
more time in the stimulus female chamber (Figure 2.5A; [F(2,40) = 20.3, p<0.001]) and
less time in the stimulus male chamber (Figure 2.5B; [F(2,40) = 13.7, p<0.001]) than did
the C females. Early postnatal treatment did not affect the time the females spent in the
middle chamber (Figure 2.5C). The 10% and 5% females also showed a preference score
greater than that of the C females (Figure 2.5D; [F(2,40) = 18.7, p<0.001]). Thus, early
estradiol treatments reduced the preference for the male and increased the preference for

the female, and these effects were seen with no significant interactions between them and

25

those of adult treatments or week of test. Also for all conditions, early postnatal

treatments did not affect the latencies to enter the male or female chambers.

In both the initial and final partner preference test, the 10% and 5% females had positive
preference scores statistically different than chance, indicating a preference for the female
stimulus animal. The C females, however, did not show a preference greater than chance
for either stimulus animal (Table 2.1). Also, the duration of time spent in the female
chamber in both the initial and final tests was significantly greater than chance for the

10% and 5% females, but not for the C females (Table 2.1).

The proportion of experimental females that displayed male-like sexual behavior directed
to the stimulus female during partner preference tests did not differ significantly between
EB and C females (Fisher’s exact probability tests). However, five out of thirty females
(four 10% females and one 5% female) that received early postnatal estradiol showed at
least one full ejaculatory reflex pattern during the preference tests, whereas no
ejaculatory patterns were shown by the C females. The proportion of experimental
females that received mounts, intromissions, or ejaculations from the stimulus male
differed significantly between early postnatal treatment groups (see Table 2.2 for the
specific comparisons). The stimulus males showed more sexual behavior toward the C

females compared to the EB females in both partner preference tests.

Efiects of Experience and Adult Treatments: Across all hormone treatments, the average

latency to enter the male chamber was significantly shorter for the final test compared to

26

the initial one (X :l: SEM: all measures are in seconds: 31.9 :1; 3.4 vs 13.6 i: 1.5; [F(1,40)
= 49.6, p<0.001]). Also, for time spent in the middle chamber, the experimental females

spent significantly more time in the middle chamber during the final tests than during the

initial tests (5(- 1 SEM: 374.9 x 15.8 vs 330.1 : 13.4; [F(1,40) = 4.8, p=0.035]).

Two measures were affected by progesterone treatment. Across all tests and conditions,

the experimental females spent less time with the stimulus female after EB + P treatment

compared to after treatment with adult EB alone (5(- :1: SEM: 517.2 1 15.9 vs 479.1 x
17.9; [F(1,40) = 6.0, p=0.019]). In addition, the experimental females spent significantly

more time in the middle chamber when treated with EB + P than when treated with adult

EB alone (I? :1: SEM: 378.3 1- 13.4 vs 326.7 1 13.1; [F(1,40) = 10.0, p=0.003]).

Both the amount of time spent with the male [F(1,40) = 5.5, p=0.024] and latency to enter
the female chamber [F(l,40) = 49.6, p<0.001] were affected by a progesterone by test

interaction. During the initial partner preference test, females treated with adult EB alone

spent more time in the stimulus male chamber than did females treated with EB + P (I? :1:

SEM: 404.1 i 17.4 vs 361.0 :t 18.6). There was also a decrease in time spent with the

male in the adult EB alone treatment group from the initial to final preference test (2? i
SEM: 404.1 1 17.4 vs 306.4 i 23.1) but no change in the EB + P treatment group. The

latency to enter the female chamber was significantly reduced from the initial to the final
test when the females received adult EB alone (If :1: SEM: 35.0 i 4.6 vs 11.6 i 1.0) but

not when progesterone was also given (if :1: SEM: 15.8 :1; 1.8 vs 12.4 -_l-_ 2.0). Adding

27

progesterone significantly reduced the latency to enter the female chamber, but only for

the initial test.

Finally, there were significant adult EB by test interactions for time spent with the female
[F(1,40) = 10.8, p=0.002], time spent with the male [F(1,40) = 5.1, p=0.030], and
preference score [F(1,40) = 10.0, p=0.003]. Individual comparisons showed that females
treated with the high adult EB dose increased the time they spent with the female from
initial to final test (2 i SEM: 478.6 1 20.8 vs6570.4 :1: 30.4), but no significant effect of
test was found for the females treated with the low adult EB dose (2? :1: SEM: 495.9 1
20.8 vs 447.8 :1: 30.4). During the final test, the high adult EB females spent more time in

the female chamber than their low dose counterparts. Females treated with the high adult

EB dose spent significantly less time with the male from initial to final test (2? :1: SEM:

392.6 :1: 22.6 vs 278.2 :1: 31.2), but females receiving the low adult EB dose remained the
same (X'- :1: SEM: 372.5 1 22.6 vs 352.2 :1: 31.2). This lead to an increase in preference
score for the females treated with the high adult EB dose (If :1: SEM: 86.0 :1: 39.1 vs

292.2 i 57.4) but no change for the low EB dose females ( I? : SEM: 123.4 :I: 39.1 vs

95.6 :1: 57.4).

Female Sexual Behavior
The 10% females spent significantly less time in the male area of the testing arena than

did the C females (Figure 2.5; [F(2,39) = 4.4, p = 0020]). Also, regardless of other

hormone treatments, all females spent less time with the male after EB + P treatment (If

i SEM: 560.0 :1: 48.5 vs 486.3 :1: 49.3; [F(1,39) = 4.1, p = 0049]).

28

Non-parametric statistics were used to analyze the remaining measures for the female
sexual behavior tests. A Mann-Whitney U test was used to compare the behaviors
displayed by the stimulus males on tests with EB females (combining the 5% and 10%
groups as explained in Methods) and with C females treated as adults with EB alone

(combining both adult doses) or with EB + P.

Male mount, intromission, and ejaculation frequencies were significantly affected by the
early postnatal treatment of the experimental females. The stimulus males displayed
fewer mounts, intromissions, and ejaculations in tests with EB females as compared to C
females (Figure 2.7). The proportion of experimental females that received mounts,
intromissions, and ejaculations by the stimulus male was analyzed using Fisher’s exact
probability test. In both tests with and without progesterone, significantly fewer EB
females received mounts, intromissions, and ejaculations compared to C females (Table

2.3).

Too few EB females (11 = 2) received at least six mounts to allow meaningful statistical
comparisons of the LQ scores of EB vs. C females. During tests with adult EB alone
treatment, one 10% female received six mounts, but showed no lordosis responses. One
5% female received six mounts during the EB + P test, but showed no lordosis responses.
In contrast, all the C females that received at least 6 mounts showed lordosis responses
with average LQs of 87% (n=12) for the EB alone (combining both adult doses) tests and

97% (n=14) for the EB + P tests.

29

The data for the proceptive behaviors were analyzed using the Mann-Whitney U test,
which revealed that EB females showed significantly fewer proceptive behaviors than C
females did (Figure 2.8). Separate statistical tests were used to compare the EB and C

females for each specific proceptive behavior.

Male-like Sexual Behavior

Nonparametric tests were used to analyze the data from the male sexual behavior tests.
No significant differences were seen between EB and C females for any behavioral
measure (Fisher’s exact probability tests). However, five out of thirty females (four 10%
females and one 5% females) that received early postnatal EB showed at least one full
ejaculatory pattern during the sexual behavior tests, whereas no ejaculatory patterns were
shown by the C females. Four of these five EB females were the same females that

showed ejaculatory patterns during the partner preference tests.

Histology

Estradiol treatment during development significantly affected the presence of corpora
lutea in the ovaries of the experimental females (Chi-Square, p<0.001; Figure 2.9).
Corpora lutea were seen in 13 of 16 C females but in none of the females treated with

early postnatal estradiol.

Early postnatal estradiol treatment also altered the number of motoneuron cells seen in

the SNB. C females showed fewer cells than did the 5% and 10% female groups (Figure

30

2.10; [F(2,37) = 14.3, p<0.001]). NO effect was seen on the number of neurons present in

the PG Of the females (5(- i SEM: 10% EB:3011.6 i 340.5 vs C: 2916.0 :1: 264.1).

31

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Table 2.2. Proportion of experimental females that received sexual behavior from the
stimulus male during the partner preference tests. C females were more likely than EB
females to receive sexual behavior from the stimulus male. Data from tests after adult
treatment with both EB alone and EB + P are shown. Data are collapsed across the two
adult EB doses. *Significantly different from cholesterol, p<0.05. **Significantly

different from cholesterol, p<0.01.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Initial Tests Adult Estradiol Alone Adult Estradiol and Progesterone
Behavior Mount Intromission Mount Intromission Ejaculation
Cholesterol 9/ 16 7/16 8/ 16 8/16 4/16
Estradiol * 6/30 1/30 4/30 3/30 0/30

Final Tests Adult Estradiol Alone Adult Estradiol and Progesterone
Behavior Mount Intromission Mount Intromission Ejaculation
Cholesterol 7/16 6/16 12/16 9/ 16 6/ 16
Estradiol ** 2/30 1/30 1/30 1/30 0/30

 

 

 

 

 

 

 

 

33

 

 

 

 

 

 

 

 

 

 

 

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34

 

 

A PNP 0 B PND l,2,4,6 IC PM!) 21 In After PLND 60

Litter Reduction Maternal Licking Capsule lRemoval Ovariectdmy Partner Preference Histology
Silastic Capsules and Grooming Weaning —Ovaries Collected Female Sexual -PG
-Cholesterol Silastic Capsules Behavior -SNB
-S% EB-C 42.5% EB-C Male-like Sexual

-10% EB-C -25% EB-C Behavior

 

 

 

 

 

 

Figure 2.1. Treatment and testing schedule for the experimental females. (A) On the day
of birth, PND O, litters were reduced to four males and four females and received Silastic
capsules of cholesterol (C, n=16) or estradiol benzoate [EB either 5% (n=14) or 10%
(n=16)]. (B) Maternal licking and grooming behavior was observed on PND 1, 2, 4, and
6. (C) At weaning on PND 21, the Silastic capsules were removed. (D) After the females
reached 60 days of age, they were ovariectomized and given Silastic capsules of either
12.5% or 25% EB. The ovaries were collected at this time. Females were tested for
partner preference, female sexual behavior, and male-like sexual behavior. After testing
was concluded, the pelvic ganglia (PG) and spinal nucleus of the bulbocavemosus (SNB)

were removed.

35

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

A Initial Partner Preference
EB Alone
Week 1
Initial Partner Preference
EB + P
Weeks 2 & 3 I Sexual and Social
Experience
1 \
B / \
Sexual Behavior | Sexual Behavior
With Female With Male
EB Alone EB Alone
Week 4 , ,
Sexual Behavior ! Sexual Behavior J
With Female With Male
EB + P EB + P
41 l
C Sexual Behavior Sexual Behavior
“5th Male With Female
EB Alone EB Alone
Week 5 A
| Sexual Behavior I Sexual Behavior J
With Male With Female
EB + P EB + P
\\l
D Final Partner Preference
EB Alone
week 6 Final Partner Preference
EB + P

 

 

 

 

 

Figure 2.2. Behavioral testing schedule for the experimental females receiving early
postnatal treatments of cholesterol (C, n=16) or estradiol benzoate [EB either 5% (n=14)
or 10% (n=16)]. Data were not collected during weeks 2 and 3 during which the animals
received sexual and social experience. EB and P represent adult hormone treatments prior
to each behavioral test. Note: Although not shown here, there were two doses (12.5% and

25%) of adult EB treatment (see text for more details).

36

 

Figure 2.3. Photomicrograph of pelvic ganglion tissue processed for Nissl staining.

Neurons were identified as darkly staining, nucleated cells (arrow). Scale bar: 100 um.

37

 

    
 
  
   

 

Early Postnatal
Treatment
1250' I 10% EB
I5% EB
1000< I Cholesterol
3
a 750 -
E—
500 -
250 -

 

 

PND] PNDZ PND4 PND6
Postnatal Day

Figure 2.4. Time (in seconds) mothers of experimental females spent licking and
grooming during the first postnatal week. No significant differences were seen among

treatment groups or days, with no significant interaction.

38

Preference Score

.p
?

300 -
200 -

oé

-100-+
-200.

A. Stimulus Female Chamber

Test _
I lnmal
IF mal

 

- B. Stimulus Male Chamber

C. Middle Chamber

D. Preference Score

 

 
  

 

_enn

l

 

 

l (3% 55/0 Cholesterol
Early Postnatal Treatment

Figure 2.5. Duration of time experimental
females spent (A) with the stimulus female,
(B) with the stimulus male, and (C) in the
middle chamber during both the initial and
final partner preference tests. Preference
score (D) is calculated as time spent with
stimulus female minus the time spent with
stimulus male. Therefore, a positive
preference score indicates more time spent
with the stimulus female. C females spend
less time with the female and more time
with the male compared with 10% and 5%
females. Also, the 10% and 5% females
show a clear preference for the stimulus

female. *Significantly different from 10%

and 5% females, p<0.001.

39

 

Adult Treatment
IEB Only
1000- I EB + P

*

 

Time (s)

 

 

 

10% 5% Cholesterol
Early Postnatal Treatment

Figure 2.6. Time experimental females spent in the male area of the observation chamber
during the female sexual behavior tests. Data for tests after adult treatment with EB alone
and EB + P are shown. Regardless of progesterone treatment, females treated with 10%
EB spent significantly less time with the male than did females treated with cholesterol.

*Significantly different from 10% females, p=0.02.

4o

 

 

 

 

 

 

25
A Early Postnatal
Treatment
20 ‘ lEstradiol
.Cholesterol
5. 15 -
3
a
8'
h
in
20 - *
a. 15 -
5
5
8'
I: 10 -
5 .
o - m

 

 

 

Mount Intromission Ej av ation
Behavior

Figure 2.7. Frequencies of mounts, intromissions, and ejaculations displayed by the
stimulus male during the female sexual behavior tests. Males showed fewer sexual
behaviors when paired with the EB females compared to the C females. (A) Test after
adult treatment with EB alone. (B) Test after treatment with EB + P. * Significantly

different from EB females, p<0.001.

41

l8

 

 

 

 

 

 

 

   

Early Postnatal
15 '* Treatment
fl Estradiol
14 ‘ , I Cholesterol
12 -
g 10-
:1
8' 8 4
In
ta
6 .-
4 .
a
2 .
o .
Hopp ing and Ear Approach
Darrin g Wi g] in g
Behavior

Figure 2.8. Frequency of proceptive behaviors shown by experimental females during the
female sexual behavior test after adult treatment with EB + P. EB females exhibited

fewer proceptive behaviors than did C females. *Significantly different from EB females,

p<0.01.

42

 

Figure 2.9 Photomicrograph of cross-sections through ovarian tissue processed for
hematoxylin and eosin staining. (A) Corpora lutea were present (arrow) in the ovaries of
C females, indicating a normal estrous cycle. (B) Ovaries of EB females showed no

corpora lutea. Scale bar: 200 um.

43

Ase

 

No. of Cells

 

 

 

[0% 5% Cholesterol
Early Postnatal 'I‘reatnmt

 

Figure 2.10. Number of motoneurons present in the SNB of experimental females. (A)
5% and 10% females showed more neurons than did C females. (B,C) Photomicrographs
of spinal cord section processed for Nissl staining at the level of the SNB. EB females
(B) had more motoneurons (arrow) than did C females (C). Scale bar: 100 um.

*Significantly different from 5% and 10% females, p<0.001.

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DISCUSSION
The overreaching hypothesis of this study was that adult partner preference in the
laboratory rat is influenced by the presence of estradiol during early development. This
idea stems from studies where blocking estrogen action during development, either
through aromatase or estrogen receptor inhibition, results in a decrease in a male rat’s

preference for an estrous female compared to control males.

Effects of Early Postnatal Treatments

Behavior: The principal finding is that increased postnatal EB exposure during
development masculinizes female partner preference. As indicated by the preference
scores, female rats that receive exogenous EB during development spent more time with
an estrous female and less time with a sexually active male than did C females. These
differences in duration were not a result of treatment differences in time spent alone, but
were due to differences in the time spent with specific stimulus animals. Also, the 5%
and 10% females showed positive preference scores significantly different from chance
indicating a preference for the female stimulus animals. This preference was a result of
spending more time in the female chamber than would be expected by chance. The early
postnatal treatments did not alter the amount of maternal licking and grooming received
by the litters; therefore, it is unlikely that the early postnatal treatment group differences
were mediated by changes in maternal care. The effect of early EB exposure on partner
preference was very robust and evident across different adult hormonal conditions and

amounts of sexual experience.

45

080
fill:

tBal

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CCUI;
Show
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treat:
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Smdj

These findings are consistent with the results of other studies that indirectly tested the
effect of estrogens on partner preference during development. Male rats treated
neonatally with the aromatase inhibitor ATD either do not show a preference for an
estrous female over a sexually active male or show a preference for the male stimulus
(Bakker et al., 1993a; Bakker et al., 1993b). Males neonatally castrated and treated with
Sci-dihydrotestosterone propionate (DHTP), a non-aromatizable androgen, exhibit a lower
preference for a stimulus female than do males that are treated with testosterone
propionate (TP) after neonatal castration (Brand and Slob, 1991a). Also, compared to
controls, male rats treated prenatally with an antiestrogen, nitromifene citrate (C1628),
show decreased preference for a female (Matuszczyk and Larsson, 1995). In addition,
when male mice lacking a functional estrogen receptor a (ERa—KO) are castrated and
treated with testosterone in adulthood, they spend less time with an estrous female than
do wild-type males (Rissman et al., 1997). Further, ERa—KO males do not prefer an
estrous female over an unreceptive female (W ersinger et al., 1997), and they approach the
odors of an estrous female less than do wild type males (W ersinger and Rissman, 2000).
Studies have also been conducted with mice in which the aromatase gene, Cyp-19, has
been knocked out (ArKO). In these studies, ArKO males show no preference for female
odors over male odors and do not show a preference for a female over an empty chamber
(Bakker et al., 2002). Further, recent work from our laboratory also shows that female
rats that receive postnatal exposure to the polychlorinated biphenyl congener 3, 4, 3’, 4’-
tetrachlorobiphenyl (PCB 77), which has been shown to have estrogenic effects (Jansen
et al., 1993; Nesaretnam et al., 1996; Seegal et al., 2005), exhibit a lower preference for a

sexually active stimulus male than do controls.

46

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Additional support for the idea that early estrogen exposure mediates a preference for
females can be found in the human literature. Diethylstilbestrol (DES) is a nonsteroidal
synthetic estrogen used through 1970 to decrease the occurrence of miscarriages. Because
it was found to increase the risk of cervical cancer in female offspring (Herbst et al.,
1971), its clinical use was discontinued. Follow-up studies suggest that DES may have
affected sexual orientation of female offspring (but see Titus-Emstoff et al. (2003) for a
report of negative results). When interviewed regarding their sexual orientation, DES-
exposed women are found to have a higher incidence of bisexuality or homosexuality

than non-exposed women (Ehrhardt et al., 1985; Meyer-Bahiburg et al., 1995).

In the present study, early postnatal EB treatment also affected the sexual behavior of the
female rats. The 10% EB dose reduced the time the female spent with the male during the
sexual behavior test, and none of the females that received postnatal EB showed lordosis
in response to male mounts. This effect on female receptivity is in agreement with other
studies showing that neonatal EB treatment decreases the frequency to show lordosis in
females and neonatally castrated males (Levine and Mullins, 1964; Whalen and Edwards,
1967; Whalen and N adler, 1963). Also, perinatal treatment with ATD enhances the
tendency of male rats to show lordosis after castration and treatment with ovarian
hormones in adulthood (Clemens and Gladue, 1978; McEwen et al., 1977; Whalen et al.,
1986), and perinatal treatment with antiestrogen, but not antiandrogen, increases the

frequency of lordosis in male rats (Matuszczyk and Larsson, 1995).

47

Slim
male
the d
Ell'tn
com
the 1-

Mid

In both the partner preference tests and the female sexual behavior tests, the EB females
were less likely than C females to receive mounts, intromissions, or ejaculations from the
stimulus male. The male’s reduced interest for the EB females could reflect multiple
factors, which are not mutually exclusive. The EB females could be less proceptive, they
could be less attractive to the males, or they could be actively avoiding the males, any of
which would lead to decreased copulatory behavior by the stimulus male. Proceptive
behaviors were measured during the female sexual behavior test after the experimental
females had received adult EB + P treatment. EB females show significantly fewer
instances of ear wiggling, hopping and darting, and approaches to the stimulus male
compared to C females. Attractivity was not measured in this study but could also be an
explanation for the low frequency of male behaviors directed toward the EB females.
Stimulus males may find the EB females less attractive than C females resulting in the
male showing less sexual behavior. Further support for this idea comes from the fact that
the differential behavior of the male was also seen during tests when only adult EB was
given, which is a hormonal condition associated with very little proceptivity even for
control females (Frye et al., 1998). Finally, avoidance behaviors were not measured, but
the lack of sexual stimulation by the male may be due to the EB females actively

avoiding contact with the males.

The argument could be made that since the EB females are not receiving sexual behavior
in the partner preference tests, the validity of the preference test is questionable.
However, Adkins—Regan (1988) made the distinction that partner preference and sexual

behavior are not necessarily linked. This outcome was also observed in other work from

48

5“

Hz

all:

this laboratory where perinatal exposure to PCBs was found to alter partner preference
without affecting sexual receptivity (Cummings, personal communication). Therefore, we
abide by the common convention (Baum, 2006) that partner preference may be studied

independently of sexual receptivity and behavior.

There were no statistically significant differences between EB females and C females in
the display of male sexual behavior. The two treatments did not differ in the frequency of
mounts or intromission patterns shown when placed with an estrous stimulus female.
Although the expression of ejaculation patterns did not reach statistical significance, it is
interesting that a total of six females treated during the early postnatal period with EB
showed at least one ejaculation pattern during either the partner preference or male-like
sexual behavior test. Four 10% females showed ejaculatory patterns in both tests, while
two 5% females showed ejaculation patterns in one of the tests. None of the C females
showed ejaculation patterns in any of the tests. Despite not reaching statistical
significance, this effect agrees with previous findings that show estrogen may play a role
in the expression of ejaculatory patterns. Brand et a]. (1991) found that compared with
control males, fewer neonatally ATD-treated males ejaculate when paired with an estrous
stimulus female. A similar decrease in male sexual behavior is also seen after neonatal

treatment with an antiestrogen (Matuszczyk and Larsson, 1995).

Histology: The experimental females treated with early postnatal BB were shown to be
anovulatory prior to ovariectomy and treatment with adult hormones. The early

postnatally treated females showed no corpora lutea present in the ovaries compared with

49

the majority of C females showing corpora lutea, and therefore, regular estrus cycles.
This effect of estradiol on ovarian function is in agreement with other studies that
demonstrate that neonatal EB treatment is sufficient to completely prevent the formation

of corpora lutea in the adult ovaries (Harris and Levine, 1965; Levine and Mullins, 1964).

The SNB is a sexually dimorphic cluster of motoneurons located in the dorsomedial
aspect of the ventral horn of the fifth and sixth segments of the lower lumbar spinal cord
in the rat. The SNB motoneurons innervate four striated perineal muscles: the sexually
dimorphic levator ani (LA) and paired bulbocavemosus (BC) muscles (Breedlove and
Arnold, 1980) and the sexually monomorphic external anal sphincter (EAS) (Sengelaub
and Arnold, 1986). The BC/LA complex is present in adult males and functions in penile
erections and ejaculations. The BC/LA muscles, however, are completely absent or

vestigial in adult females (Breedlove and Arnold, 1980; Cihak et al., 1970).

In the present study, early postnatal estradiol treatment increased the number of
motoneurons seen in the SNB of the experimental females. This finding conflicts with the
current literature on SNB development. The SNB system has previously been shown to
be sensitive to steroid hormones. For example, the motoneurons accumulate radioactivity
after injection with tritiated testosterone and the non-aromatizable androgen,
dihydrotestosterone, but not estradiol (Breedlove and Arnold, 1980). Persistence of the
neuromuscular system postnatally is androgen dependent. Exogenous perinatal androgen
treatment can affect the development of the SNB system in the female. If females are

treated with testosterone propionate both pre- and postnatally, they display the same

50

level
offe:
and l
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thin

The

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developmental patterns as male pups (N ordeen et al., 1985). Perinatal androgen treatment
of females results in an increase in the number of SNB motoneurons, the cell soma size,
and the number of target muscle fibers (Breedlove and Arnold, 1983). Treatment with
estradiol benzoate does not masculinize the SNB in female pups suggesting that

androgens, and not estrogens, control the development of the SNB system (Breedlove et

al., 1982).

The reason for the discrepancy may lie in differences in the treatment paradigm.
Breedlove et al (1982) limited neonatal estradiol treatment to one injection on PND 1. In
the present study, EB was received constantly through the Silastic capsule for 21 days.
The increase in dose and length of treatment may have different effects on the SNB
system. The current hypothesis regarding SNB development is that androgens act at the
BC/LA muscles preventing them from involution, thereby rescuing the SNB
motoneurons indirectly. Also, it has been shown that neonatally treating either castratred
males (McAbee and Doncarlos, 1999) or females (Suzuki and Nishihara, 2002) with
estrogens increases the expression of androgen receptor (AR) mRNA in the
hypothalamus. Since measurable levels of testosterone are present in the plasma of
female rats postnatally (Weisz and Ward, 1980), perhaps the early EB treatment is
increasing the number of AR present in the BC/LA muscles, thereby allowing the
testosterone present to act on the increased receptors, saving the SNB motoneurons from
cell death. The increase in motoneuron number seen, however, may be playing a role in
the expression of male-like ejaculatory patterns shown by the females treated with the

early postnatal estradiol. The early postnatal treatments did not alter the amount of

51

maternal licking and grooming received by the litters; therefore, these data suggest a
direct effect of EB on motorneuron number rather than an increase seen due to maternal

mediation.

The PG is a sexually dimorphic structure that in the male rat contains 2 to 4 times more
cells than in the female (Greenwood et al., 1985). Gonadal steroids have been shown to
have an effect on the number of cells present in the PG. Preliminary work in our
laboratory has shown that neonatal treatment of female pups with one injection of
testosterone propionate (50 pg) on PND 3 resulted in a significant increase in the number
of adult PG cells (Fang and Clemens, unpublished results). Another study looked at the
number of cells in Tfm males. The number of cells in the PG of Tfm males was
significantly less than their wild-type brothers and did not differ from normal females
(Fang, Jordan, Breedlove, and Clemens, unpublished results). These studies indicate the
importance of androgens in the development of the male PG. However, it is not clear

what mechanism is responsible for these androgenic effects.

The results from the present study seem to indicate that the development of the PG does
not rely on the estrogenic metabolite of testosterone. Females treated with either the 5%
or 10% dose of early postnatal EB showed the same number of neurons present in the PG

as did the C females.

52

Effects of Experience and Adult Treatments

Although the alteration of adult female partner preference and sexual behavior by early
postnatal EB treatment did not change across adult hormone or testing conditions, some
effects of adult treatment and experience were evident. The most salient of these effects
was that all experimental females, regardless of hormonal condition, ‘decreased their
latency to enter the male chamber and increased the time spent in the middle chamber
from the initial to the final partner preference test. This effect may be due to the sexual
and social experience the females received during the sexual behavior tests since sexual
experience can play a role in the display of partner preference. Some studies show that
hormonally primed females display a preference for the male stimulus both before and
after sexual experience (Dejonge et al., 1986; Slob et al., 1987). Others find a change in
preference between naive tests and experienced tests, such that the females show no
preference initially but after sexual experience they prefer the male (Matuszczyk and
Larsson, 1991). These different outcomes may be due to the different methods used by
each study. Some experiments use short term treatment (one injection) of EB (Dejonge et
al., 1986), while others use long term (Silastic capsules or repeated injections; present
study, Matuszczyk and Larsson, 1991; Slob et al., 1987). Also, the partner preference
tests differ in terms of whether the experimental female is allowed to interact with the

stimulus animals.

Also, compared to treatment with EB alone, adult EB + P treatment decreased the amount
of time the experimental females spent within the stimulus animals’ chambers during the

partner preference tests, while increasing the time spent in the middle chamber. In

53

addition, EB + P treatment decreased the amount of time the experimental females spent
in the male chamber during the female sexual behavior test compared to treatment with
EB alone. Although these effects were seen across all the early postnatal treatment
groups, the mechanism behind these effects may be different for C versus EB females,
since sexual behavior is facilitated by progesterone only in the case of the C females.
Thus, in the C females the addition of progesterone treatment increases sexual receptivity
and the likelihood of a female-paced copulatory bout (Dejonge et al., 1986; Frye et al.,
1998). During a female-paced sexual behavior test, a receptive female would approach
the stimulus male, be mounted, and then leave the male chamber and enter the escape
chamber leading to a decrease in the overall time spent with the male. Similarly, in a
partner preference test, a receptive female would enter the male chamber, be mounted,
and then move into the middle chamber, thereby decreasing the time spent in the stimulus
chambers and increasing the duration in the middle chamber. Therefore, the differences
seen for both tests of sexual behavior and partner preferences between the EB and the EB
+ P conditions can be explained for the C females as a consequence of the enhanced

receptivity induced by the addition of progesterone in this group.

While enhanced receptivity and the pacing of copulation may account for the increase in
time the C females spent in the escape chambers when given progesterone in addition to
EB, a similar explanation cannot be made for the females that received early postnatal
EB, since they did not receive any intromissions or ejaculations, and evidently were not
pacing copulatory bouts. Failure of the postnatally estrogenized females to show sexual

receptivity following progesterone treatment is consistent with earlier reports showing

54

that perinatal steroid treatments interfere with progesterone responsiveness. A major
effect of perinatal treatment with aromatizable androgens is to eliminate or severely
reduce progesterone enhancement of sexual receptivity (Clemens, 1970; Davidson and
Levine, 1969). But the reduction in the amount of time the EB females in the current
study spent with the stimulus animals following adult EB + P treatment may reflect other
actions of progesterone different from the facilitation of female receptivity. For example,
progesterone has been shown to have anesthetic or tranquilizing effects (Bixo and
Backstrom, 1990; Merryman et al., 1954; Selye, 1941) that use a progesterone receptor
(PR)-independent mechanism of action (Reddy and Apanites, 2005). Such effects might
easily account for the female’s tendency to stay in the middle chamber in the partner
preference test and the escape chamber in the sexual behavior test in the absence of

progesterone-induced sexual receptivity.

The effect of adult progesterone treatment on the amount of time spent in the male
chamber showed an interaction with sexual experience. Sexually na‘r’ve experimental
females treated in adulthood with EB + P spent less time in the male chamber than did
females treated with EB alone. However, this effect of progesterone treatment was absent

on the final test, after the females had received sexual experience.

In the control females, the decrease in time spent in the male chamber from initial to final
test shown by females treated with adult EB alone may be due to dopamine action on the
PR. Adult EB treatment has been shown to increase the number of PR in hypothalarnic

areas important for the expression of female sexual behavior (Maclusky and McEwen,

55

1978; Maclusky and McEwen, 1980). It has also been shown that PR can be activated by
hormonally-independent mechanisms. Depamine agonists have been shown to mimic the
facilitating effect of progesterone on female sexual behavior, and these effects are
blocked by treating with progesterone receptor antagonists or antisense oligonucleotides
to PR mRNA (Mani et al., 1994). Sexual experience is known to increase dopamine
levels in the brain of female rats (Jenkins and Becker, 2003; Mermelstein and Becker,
1995; Pfaus et al., 1995). It is possible that the progesterone treatment of control females
during the initial test increases the female’s sexual motivation, causing her to pace the
copulatory bout, and by doing so decrease the time spent in the male chamber compared
to females that received EB alone. However, when the control females are returned to the
preference chamber for the final test after sexual experience, the females treated with
adult EB alone may have an increase in dopamine levels because previous interactions
with a male have been rewarding for her. This increase in dopamine could act on PR,
mimicking the effects of progesterone treatment and leading to pacing behavior similar to
that of the EB + P-treated females, thus resulting in a decrease in the time spent in the

male chamber.

The explanation provided above for the differential effects of progesterone during the
initial and final partner preference tests does not generalize to the females that received
early postnatal EB. In the early postnatally treated females, progesterone does not
facilitate sexual receptivity, and hence their sexual experience during the sexual behavior

tests are unlikely to be rewarding and in fact may have been aversive. Consequently, this

56

experience may have caused the early postnatally estrogenized females treated with adult

EB to avoid the stimulus male chamber during the final partner preference test.

During the initial partner preference test, females treated with adult EB alone took longer
to enter the female stimulus chamber than did females treated with EB + P. However, by
the last test the two adult hormone groups were similar on this measure. The progesterone
treatment may be decreasing anxiety behaviors in the females (Frye and Walf, 2004), but
this effect may be salient only during the initial test. In the group treated with adult E
alone, the social and sexual experience the experimental females received during the
sexual behavior tests may have caused a reduction in fear of the novel stimulus animals
in the partner preference chamber. Therefore, during the final test, experienced females
showed less fear, and thus shorter latencies to enter the female chamber, even without

progesterone treatment.

Overall

The most robust finding of this study was that postnatal EB treatment alters female
partner preference and sexual behavior. Estradiol seems to have a masculinizing and
defeminizing effect. Females treated with postnatal EB preferred to spend more time with
an estrous female and less time with a sexually active male than did C females. Also, EB
females showed less female sexual behavior, receptivity, proceptivity, and possibly
attractivity to the males, while occasionally showing ejaculatory patterns when placed

with an estrous female. These findings lead to the suggestion that estrogens during the

57

postnatal critical period plays a role in the development of male partner preference and

sexual behavior.

58

CHAPTER THREE:
HYPER-ANDROGEN EXPOSURE DURING DEVELOPMENT ALTERS ADULT
PARTNER PREFERENCE AND REPRODUCTIVE BEHAVIOR IN

GONADALLY INTACT MALE RATS

INTRODUCTION
As previously described, exposure to testosterone during development organizes both the
brain and behavior of male rats (Baum, 1979). However, there are also studies showing
that high levels of exogenous testosterone administered to an intact male early in life may
alter adult behavior (Pollak and Sachs, 1975). In the rat, neonatal administration of
testosterone propionate to a castrated male causes both defeminization and
masculinization of behavior (Beach et al., 1969). However, if an intact male is treated
neonatally with testosterone, there is a disruption of normal male sexual behavior (Pollak
and Sachs, 1975). There is a chance the hyper-androgen treatment is altering the male’s
partner preference, thereby decreasing the male’s interest in a female partner. Therefore,
the intact male rats that receive high androgen exposure may be exhibiting a suppression

of male sexual behavior as a result of an altered partner preference.

Prenatal hyper-androgen exposure may also play a role in human sexual orientation.
Homosexual men have been shown to have larger genitalia (Bogaert and Hershberger,
1999), more masculine auditory evoked potentials (McFadden and Champlin, 2000), and

more masculine 2D:4D digit length ratios (Rahman, 2005; Rahman and Wilson, 2003;

59

Robinson and Manning, 2000; reviewed in Chapter One) than heterosexual men

indicating a higher level of androgen during development.

In the present study, the effect of exogenous testosterone on adult behavior was tested by
treating intact male rats during either the early postnatal period or prenatal period with
testosterone propionate and examining their adult partner preference. In addition, the
effects of early postnatal exposure to testosterone on serum testosterone and estradiol
levels and cell number in the spinal nucleus of the bulbocavemosus and pelvic ganglia

were also measured.

METHODS

Animals

Experimental Males (Experiments 1-3): Time-mated pregnant Long-Evans rats (Charles
River, Raleigh, NC) were housed individually with ad lib food and water in plastic cages
(45.5 x 24 x 21 cm) in a 14:10-hr light dark cycle with lights on at 01:00. For nest
building material thirty, one-inch paper towel strips were given to the dams on GD 20. A
subset of the male offspring of these dams became the experimental males of this study
(see below). In Experiments 1 and 2, on the day of birth, PND O, the litter was reduced to
four male and four female pups (Figure 3.1A). For litter reductions, the AGD for each
pup was measured, and since the AGD is shorter in females than in males, the four
shortest and the four longest were retained. In Experiment 3, since prenatal testosterone

increases AGD of both sexes, the seven animals with the largest AGD were kept to

60

maximize the chance of picking a male and the animal with the smallest AGD was kept

in an attempt to prevent forming an all-male litter (Figure 3.2B).

Experimental Male Hormone Treatments (Experiments 1,2): Silastic capsules (Dow
Corning; inner diameter 1.47 mm; outer diameter 1.96 mm; length 5 mm) were used to
administer either testosterone propionate (Sigma; TP) or cholesterol (Sigma; C)
treatments on the day of birth (Figure 3.1A). Capsules were implanted subcutaneously
(s.c.; one treatment per litter) through a small incision on the back of the animal while the
pups were under ice anesthesia on PND 0. These animals are referred to as TP or C
males, respectively. The incision was closed using superglue (Loctite). Following
surgery, pups were warmed under a heat lamp until the incision was dry (approximately
one hour) and then returned to their mothers. Capsules were left in place for 3 weeks. On
PND 21, at weaning, the pups were anesthetized with isoflurane (Isoflo, Abbot
' Laboratories), and the implants were removed through an incision made near one end of
the capsule (Figure 3.1B). The incision was closed with an Auto Clip (Clay Adams) and
covered with First Aid Cream (Johnson and Johnson). Pups were then housed with same-
sex littermates. Only male pups were used in this Experiment. Behavioral tests began
after the animals reached 90 days of age. (Experiment 1: TP n=18, C n=14; Experiment

2: TP n=19, C n=20; Figure 3.1C).

Experimental Male Hormone Treatments (Experiment 3): Pregnant dams were treated
with either TP in sesame oil (2 mg/0.l ml/day) or oil vehicle on GD 16-20 (Figure 3.2A).

Pups were weaned on PND 21 (Figure 3.2C) and housed with same-sex littermates until

61

90 days of age when behavioral tests were conducted (Figure 3.2D). These males are
referred to as TP (n=9) and Oil (n=13) males, respectively. Only male pups were used in

this Experiment. Behavioral tests began after the animals reached 90 days of age.

Stimulus Females (Experiments 1-3): Sexually experienced, gonadally intact, adult Long
Evans female rats at least 60 days old (Charles River, Raleigh, NC) were used as stimulus
animals for the behavioral tests. Stimulus females were implanted with a Silastic capsule
containing 25% EB-C mixture prior to testing but were not ovariectomized. The capsules
were implanted s.c. while the animals were anesthetized with isoflurane; the incision was
closed with an Auto Clip and covered with First Aid Cream. After 4 weeks, the capsules
begin to lose efficacy because of connective tissue growth around the capsule (personal
observation). The capsules were removed and reimplanted via a new incision in the neck.
Stimulus females were injected s.c. with 0.5 mg progesterone four hours prior to partner

preference and sexual behavior testing.

Stimulus Males (Experiments 1,3): Sexually experienced, gonadally intact, adult Long
Evans male rats at least 90 days old (Charles River, Raleigh, NC) were used as stimulus

animals for the behavioral tests.

Stimulus Males (Experiment 2): The stimulus males in Experiment 2 received ATD
(Sigma) postnatally. Male neonates were treated with Silastic capsules containing ATD
on PND 0 through 21. Procedures were the same as those used for the early postnatal TP

and C treatments. This treatment was used because Experiment I attempted to determine

62

if the experimental males would show sexual behavior with another male. However,
during the social and sexual behavior tests the experimental and stimulus males often
fought. It was also found that during the final partner preference test of Experiment 1,
both the TP and the C males spent more time alone in the middle chamber than would be
expected by chance (data not shown). The social and sexual tests with the stimulus male
may have been aversive to the experimental males, causing them to remain in the middle
chamber. In an attempt to decrease the aggression shown between the experimental and
stimulus males, ATD was used. Males treated postnatally with ATD have been shown to

display lordosis when paired with another male (Baker et al., 1996).

Animals were maintained in a facility accredited by the Association for Assessment and
Accreditation of Laboratory Animal Care, and all experimental procedures were

approved by the Michigan State University Animal Care and Use Committee.

Behavioral Testing (Experiments 1-3)
Partner Preference: Tests for partner preference were conducted as described in Chapter

Two.

Social and sexual behavior: Tests for social and sexual behavior displayed by the
experimental males were conducted in a Plexiglas observation chamber (46 x 58 x 51
cm). During the test, the experimental male had unrestricted access to the stimulus
animal. The tests lasted 25 minutes or until ejaculation, whichever occurred first.

Behavioral testing took place under dim red light illumination in the middle part of the

63

dark phase of the light-dark cycle. Video recordings of these tests were analyzed to
determine frequency of mounts, intromissions, and ejaculatory patterns shown by the

experimental males and the latency to show these behaviors.

Testing Schedule: The six-week testing schedule is summarized in Figure 3.3. The initial
partner preference of the male was tested in week 1. Each experimental male received
social and sexual experience with both male and female stimulus animals during weeks 2
and 3, but data were not collected. During experience weeks, experimental males were
partnered with stimulus animals for 30 minutes during which time sexual behavior could
occur. During week 4, half of the experimental males were tested for social and sexual
behavior with a male and the other half with a female. The sex of the stimulus animals
was switched for week 5. Sexual behavior during weeks 4 and 5 was recorded and

scored. In week 6, the male’s final partner preference was evaluated.

Histology (Experiment 1)
Pelvic Ganglia and Spinal Nucleus of the Bulbocavernosus: Tissue removal and
histological processing were conducted as described in Chapter Two. A subset of males

from Experiment 1 were analyzed for cell counts (Figure 3.1C).

Hormone Analysis (Experiments 1,2)
Prior to perfusion, blood was collected intracardially into heparinized tubes (Figure
3.1C). The samples were kept on ice for 30 minutes, and then centrifuged at 8° C for 20

minutes at 3000 rcf. The supernatant was removed and stored at -20° C.

64

A solid-phase radioimmunoassay that uses 125iodine-labeled steroid, which competes with
steroid in the sample for antibody sites in a polypropylene tube, was used to determine
the amount of hormones in the samples. Testosterone and estradiol were measured using
the Coat-A-Count Testosterone (Siemens) and Active Estradiol (Diagnostic Systems
Laboratories, Inc.) radioimmunoassay kits, respectively. After incubation for 3 hours
(testosterone) or 2 hours (estradiol) at 37° C, the tube was decanted and counted in a
gamma counter. The quantity of hormone present in the sample was determined from a
calibration curve. Radioimmunoassays were completed by the Diagnostic Center for
Population and Animal Health at Michigan State University. A subset of males from both

Experiment 1 and Experiment 2 were used for the radioimmunoassays.

Analysis

The data for the behavioral measures during the partner preference tests were analyzed
using a 2 x 2 (perinatal treatment x initial or final test) AN OVA with repeated measures
on the second factor. Preference scores and duration of time spent in the male chamber
during the partner preference tests were also analyzed within each early postnatal
treatment group using a one-sample t—test. This test was used to determine if the animals
showed a preference for one stimulus animal above chance (preference score = O) or
spent more time with the male above chance (duration = 400 seconds). The data for the
behavioral measures during the social and sexual behavior tests, histological cell counts,

and hormones assays were analyzed using an independent samples t-test.

65

For some variables, the data did not meet homogeneity of variance assumptions, even
after the prescribed transformations (i.e. square root). For these measures, nonparametric
statistics were used for analysis. The Mann-Whitney U and the Fisher’s exact probability

tests were used for these analyses.

RESULTS
Experiment 1 — Early Postnatal TP
Partner Preference: Early postnatal treatment with testosterone altered the partner
preference of the male experimental animals. The TP males spent more time in the
stimulus male chamber during both partner preference tests than did the C males (Figure
3.4; [F(1,27) = 18.0, p<0.001]. Also, the C males showed a higher preference score than

that of the TP males (Figure 3.5; [F(1,27) = 10.9, p=0.003]).

In both the initial and final partner preference test, the C males had positive preference
scores significantly different from chance, indicating a preference for the female stimulus
animal. The TP males, however, did not show a preference greater than chance for either
stimulus animal (Table 3.1). Also, the duration of time spent in the male chamber in both
the initial and final tests was significantly less than chance for the C males, but not for the

TP males (Table 3.1).

The latency to enter the stimulus female chamber [F(1,27) = 12.7, p=0.001] and time
spent with the male [F(1,27) = 4.2, p=0.05] were affected by a main effect of week of

test. Regardless of early postnatal hormone treatment, the males decreased their latency

66

to enter the female chamber from initial to final test (X— :1: SEM: all measures in seconds:

39.3 :1: 4.2 vs. 23.2 i 2.8). They also decreased the amount of time spent in the stimulus

male chamber from initial to final test or a». SEM: 335.2 1 22.7 vs. 252.0 1 35.1).

Non—parametric statistics were necessary to analyze the amount of time spent in the

middle chamber. TP males spent less time alone in the middle chamber during the initial

partner preference test than during the final test (5(- :1: SEM: 366.8 i 41.8 vs. 472.6 :1:

47.6).

The proportion of males that showed sexual behavior with the stimulus female during the
partner preference test was analyzed using the Fisher’s exact probability test. More C
males showed mounts and intromissions during the initial test, and more mounts,
intromissions, and ejaculations during the final test compared with the TP males (Table

3.2). No differences were seen in behaviors shown with the stimulus male.

Social and Sexual Behavior: Early postnatal testosterone treatment altered the expression
of male sexual behavior when paired with a stimulus female. TP males showed fewer
mounts, intromissions, and ejaculations during the test than did C males (Figure 3.6;
Mann-Whitney). Also, the proportion of males that showed behaviors when paired with a
female was higher in the C male group than in the TP male group (Table 3.3). No

significant differences were seen in behaviors shown when paired with the stimulus male.

67

Hormone Assays: The amount of circulating testosterone was affected by the early
postnatal testosterone treatment during early life (t = -2.7, p=0.012). The C males had
significantly higher circulating testosterone levels than did the TP males (Figure 3.7). To
rule out the possibility that changes in behavior were seen due to different levels of
circulating testosterone, a correlation was run between serum testosterone levels and
preference score. No correlation between the two measures was seen. Circulating levels

of estradiol were not affected by the early hormone treatments.

Histology: Early postnatal testosterone did not have an effect on cell number in either the

PG (2? :1: SEM: TP: 6261.7 :1: 432.2; C: 5983.4 :1: 635.5; p>0.05) or the SNB ()7 a: SEM:

TP: 75.2 i 3.3; C: 86.0 i 4.4; p>0.05).

Experiment 2 - Early Postnatal TP and ATD Stimulus Male

Partner Preference: Treatment with testosterone early in development altered the partner
preference of the male experimental animals. The TP males spent more time in the ATD
stimulus male chamber during the partner preferences tests than did the C males (Figure
3.4; [F(1,31) = 4.1, p=0.05]). The data for time spent in the male chamber were subjected

to a square root transformation.

In both the initial and final partner preference test, the C males had positive preference
scores statistically different than chance, indicating a preference for the female stimulus
animal. The TP males, however, did not show a preference greater than chance for either

stimulus animal (Table 3.1). Also, the duration of time spent in the ATD male chamber in

68

the final test was significantly less than chance for the C males, but not for the TP males

(Table 3.1).

Social and sexual behavior also altered the partner preference of the experimental males
regardless of hormone treatment. Across both hormone treatments, the average latency to
enter the female chamber was significantly shorter for the final test compared to the
initial one (if 1 SEM: 20.1 i 2.0 vs 55.5 i 6.4; [F(1,31) = 38.2, p<0.001]). The latency
to enter the ATD male chamber was also shorter for the final test than for the initial test
(.3? :1: SEM: 29.3 i 3.4 vs 76.4 i 18.6; [F(1,31) = 5.8, p=0.02]). The experimental males
spent less time in the middle chamber during the initial partner preference test than
during the final test (2?: SEM: 311.1 j; 21.3 vs 382.1 i 24.9; [F(l,31) = 5.6, p=0.02]),
and they spent more time in the ATD male chamber during the initial test compared with
the final test (if: SEM: 345.6 _-I_- 22.2 vs 245.8 i 29.0; [F(1,31) = 14.7, p=0.001]).
Finally, the males had a lower preference score during the initial test than during the final

test (2'? i SEM: 197.6 i 50.0 vs 323.9 i 64.3; [F(1,31) = 6.4, p=0.01]).

The proportion of males that showed sexual behavior with the stimulus female during the
partner preference test was analyzed using the Fisher’s exact probability test. More C
males showed mounts, intromissions, and ejaculations during the final test compared with
the TP males (Table 3.2). No differences were seen in behaviors shown with the stimulus

male.

69

Social and Sexual Behavior: Early postnatal testosterone treatment altered the expression
of male sexual behavior when paired with a stimulus female. TP males showed fewer
mounts, intromissions, and ejaculations during the test than did C males (Figure 3.6;
Mann-Whitney). Also, the proportion of males that showed behaviors when paired with a
female was higher in the C male group than in the TP male group (Table 3.3). No

significant differences were seen behaviors shown when paired with the stimulus male.

Experiment 3 — Prenatal TP

Partner Preference: In initial and final partner preference tests, both the C males and the
prenatal TP males had positive preference scores statistically different from chance,
indicating a preference for the female stimulus animal (Table 3.4). Also, the duration of
time spent in the male chamber in the initial and final tests for the C males, and the final

test for the TP males was significantly less than chance (Table 3.4).

Social and sexual experience altered the partner preference of the experimental males

regardless of hormone treatment. The latency to enter the female chamber was longer

during the initial partner preference test compared to the final (I? 1 SEM: 51.0 i 11.4 vs.
14.4 :1: 3.1; [F(l,12) = 24.9, p<0.001]). The data for latency to enter the female chamber
was subjected to a square root transformation. The duration of time spent in the female
chamber was higher during the final test than during the initial (if :1: SEM: 813.6 i 51.5
vs. 655.7 :I: 40.8; [F(l,12) = 8.0, p=0.015]). The duration of time spent in the male
chamber also decreased from initial to final test (2? 1 SEM: 316.8 :1: 24.8 vs. 100.7 1

23.6; [F(l,12) = 75.8, p<0.001]). Finally, the males showed an increased preference score

70

from the initial test to the final test (5(- 1 SEM: 338.8 i 63.2 vs. 712.9 1 71.9; [F(l,12) =

24.8, p<0.001]).

The latency to enter the stimulus male chamber was affected by a test by prenatal
treatment interaction [F(l,12) = 14.2, p=0.003]. The Oil males decreased the latency to
enter the chamber from initial to final test (2? a; SEM: 79.8 1 13.5 vs. 17.9 a 6.3), but the
TP males showed no change (2? i SEM: 23.3 i 13.5 vs. 35.9 :l: 6.3). Also, during the

initial test, the Oil males took longer to enter the male chamber than did the TP males (5(-
:1: SEM: 79.8 i 13.5 vs. 23.3 :t 13.5). The data for latency to enter the male chamber was

subjected to a square root transformation

Prenatal testosterone treatment only had an effect on one sexual behavior measure during
the partner preference tests. During the final test, the TP males showed fewer
intromissions than did the Oil males (Mann-Whitney U; 5(- 1 SEM: 6.3 i 2.8 vs. 19.4 :1:

2.8).

Social and Sexual Behavior: No behaviors measured during the social and sexual

behavior tests were affected by the prenatal testosterone treatment.

71

TABLES AND FIGURES

 

 

 

 

 

 

 

 

 

 

 

 

 

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Table 3.2. Proportion of experimental males that displayed sexual behavior with the
stimulus female during the partner preference tests (Experiments 1 and 2). In Experiment
1, the experimental males were exposed to early postnatal treatments and stimulus males
were untreated. In Experiment 2, experimental males were exposed to early postnatal
treatments and stimulus males were treated with ATD. C males were more likely to
display male sexual behavior with the stimulus female than were TP males in both
partner preference tests during Experiment 1, and in the final partner preference test
during Experiment 2. *Significantly different from cholesterol, p<0.02, **Significantly

different from cholesterol, p<0.001

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Experiment 1
Initial Test Final Test
Behavior Mount Intromission Mount Intromission Ejaculation
Cholesterol 6/ 12 4/ 12 10/14 10/14 8/14
Testosterone* 1/ 17 0/ 17 3/ 18 2/ 18 1/ 18
Experiment 2
Initial Test Final Test
Behavior Mount Intromission Mount Intromission Ejaculation
Cholesterol 8/ 19 5/ 19 15.17 15/17 12/17
Testosterone 5/18 1/18 2/18** 2/18** 1/18**

 

 

 

 

 

 

 

73

Table 3.3. Proportion of experimental males that displayed sexual behavior with the
stimulus female during the social and sexual behavior tests (Experiments 1 and 2). In
Experiment 1, the experimental males were exposed to early postnatal treatments and
stimulus males were untreated. In Experiment 2, experimental males were exposed to
early postnatal treatments and stimulus males were treated with ATD. C males were more
likely to display male sexual behavior with the stimulus female than were TP males in

both experiments. *Significantly different from cholesterol, p<0.002

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Experiment 1
Social and Sexual Behavior Test
Behavior Mount Intromission Ejaculation
Cholesterol 11/13 1 1/ 13 9/ 13
Testosterone* 5/18 4/18 2/ 18
Experiment 2
Social and Sexual Behavior Test
Behavior Mount Intromission Ejaculation
Cholesterol 19/21 19/21 16/21
Testosterone* 6/20 6/20 5/20

 

 

 

 

 

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Litter Reduction Capsule IRemoval Partner P'reference Histology Hormones

 

 

 

Silastic Capsules Weaning Social and Sexual -PG ~Testosterone
-Cholesterol Behavior Tests -SNB -Estradiol
-Testosterone -With Male

Propionate -With Female

 

Figure 3.1. Treatment and testing schedule for the experimental males (Experiments 1
and 2). In Experiment 1, the experimental males were exposed to early postnatal
treatments and stimulus males were untreated. In Experiment 2, experimental males were
exposed to early postnatal treatments and stimulus males were treated with ATD. (A) On
the day of birth, PND O, litters were reduced to four males and four females and received
Silastic capsules of cholesterol or testosterone propionate (Experiment 1: TP n=18, C
n=14; Experiment 2: TP n=19, C n=20). (B) At weaning on PND 21, the Silastic capsules
were removed. (D) After the males reached 90 days of age, they were tested for partner
preference and social and sexual behavior with both male and female stimulus animals.
After testing was concluded, the pelvic ganglia (PG) and spinal nucleus of the
bulbocavemosus (SNB) were removed. Blood samples were also taken to analyze plasma

testosterone and estradiol concentrations.

76

 

 

the

fen

 

 

A GD 16-20 B PNP O C PND 21 D After PND 9O
Injectibns Litter R'eduction Wea'ning Partner Plreference
-Oil Social and Sexual
-Testosterone Behavior Tests

Propionate -With Male
-W1th Female

 

 

 

 

 

 

Figure 3.2. Treatment and testing schedule for the experimental males (Experiment 3). In
Experiment 3, experimental males were exposed to prenatal treatments and stimulus
males were untreated. (A) Pregnant dams were treated with either oil (n=13) or
testosterone propionate (n=9) on GD 16-20. (B) On the day of birth, PND O, litters were
reduced. (C) Pups were weaned on PND 21. (D) After the males reached 90 days of age,
they were tested for partner preference and social and sexual behavior with both male and

female stimulus animals.

77

M

We

We

We

We

Fig
Ex;
stin
earl
CXp
um:

ICC”:

 

Week I lnitial Partner Preference

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1
Weeks 2&3 Sexual and Social
Experience
Week 4 Sexual Behavior Sexual Behavior
WIth Female With Male
l [

, Sexual Behavior Sexual Behavior

Week 5 ,
With Male Mth Female

Week 6 Final Partner Preference

 

 

 

Figure 3.3. Behavioral testing schedule for the experimental males (Experiments 1-3). In
Experiment 1, the experimental males were exposed to early postnatal treatments and
stimulus males were untreated. In Experiment 2, experimental males were exposed to
early postnatal treatments and stimulus males were treated with ATD. In Experiment 3,
experimental males were exposed to prenatal treatments and stimulus males were
untreated. Data were not collected during weeks 2 and 3 during which the animals

received sexual and social experience.

78

Ch
CD
CD

 

A B Test
I Initial
500 - - I Final

 

 

 

 

      

Testosterone Cholesterol Testosterone Cholesterol
Early Postnatal Trauma Early Postnatal Treatment

Figure 3.4. Time spent with in the stimulus male chamber during the partner preference
tests (Experiments 1 and 2). (A) In Experiment 1, the experimental males were exposed
to early postnatal treatments and stimulus males were untreated. (B) In Experiment 2,
experimental males were exposed to early postnatal treatments and stimulus males were
treated with ATD. In both experiments the TP males spent more time in the stimulus
male chamber than did the C males. *Significantly different from TP males, p<0.05.

“Significantly different from TP males, p<0.001.

79

 

 

 

 

 

 

 

 

 

700
nm
600‘ IFinal
500- *
i
3 400 -
a
E 300 -
E
200 -
100 q
0 .
Testosterone Cholesterol
Early Postnatal Treatment

Figure 3.5. Preference scores of the experimental males during the partner preference
tests (Experiment 1). In Experiment 1, the experimental males were exposed to early
postnatal treatments and stimulus males were untreated. Preference score is calculated as
time spent with stimulus female minus the time spent with stimulus male. The C males
showed a higher preference score than did the TP males in the partner preference tests in

Experiment 1. *Significantly different from TP males, p<0.01.

80

20

 

 

A B Early Postnatal
Treatment

I Testosterone
I Cholesterol

 
  

 

 

15-

 

10-

Frequency

5.1

 

 

 

                 

Mount Intromission Ejaculation Mount Intromission

Behavior Behavior

Ejaculation

Figure 3.6. Frequencies of mounts, intromissions, and ejaculations displayed by the
experimental males during the social and sexual behavior tests when paired with a
stimulus female (Experiments 1 and 2). (A) In Experiment 1, the experimental males
were exposed to early postnatal treatments and stimulus males were untreated. (B) In
Experiment 2, experimental males were exposed to early postnatal treatments and
stimulus males were treated with ATD. TP males showed fewer sexual behaviors

compared to the C males. *Significantly different from TP males, p<0.03.

81

 

Testosterone (ng/ml)
U)

 

 

 

Testosterone Cholesterol
Early Postnatal Treatment

Figure 3.7. Levels of circulating testosterone in a subset of males from Experiments 1 and
2. In Experiment 1, the experimental males were exposed to early postnatal treatments
and stimulus males were untreated. In Experiment 2, experimental males were exposed to
early postnatal treatments and stimulus males were treated with ATD. C males had higher
levels of plasma testosterone than did TP males. *Significantly different from TP males,

p=0.012.

82

DISCUSSION
Early Postnatal Hormone Effects
Regardless of the treatment of the stimulus male (Experiments 1 and 2), early postnatal
testosterone treatment increased the amount of time the TP males spent in the stimulus
male chamber compared to that of the C males. The C males spent less time in the male
chamber than was expected by chance, but the TP males did not. This resulted in the C
males showing a distinct preference for the stimulus female, whereas the TP males did

not show a preference for a stimulus animal in either Experiment 1 or 2.

In the human literature, the idea arises that hyper-androgen exposure early in
development may lead to an increase probability of homosexuality (Rahman, 2005;
Rahman and Wilson, 2003; Robinson and Manning, 2000; Williams et al., 2000). In the
present study, a change in sexual preference for a stimulus female to a stimulus male by
the TP males is not seen, but there is a disruption of the clear preference for the female
shown by the C male controls. A preference for the female is not seen because the TP
males are spending as much time with the stimulus males as they are with the stimulus
females. The TP males also spent as much time with the female as did the cholesterol
males. Thus, the TP males’ interest in the female did not decline, but their interest in the
male increased Therefore, in terms of preference, the early testosterone treatment appears

to have caused the males to become bisexual in their preference.

Early postnatal TP treatment also disrupted male sexual behavior. In both Experiments 1

and 2 during the final partner preference test and during the social and sexual behavior

83

tests, the C males were more likely to show copulatory behavior with a stimulus female
than were the TP males. The disruption of male sexual behavior is in agreement with
other studies that find perinatal treatment of intact male rats (Diamond et al., 1973;
Piacsek and Hostetter, 1984; Pollak and Sachs, 1975; Zadina et al., 1979), ferrets (Baum
and Schretlen, 1975), and hamsters (Vomachka et al., 1981) with TP reduced male
copulatory behavior in adulthood. It is difficult to determine from these data if the early
hormone treatment disrupted normal male sexual behavior or if the TP males are less
interested in the stimulus female. It is possible that the TP males do not show a
preference for the female because they have copulatory deficits. However, it is equally
likely that they are less motivated to copulate and, therefore, do not show a preference for

the female.

Circulating testosterone levels were also affected by early postnatal hormone treatment in
the present study. C males had higher levels of serum testosterone than did the TP males.
In a previous study, male hamsters given TP early in development also showed a
decrease in adult serum testosterone levels (Vomachka et al., 1981), and numerous
studies have linked neonatal estrogen treatment to decreased adult testosterone
production (for review, Delbes et al., 2007). One hypothesis concerning the effect of
neonatal steroid treatment on adult hormone levels is that early estrogen may have a
direct inhibitory effect on the enzymes that produce testosterone (Goyal et al., 2004;
Goyal et al., 2003; Majdic et al., 1996; Tsaimorris et al., 1986). It also has been shown
that early treatment with estrogens decreases AR-ir and ERa-ir in the testis in adulthood

(Shibayama et al., 2001; Warita et al., 2006). While the mechanism concerning how early

84

steroids affect adult testosterone levels needs further elucidation, it appears that neonatal
hormone treatment permanently alters the sensitivity of the hypothalamic-pituitary-

gonadal axis and reduces the responsiveness of adult Leydig cells.

It is unlikely, however, that the behavioral differences in the display of partner preference
and sexual behavior seen in the males treated with early postnatal testosterone are due to
lower levels of serum testosterone that controls. In the present study, no correlation was
seen between serum testosterone level and preference score. Also, plasma testosterone
concentrations as low as 0.7 ng/ml have been shown to maintain male sexual copulatory
measures in castrated adult rats (Damassa et al., 1977), which is the mean concentration
of serum testosterone seen in the early testosterone-treated males in this study. Portillo et
al (2006) showed that sexually sluggish male rats (duds) did not have testosterone
concentrations different from copulating males and that both groups showed male-typical
partner preferences. Finally, ATD males show a nocturnal rhythmicity in their partner
preference, preferring a stimulus male early in the dark phase and an estrous female late
in the dark phase, whereas control males prefer an estrous female throughout (Bakker et
al., 1995). Adult castration and testosterone replacement did not affect partner preference
expression indicating that the ATD male preference was not due to individual differences
in circulating testosterone. Therefore, circulating testosterone levels do not appear to

correlate with male behavior.

In the human literature, results from studies of sexual orientation and circulating hormone

levels are inconsistent. Some studies find no effect of orientation on plasma testosterone

85

levels, some find testosterone levels to be higher in homosexual men than heterosexual
men, and some find testosterone levels to be higher in heterosexual men than homosexual
men (for review, Banks and Gartrell, 1995). No definite conclusions about the
relationship between human circulating steroid levels and sexual orientation can be
drawn at this time. In the present study low circulating testosterone levels were associated
with altered partner preference behavior and reduced male sexual behavior; however, the
extent to which these low levels contributed to this altered preference cannot be

determined from these data.

Early postnatal testosterone treatment did not affect the number of motoneurons in the
SNB or neurons in the PG. These areas appear not to be affected by increased levels of

exogenous androgens postnatally.

Prenatal Hormone Effects

Prenatal TP treatment did alter a few behaviors measured during the behavior tests, but it
did not have a large effect on adult partner preference or sexual behavior. Both the Oil
and TP males showed a positive preference score during both tests, and during the social
and sexual behavior tests, the prenatal TP treatment did not affect any of the behaviors

measured.

In both partner preference tests the Oil males spent less time with the stimulus male than
was expected by chance, but the TP males did so only during the final test. Also, Oil

males decreased their latency to enter the male chamber from initial to final test, whereas

86

the TP males remained the same, and therefore, during the initial test, the Oil males took
longer to enter the male chamber than did the TP males. These interactions may imply
that the Oil males were less interested in the stimulus male than the TP males during the
initial partner preference. However, no statistical differences were seen between the
treatment groups when analyzing the amount of time spent with the male. Therefore,
although the Oil males spent less time than chance with the stimulus male, the Oil males
were not significantly different from the TP males. Finally, in the final partner preference
test, the Oil males showed more intromissions with the stimulus female than did the '1‘?
males. However, no other sexual behavior measures were altered by the early hormone
treatment, including ejaculation frequency during the final test or any measures during

the sexual behavior test with a female.

Experience Effects

During the partner preference tests, regardless of perinatal hormone treatment, a number
of measures were affected by the experience received during the social and sexual
behavior tests. In Experiment 1, the males decreased the time to enter the female chamber
and decreased the amount of time spent in the male chamber from initial to final test.
Also, the TP male increased the amount of time spent in the middle chamber from initial
to final test. In Experiment 2, the latency to enter both stimulus animal chambers
decreased from initial to final test, as did the amount of time spent in the male chamber.
The males increased the amount of time spent in the middle chamber from initial to final
test and showed a higher preference score in the final test. In Experiment 3, the males

decreased the time to enter the female chamber and decreased the amount of time spent in

87

the male chamber from initial to final test. They also increased the amount of time spent

with the female and increased their preference score from initial to final test.

These changes can be explained by the experience the males received between the two
partner preference tests. Sexual experience has been shown to lead to increased
preference for a female over a male stimulus animal (Matuszczyk and Larsson, 1994).
Therefore, in the present study, the sexual experience would lead to a decreased latency
to enter the female chamber (Experiments 1, 2, 3), a reduced amount of time spent with
the male (Experiments 1, 2, 3), an increased preference score (Experiments 2, 3), and

increased time spent with the stimulus female (Experiment 3).

In experiments 1 and 2, the increase seen in the amount of time spent in the middle
chamber from initial to final test is also probably due to the social and sexual experience
received. In Experiment 1, we thought the tests with a stimulus male were probably
aversive to the experimental males because in the final test, both treatment groups spent
more time alone in the middle chamber than was expected by chance (data not shown).
The change in stimulus males was an attempt to prevent this aversion. However, the
experimental animals still spent more time-alone‘during the final partner preference test
in Experiment 2. The experience with the ATD stimulus males may have been aversive to
both treatment groups in Experiment 2. It is also possible that they interaction with ATD
males was aversive to the C males, whereas the sexual tests with a female were aversive
to the TP males, which could also explain the increase in middle chamber duration seen

in Experiment 1.

88

Selection of Experimental Males

It should be noted that the experimental males used in the present study were not selected
randomly at birth. The males with the largest AGD were kept on PND 0 when the litter
was reduced. The AGD is an indication of androgen exposure perinatally, and therefore

the males used were selected on the basis of high androgen responsiveness.

Overall

The early postnatal testosterone treatment caused the males to increase the time spent
with the stimulus male without affecting the time spent with the stimulus female. This
resulted in the testosterone treated males showing no preference for either animal, and no
sexual behavior was seen with either stimulus animal. Exogenous testosterone during
early development also decreased adult levels of circulating testosterone compared to
controls. Prenatal testosterone treatment did not significantly alter either partner

preference or sexual behavior.

89

CHAPTER FOUR:
ESTROGEN IS NOT RESPONSIBLE FOR THE EFFECTS OF HYPER-
ANDROGEN EXPOSURE DURING DEVELOPMENT ON ADULT PARTNER
PREFERENCE AND REPRODUCTIVE BEHAVIOR IN GONADALLY INTACT

MALE RATS

INTRODUCTION
In the previous chapter, I reported that early postnatal treatment of male rats with
testosterone propionate altered the expression of adult partner preference. Males treated
during early development with testosterone spent more time in the stimulus male
chamber during the preference tests than did cholesterol-treated controls. Since
testosterone can have its effects by acting directly on the androgen receptor, or by being
metabolized into dihydrotestosterone and estradiol, further studies are needed to
determine the mechanism by which the early testosterone treatment is changing the adult

behavior.

Early postnatal estrogen treatment of males has effects on adult behavior that are similar
in some ways to those seen after postnatal treatment with testosterone. For example,
neonatal estradiol exposure, like testosterone treatment (Pollak and Sachs, 1975), has
been shown to disrupt male sexual behavior in adulthood (Diamond et al., 1973; Harris
and Levine, 1965; Whalen, 1964). Also, early estrogen exposure has been shown to
decrease adult levels of circulating testosterone (Atanassova et al., 1999; Delbes et al.,

2007; Goyal et al., 2004; Goyal et al., 2003; Shibayama et al., 2001; Warita et al., 2006),

90

a measure that was also altered by early testosterone administration in the previous study.
As reported in Chapter Two, early postnatal estrogen masculinizes partner preference of
adult females in the same manner as neonatal testosterone treatment (Meyerson and
Lindstrom, 1973). From these comparisons, it is reasonable to suggest that the effects of
early postnatal testosterone treatment, seen in the previous chapter, are due to the action
of the estrogenic metabolite of testosterone. Therefore, to determine if the alteration of
adult male partner preference by hyper-androgen exposure early in development is
mediated by the estrogen metabolite, intact male rats were exposed to early postnatal

estradiol treatments and adult behaviors were examined.

NIETHODS

Animals

Experimental Males: Time—mated pregnant Long-Evans rats (Charles River, Raleigh,
NC) were housed individually with ad lib food and water in plastic cages (45.5 x 24 x 21
cm) in a 14:10—hr light dark cycle with lights on at 01:00. For nest building material
thirty, one-inch paper towel strips were given to the dams on GD 20. A subset of the male
offspring of these dams became the experimental males of this study (see below). On the
day of birth, PND 0, the litter was reduced to four male and four female pups (Figure
4.1A). For litter reductions, the AGD for each pup was measured, and since the AGD is

shorter in females than in males, the four shortest and the four longest were retained.

91

Stimulus Animals: Sexually experienced, gonadally intact, adult Long Evans rats (Charles
River, Raleigh, NC) were used as stimulus animals for the behavioral tests (females at

least 60 days old; males at least 90 days old).

Animals were maintained in a facility accredited by the Association for Assessment and
Accreditation of Laboratory Animal Care, and all experimental procedures were

approved by the Michigan State University Animal Care and Use Committee.

Hormone Treatments

Postnatal hormone treatments were the same as those used in Chapter Two (Figure
4.1A,C). Only male pups were used in this Experiment. Behavioral tests began after the
animals reached 90 days of age (Figure 4.1D). While these estradiol treatments are most
likely outside the physiological range of estrogen exposure, this study was designed to
provide further details about long-lasting effects of early exposure to estrogen on males.
A number of studies show that early exposure to estrogenic environmental contaminants

disrupts normal male sexual development (for review, Delbes et al., 2006).

Stimulus females hormone treatments were the same as those used in Chapter Two.

Maternal Licking and Grooming

Maternal behavior was analyzed as described in Chapter Two (Figure 4.1B).

92

Behavioral Testing
Partner Preference: Tests for partner preference were conducted as described in Chapter

Two.

Social and sexual behavior: Tests for social and sexual behavior were conducted as

described in Chapter Three.

Testing Schedule: Testing schedule was the same as described in Chapter Three (Figure

4.2).

Histology
Pelvic Ganglia and Spinal Nucleus of the Bulbocavernosus: Tissue harvesting,
sectioning, staining and analysis were conducted as described in Chapter Three (Figure

4.1D). One male from each litter was used for analysis.

Hormone Analysis

Hormone Analysis was conducted as described in Chapter Three (Figure 4.1D).

Analysis

Maternal licking and grooming data were analyzed using a 3 x 4 (early postnatal
treatment x day) AN OVA with repeated measures on the second factor. The data for the
behavioral measures during the partner preference tests were analyzed using a 3 x 2

(early postnatal treatment x initial or final test) ANOVA with repeated measures on the

93

second factor. Preference scores and duration of time spent in the male chamber during
the partner preference tests were also analyzed within each early postnatal treatment
group using a one-sample t-test. This test was used to determine if the animals showed a
preference for one stimulus animal above chance (preference score = 0) or spent more
time with the male above chance (duration = 400 seconds). The data for the behavioral
measures during the social and sexual behavior tests, histological cell counts, and

hormones assays were analyzed using a one-way AN OVA by early postnatal treatment.

For some variables, the data did not meet homogeneity of variance assumptions, even
after the prescribed transformations (i.e. square root). For these measures, nonparametric
statistics were used for analysis. In these situations, data from the 5% and 10% males
were collapsed into a early postnatal estradiol treatment group (EB males) because no
significant differences were found between the two postnatal estradiol doses for any of
the variables analyzed using nonparametric statistical tests. The Mann-Whitney U and the

Fisher’s exact probability tests were used for these analyses.

RESULTS
Maternal Licking and Grooming
No significant differences in maternal licking and grooming were seen among treatment

groups (p=.292) or days (p=0.078), with no significant interaction (p=0.147; Figure 4.3).

Partner Preference
During the initial test the 5% males had a lower preference score than the other two

treatment groups (Figure 4.4B), and they did not show a preference for either the male or

94

female stimulus animal. Both the C and 10% males, though, showed a preference score
significantly higher than expected by chance, indicating a preference for the female
(Table 4.1). During the final test all groups showed a preference for the female (T able
4.1) with both the 5% and C males showing a significant increase in preference score as
indicated by an early postnatal treatment by week of test interaction [F(2, 31) = 3.9,
p=0.03], (Figure 4.4B). In addition, the duration of time spent in the stimulus male
chamber was significantly less than expected by chance for the 10% and C males in both

the initial and final tests, and for the 5% males during the final test (Table 4.1).

The changes seen in preference score were the result of alterations in the duration of time
spent with the stimulus male, which was also affected by an early postnatal treatment by
week of test interaction [F(2,31) = 11.2, p<0.001]. Both the 5% males and the C males
decreased the amount of time they spent in the stimulus male chamber from the initial to
final test, but the 10% males remained the same (2? :1: SEM: all measures are in seconds:
C males: 235.3 4.- 31.1 vs. 63.0 i 21.8; 5% males: 399.6 :1: 79.1 vs 155.5 :1: 32.4; 10% _
males: 189.9 :l: 55.7 vs 198.9 :1: 43.3). During the initial test, the 5% males spent more
time with the stimulus male than did the 10% males. During the final test, the 10% males
spent more time with the stimulus male than did the C males (Figure 4.4A). Duration of
time spent with the stimulus male was subjected to a square root transformation before
analysis.

The latency to enter the stimulus female chamber [F(1,31) = 6.0, p=0.02] and time spent
with the female [F(1,3 l) = 5.6, p=0.025] were both affected by a main effect of week of

test. Regardless of early postnatal hormone treatment, the males decreased their latency

95

to enter the female chamber from initial to final test (5(- i SEM: 59.0 i 74.0 vs. 23.1 i

14.1), and they also spent more time in the stimulus female chamber (X i SEM: 475.2 :I:

35.8 vs. 578.0 1 36.1)

Non-parametric statistics (Mann-Whitney U) were used to compare the male sexual
behaviors displayed during the partner preference tests by the EB males (combining the
5% and 10% groups as explained in Methods) and the C males. No significant differences
were seen in the display of male sexual behavior in the initial partner preference test. In
the final partner preference test mount, intromission, and ejaculation frequencies were
affected by early postnatal estradiol treatment. EB males showed decreased sexual
behavior with the stimulus female compared to the C males (Figure 4.5). Also, the
proportion of C males that showed mounts, intromissions, and ejaculations during the
final test was higher than that of the EB males (Table 4.2). No differences were seen in

behaviors shown with the stimulus male.

Social and Sexual Behavior

Early postnatal estradiol treatment altered the expression of male sexual behavior when
paired with a stimulus female. C males showed more intromissions and ejaculations
during the test than did EB males (Figure 4.6; Mann-Whitney U). Also, the latency to
show a mount or intromission were longer for the EB males that for the C males (Figure
4.7; Mann-Whitney U). Finally, the proportion of males that showed behaviors when

paired with a female was higher in the C male group than in the EB male group (Table

96

4.2). No significant differences were seen in behaviors shown when paired with a

stimulus male.

Histology

Early postnatal estradiol treatment altered the number of neurons seen in the PG of the
experimental males [F(2,18) = 5.1, p=0.02]. Males treated postnatally with 10% estradiol
had fewer cells in the PG than did males treated with cholesterol (Figure 4.8). Early
postnatal estradiol treatment did not affect the number of motoneurons present in the

SNB.

Hormone Assay

The amount of circulating testosterone was affected by the early postnatal estradiol
treatment (Kruskel-Wallis, p=0.001). The C males had significantly higher circulating
testosterone levels than did either the 10% or 5% males (Figure 4.9). Circulating levels of

estradiol were not affected by the early hormone treatment.

97

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98

Table 4.2. Proportion of experimental males that displayed sexual behavior with the
stimulus female during the final partner preference test and during the social and sexual
behavior test. C males were more likely to display mounts, intromissions, and
ejaculations than were EB males. *Significantly different from cholesterol, p<0.01.

**Significantly different from cholesterol, p<0.001

 

Final Partner Preference Test Social and Sexual Behavior Test

 

 

 

 

Behavior Mount Intromission Ejaculation Mount Intromission Ejaculation
Cholesterol 14/15 14/15 12/15 14/15 14/15 12/15
Estradiol 2/19** 2/19** 0/19** 8/17* 8/17* l/17*

 

 

 

 

 

 

 

99

 

 

I
A PNP O B PND l,2,4,6 C PNII) 21 D AfierPlND 90

 

Litter Reduction Maternal Licking Capsule IRemoval Partner Preference Histology Hormones

 

 

 

 

 

Silastic Capsules and Grooming Weaning Social and Sexual -PG -Testosterone
-Cholesterol Behavior Tests -SNB -Estradiol
-S% EB-C -With Male

-lO% EB-C -VV1th Female

 

Figure 4.1. Treatment and testing schedule for the experimental males. (A) On the day of
birth, PND O, litters were reduced to four males and four females and received Silastic
capsules of cholesterol (C, n=15) or estradiol benzoate [EB either 5% (n=10) or 10%
(n=9)]. (B) Maternal licking and grooming behavior was observed on PND 1, 2, 4, and 6.
(C) At weaning on PND 21, the Silastic capsules were removed. (D) After the males
reached 90 days of age, they were tested for partner preference and social and sexual
behavior with both male and female stimulus animals. After testing was concluded, the
pelvic ganglia (PG) and spinal nucleus of the bulbocavemosus (SNB) were removed.
Blood samples were also taken to analyze plasma testosterone and estradiol

concentrations.

100

 

Week I lnitial Partner Preference

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

l
Weeks 2&3 Sexual and Socral
Experience
Week 4 Sexual Behavior Sexual Behavior
With Female Mth Male
1 1
Week 5 Sexual Behavior Sexual Behavior
With Male With Female

Week 6 Final Panner Preference

 

 

 

Figure 4.2. Behavioral testing schedule for the experimental males. Data were not

collected during weeks 2 and 3 during which the animals received sexual/social

experience.

101

 

    
 

 
 
 

 

Early Postnatal
Treatment
1250- l10% PB
l5% EB
1000 -« I ChOlcsterol
3
Q .
E 750
l-
500 -
250 -
0 .

 

 

PND] PNDZ PND4 PND6
Postnatal Day

Figure 4.3. Time (in seconds) mothers of experimental males spent licking and grooming
during the first postnatal week. No significant differences were seen among treatment

groups or days, with no significant interaction.

102

 

 

A. Stimulus Male Chamber Test
Ilnitial
6001 IFinal

 

 

 

500- *#

Time (s)

     

B. Preference Score

 

 

 

 

10% 5% Cholesterol
Early Postnatal Treatment

Figure 4.4. (A) Duration of time experimental males spent in the stimulus male chamber
and (B) preference scores. Preference score is calculated as time spent with stimulus
female minus the time spent with stimulus male. The 5% and C males increased their
preference score and decreased the time spent with the male from initial to final test,
while the 10% males remained the same. *Significantly different from final test (within
treatment group). #Significantly different from 10% males (within test). ASignificantly

different from 10% and C males (within test).

103

 

 

Early Postnatal
Treatment

I Estradiol

I Cholesterol

 

 

Frequency

 

 

 

Mount Intromission Ejaculation
Behavior

Figure 4.5. Frequencies of mounts, intromissions, and ejaculations displayed by the
experimental males during the final partner preference test. EB males showed fewer

sexual behaviors compared to the C males. *Significantly different from EB males,

p<0.001.

104

50

 

 

Early Postnatal
Treatment

40_ I Estradiol

I Cholesterol

 

 

 

 

 

Mount Intromission Ejaculation
Behavior

Figure 4.6. Frequencies of mounts, intromissions, and ejaculations displayed by the
experimental males during the social and sexual experience test when paired with a
female. EB males showed fewer intromissions and ejaculations compared to the C males.

*Significantly different from EB males, p<0.001.

105

 

 

 

 

1250‘ Early Postnatal
Treatment
IEstradiol
1000. I Cholesterol
1; 750'
I;
E
E" 500-

 

 

   

Mount Intromission
Latency Latency
Behavior

Figure 4.7. Mount and intromission latencies shown by the experimental males during the
social and sexual experience test when paired with a female. EB males showed longer

latencies to display a behavior than did C males. *Significantly different from EB males,

p<0.05.

106

mm
lvvv

 

a
7500-

   
 
 

N umber of Cells
3 O\
.8 §

§

l500-

 

 

10% 5%
Early Postnatal Treatment

Cholesterol

Figure 4.8. Number of neurons present in the PG of experimental males. C males had

more PG neurons than did the 10% males. *Significantly different from 10% males,
p=0.02.

107

J}

 

34

Testosterone (rig/ml)
N

 

 

   

10% 5% Cholesterol
Early Postnatal Treatment

Figure 4.9. Levels of circulating testosterone in the experimental males. C males had

higher levels of plasma testosterone than did the 10% or 5% males. *Significantly

different from 10% and 5% males, p=0.001.

108

DISCUSSION
Early Postnatal Hormone Effects
Estradiol treatment during early development affected adult male partner preference in
different ways depending on the dose used. The low dose of estradiol seemed to alter
naive partner preference, while the high dose of estradiol seemed to remove the effect of

sexual experience on preference.

Control males showed a strong preference for the stimulus female in both partner
preference tests, and this preference increased significantly from the initial to the final
test. Males that received the high dose (10%) of EB also showed a preference for the
female in both tests, but this preference did not increase over testing. In contrast, males
receiving a low dose (5%) of EB during early development did not show an initial
preference for either the male or female stimulus animal, but during the final test they
displayed a preference for the stimulus female. The early postnatal treatments did not
alter the amount of maternal licking and grooming received by the litters; therefore, it is
unlikely that the early postnatal treatment group differences were mediated by changes in

maternal care.

One explanation for this lack of effect of experience seen in the 10% males could be due
to the fact that the 10% males did not copulate during the sexual behavior tests. However,
the 5% males did not show any male sexual behavior, either, and they continued to
display an increase in preference for the female in the final test. Another, and perhaps

more likely explanation, could be the health of the 10% males. Prior to the start of the

109

behavioral studies, six males treated with the high dose of estrogen became ill and died.
Upon examination of two of these males, it was found that the animals had severe kidney
and bladder infections. These infections were only found in the males treated with the
high dose of estradiol, and no other animals in the colony were found to be ill at that
time. The early postnatal estrogen exposure likely had damaging effects on the male’s
urinary and renal systems as has been shown in other studies (Lehtimaki et al., 1996;
Streng et al., 2001). Although the remaining males used in the study remained outwardly
healthy, and their behavior seemed normal, there is a chance that the behavior seen in

both the 5% and the 10% males was altered by subclinical conditions.

It seems that early postnatal estradiol treatment affected naive adult partner preference in
the 5% males, but these results do not correspond to those seen when males were treated
during early development with testosterone. Therefore, the effects on adult male partner
preference seen with early postnatal testosterone treatment most likely are due either to
androgen effects alone, or to a combination of androgen and estrogen effects, but not

estrogen effects alone.

Early postnatal estradiol also disrupted male sexual behavior in both the final partner
preference test and the social and sexual experience tests when paired with a stimulus
female. The outcomes of the present study are similar to those seen in Chapter Three, and
therefore the disrupting effects of testosterone on male sexual behavior could be
attributable to the action of the estrogen metabolite. This also agrees with results seen in

other studies. In addition to the studies that show that perinatal testosterone treatment to

110

intact rodents reduces male sexual behavior (Baum and Schretlen, 1975; Diamond et al.,
1973; Piacsek and Hostetter, 1984; Pollak and Sachs, 1975; Vomachka et al., 1981;
Zadina et al., 1979), other studies have found similar outcomes after perinatal treatment

with estrogen (Diamond et al., 1973; Harris and Levine, 1965; Whalen, 1964).

The PG is a sexually dimorphic, mixed sympathetic and parasympathetic ganglion that
innervates the reproductive organs, bladder, and colon of the male rat (Dail et al., 1975;
Keast, 1995; Keast et al., 1989; Keast and Degroat, 1989). Little is known, however,
about the development of this autonomic ganglion. In the present study, the high dose of
early postnatal estradiol decreased the number of cells present in the adult PG compared
to controls. The estradiol could be affecting the PG directly during development, either
preventing neurogenesis of the PG neurons or increasing the amount of cell death. The
neurons in the pelvic ganglia have been shown to express both ERa and ERB as well as
the aromatase enzyme (Purves-Tyson et al., 2007). However, estrogen does not seem to
play a role in the normal development of the PG. Studies show that males with the
testicular'feminizing mutation, a mutation of the androgen receptor, have fewer neurons
than do controls (Fang, Breedlove, and Clemens, unpublished results). It was also
reported in Chapter Two that early postnatal estrogen treatment did not increase PG cell

number in adult females.

Another explanation for the effects on the PG could be an indirect action of the estradiol
on PG cell number. Neonatal estrogen treatment has been shown to have significant

effects on the male urogenital system, specifically treatment decreases penis size (Goyal

lll

et al., 2004), testes weight (Atanassova et al., 1999), and prostate size (vomSaal et al.,
1997). The PG is known to innervate all of these reproductive organs in the male. If the
major target organs of the ganglion have been reduced in size, thereby decreasing the

level of innervation, it is likely there would be fewer neurons remaining in the adult PG.

Circulating testosterone levels were also affected by early postnatal hormone treatment in
the present study. C males had higher levels of serum testosterone than did the 5% and
10% males. Therefore, it seems likely that the change in adult plasma testosterone levels
seen in Chapter Three after postnatal testosterone treatment, is mostly likely an effect of
the estradiol metabolite. Numerous studies have linked neonatal estrogen treatment to
decreased adult testosterone production (Atanassova et al., 1999; Delbes et al., 2007;
Goyal et al., 2004; Goyal et al., 2003; Shibayama et al., 2001; Warita et al., 2006). As
previously mentioned, early estrogen is thought to have a direct inhibitory effect on the
enzymes that produce testosterone (Goyal et al., 2004; Goyal ct al., 2003; Majdic et al.,
1996; Tsaimorris et al., 1986), and it has been shown that early treatment with estrogens
decreases AR-ir and ERa-ir in the testis in adulthood (Shibayama et al., 2001; Warita et
al., 2006). The mechanism for how early steroids affects adult testosterone levels needs
further elucidation, but it appears that neonatal hormone treatment permanently alters the

sensitivity of the hypothalamic-pituitary-gonadal axis.

Experience Effects
Regardless of early postnatal hormone treatment, the experimental males decreased the

time to enter the female chamber and increased the amount of time spent with the female

112

from initial to final partner preference test. Similar to the discussion of experience effects
in Chapter Three, these changes can be explained by the sexual and social experience the
males received between the two partner preference tests. Sexual experience has been
shown to lead to increased preference for a female over a male stimulus animal
(Matuszczyk and Larsson, 1994), which would explain the decreased latency to enter the

female chamber, as well as the increase in time spent with the female.

Limitations

It should be mentioned that there were apparent limitations associated with the methods
of the present study, which make interpretation of the data difficult. First, the doses of
estrogen used were well above physiologically relevant levels and most likely higher than
the level of exogenous estrogen seen after aromatization in the previous study with TP.
However, the doses of estradiol used were based on developing a positive estrogen
control for evaluating estrogenic environmental agents, such as polychlorinated
biphenyls. Another issue that arose was the health status of the estradiol-treated males.
The high dose of estradiol most likely disrupted the development of the urogenital
system, which lead to some males becoming severely ill with kidney and bladder

infections.

Overall
Early estradiol exposure affected the initial preference of males treated with a low dose,
while reducing the effect of sexual experience on preference in males treated with a high

dose. However, these results may be confounded by the health status of the males. The

113

changes in adult male partner preference observed in the present study are not
comparable to the alteration seen after early postnatal testosterone treatment. Therefore,
based on the present study, estradiol alone does not seem to be responsible for the hyper-
androgenization effects on partner preference. However, the dosages of estradiol used in
the current study were certainly super-physiological and led to sever disruption of genital
physiology and anatomy. Quite possibly lower doses of estrogen that do not result in such
severe disruptions might have more subtle masculinizing effects that parallel those seen

with testosterone treatments.

114

CHAPTER FIVE:
NATURAL VARIATION IN MATERNAL LICKIN G AND GROOMING DOES
NOT ALTER ADULT PARTNER PREFERENCE EXPRESSION IN EITHER

MALE OR FEMALE RATS

INTRODUCTION
Rat dams have been shown to discriminate between male and female pups, showing more
anogenital licking and grooming towards male pups in a litter (Moore and Morelli, 1979).
Studies have shown that neonatal hormone treatments can alter the display of maternal
behavior. Testosterone, estradiol, or dihydrotestosterone injections to female rat pups on
the day of birth increased the amount of maternal licking and grooming the females
received (Moore, 1982). The level of maternal licking and grooming was equivalent for
treated female and male pups and greater than that of untreated females. Therefore, early

postnatal hormone manipulations can alter the behavior of the mother.

Alterations in maternal behavior in the rat have been shown to influence a number of
behavioral measures, endocrine responses, and neural development in offspring. Maternal
licking and grooming, specifically, can affect a range of offspring measures including
offspring maternal care (Champagne et al., 2001; Francis et al., 1999), sexual behavior
(Cameron et al., 2008; Moore, 1984), cognitive ability (Liu et al., 2000), hypothalamic-
pituitary-adrenal stress responses (Liu et al., 1997), motoneuron number (Moore et al.,
1992), and neural receptor number and methylation (Caldji et al., 1998; Champagne et

al., 2006; Liu et al., 1997; Weaver et al., 2004).

115

Natural variations in maternal care have been shown to affect steroid receptor number
and feedback responses in offspring. Offspring raised by high licking and grooming dams
show decreased corticosterone responses to restraint stress as adults compared to animals
raised by low licking and grooming dams (Liu et al.). The high licking and grooming
offspring also show decreased corticotropin—releasing hormone mRNA and increased
glucocorticoid receptor mRNA in the hypothalamus. These changes lead to enhanced

glucocorticoid feedback sensitivity and a decreased response to stress.

In our laboratory, recent data indicate that maternal licking and grooming do not play a
role in the expression of adult partner preference (Cummings et al., 2008). Cummings et
al (2008) found no correlation between amount of licking and grooming and preference
score. However, these data were analyzed within groups of animals treated with PCBs.
Although early postnatal estradiol treatment did not alter maternal licking and grooming
in the earlier studies, the fact that variations in maternal care have been shown to have
powerful effects on the offspring’s behaviOr and neuroendocrine responses made it
necessary to definitively rule out any possibility that results seen in the previous chapters
could be attributed to changes in maternal care. Therefore, maternal licking and grooming
was studied without other early postnatal treatments to determine if maternal care could
alter adult partner preference in the laboratory rat. In this study, naturally occurring
variations in licking and grooming displayed by rat dams were measured, and their

effects on adult partner preference and reproductive behavior were analyzed.

116

METHODS

Animals

Experimental Animals: Time-mated pregnant Long-Evans rats (Charles River, Raleigh,
NC) were housed individually with ad lib food and water in plastic cages (45.5 x 24 x 21
cm) in a 14:10—hr light dark cycle with lights on at 01:00. For nest building material
thirty, one-inch paper towel strips were given to the dams on GD 20. A subset of the
offspring of these dams became the experimental animals of this study (see below). On
the day of birth, PND O, the litter was reduced to four male and four female pups. For
litter reductions, the AGD for each pup was measured, and since the AGD is shorter in
females than in males, the four shortest and the four longest were retained. The pups were

Weaned on PND 21 and were then housed with same-sex littermates.

After animals reached 60 days of age, two females from each litter were ovariectomized
and implanted with a Silastic capsule containing estradiol benzoate (Sigma; EB) mixed
with cholesterol (Sigma; C) to achieve 25% EB-C mixtures. The capsules were implanted
subcutaneously (s.c.) while the animals were anesthetized with isoflurane; the incision
was closed with an Auto Clip and covered with First Aid Cream. After 4 weeks, the
capsules begin to lose efficacy because of connective tissue growth around the capsule
(personal observation). The capsules were removed and reimplanted via a new incision in
the neck. For all tests the females were injected s.c. with 0.5 mg progesterone (Sigma; P,

in sesame oil) four hours prior to data collection.

117

Two male offspring from each litter were tested after they reached 90 days of age. Males

were tested gonadally-intact.

Stimulus Animals: Sexually experienced, gonadally intact, adult Long Evans rats (Charles
River, Raleigh, NC) were used as stimulus animals for the behavioral tests (females at

least 60 days old; males at least 90 days old).

Stimulus females were implanted with a Silastic capsule containing 25% EB-C mixture
prior to testing but were not ovariectomized. It is the practice of this laboratory to not
ovariectomize stimulus females for testing procedures. The hormone treatments used
reliably induce sexual receptivity while avoiding the trauma of an unnecessary surgery.
Stimulus females were injected s.c. with 0.5 mg P four hours prior to partner preference

and sexual behavior testing.

Animals were maintained in a facility accredited by the Association for Assessment and
Accreditation of Laboratory Animal Care, and all experimental procedures were

approved by the Michigan State University Animal Care and Use Committee.

Maternal Licking and Grooming

Maternal behavior was videotaped during the last hour of the light phase and the first
hour of the dark phase of the light-dark cycle on PND 1, 2, 4, and 6. These recordings, as
well as those for all other behavioral tests, were analyzed using The Observer 5.0

(Noldus), a behavioral data acquisition computer program. The amount of time the dam

118

spent licking and grooming (LG) the litter was determined. Experimental offspring were
divided into 3 LG groups dependent on the level of LG received during development.
Dams that fell below one standard deviation from the mean are referred to as Low LG
mothers, whereas dams that were above one standard deviation from the mean are

referred to as High LG mothers. All other dams are called Mid LG mothers.

Behavioral Testing
Partner Preference: Tests for partner preference were conducted as described in Chapter

Two.

Female sexual behavior: When female offspring were tested for sexual behavior, a
barrier with four holes (4.5 x 4.5 cm) was placed in a Plexiglas observation chamber (46
x 58 x 51 cm), which divided it into a female escape chamber (46 x 22 x 51 cm) and a
male chamber (46 x 36 x 51 cm). The holes in the barrier were too small for the male to
get through but gave the female free access to both chambers. The tests lasted 30 minutes
or until the female exited and returned after an ejaculation, which ever occurred first.
Behavioral testing took place under dim red light illumination in the middle part of the
dark phase of the light-dark cycle. The test was videotaped, and frequency of male
mounts, intromissions and ejaculations were scored, as was the latency to show these
behaviors. The latency of the experimental female to approach the male, the amount of
time the female spent in male chamber, and sexual receptivity were also recorded.
Receptivity was measured using lordosis quotient (LQ), which was calculated for all

females who received at least seven mounts. For the first 10 mounts (including

119

intromissions or ejaculations) female responses were scored as a 0 (no lordosis) or 1
(lordosis), from which a percentage of number of lordoses per test was calculated.
Finally, the frequency of exits and latency to return to the male after mounts,

intromissions, and ejaculations were calculated.

Male sexual behavior: Male offspring were tested for male sexual behavior in a Plexiglas
observation chamber (46 x 58 x 51 cm). During the test, the experimental male had
unrestricted access to the female stimulus animal. The tests lasted 30 minutes or until the
first intromission after an ejaculation, whichever occurred first. Behavioral testing took
place under dim red light illumination in the middle part of the dark phase of the light-
dark cycle. Video recordings of these tests were analyzed to determine frequency of
mounts, intromissions, and ejaculations shown by the experimental males and the latency

to show these behaviors.

Testing Schedule: Experimental animals were tested once a week for 5 weeks. The initial
partner preference of each animals was tested in week 1. Then each experimental animal
received sexual experience (female offspring with intact, adult stimulus males; male
offspring with hormonally primed stimulus females) during weeks 2 and 3, but data were
not collected. During experience weeks, experimental animals were partnered with
stimulus animals for 30 minutes during which time sexual behavior could occur. During
week 4, the experimental animals were tested for sexual behavior (female offspring with

intact, adult stimulus males; male offspring with hormonally primed stimulus females),

120

and the test was recorded and scored. In week 5, the animal’s final partner preference was

evaluated.

Analysis

The data for the behavioral measures during the partner preference tests were analyzed
using a 3 x 2 (LG treatment group x initial or final test) AN OVA with repeated measures
on the second factor. Preference scores and duration of time spent in the male chamber
(for experimental female offspring) or female chamber (for experimental male offspring)
during the partner preference tests were also analyzed within each LG treatment group
using a one-sample t-test. This test was used to determine if the animals showed a
preference for one stimulus animal above chance (preference score = 0) ‘or spent more
time with the stimulus animal above chance (duration = 400 seconds). The data for the
behavioral measures during the sexual behavior tests were analyzed using a one way

AN OVA by LG treatment group.

RESULTS
Female Offspring
Partner Preference: The time spent in the stimulus male chamber was affected by a week
of test by LG treatment interaction (Figure 5.1; [F(2,70) = 3.5, p<0.036]). The Low LG
females increased the amount of time spent with the stimulus male from initial to final
test, but there was no change in duration for the Mid or High LG females. Also, in the

final partner preference test, the Low LG females spent more time in the male chamber

121

than did the Mid LG females. The data for time spent in the male chamber were subjected

to a square root transformation.

The sexual experience received between the partner preference tests affected the latency
to enter the female chamber [F(1,70)=14.6, p<0.001], latency to enter the male chamber
[F(1,70)=21.5, p<0.001], and the display by the stimulus male of mounts [F(1,70)=7.2,
p=0.009], intromissions [F(1,70)=32.2, p<0.001], and ejaculations [F(1,70)=55.9,
p<0.001]. Regardless of LG treatment, the female offspring decreased from initial to final

test the time it took to enter both the female stimulus chamber (2? :1: SEM: all measures

are in seconds: 24.5 i 3.1 vs 12.7 j; 1.9) and the male stimulus chamber ()7 :1: SEM: 20.2

i 2.3 vs 8.8 i 1.2). The stimulus male also increased from initial to final test the number
of mounts (if : SEM: 1.5 1 0.2 vs 2.7 1 0.4), intromissions (if :1: SEM: 3.8 a: 0.4 vs 8.1 s:

0.9), and ejaculations (Y 1 SEM: 0.4 i 0.1 vs 1.3 :l: 0.1) shown with the experimental

female offspring.

In both the initial and final partner preference tests, all three treatment groups had a
negative preference score significantly different from chance, indicating a preference for
the male stimulus animal (Table 5.1). Also, the duration of time spent in the male
chamber was significantly greater than chance for the Mid LG and High LG females in
both the initial and final tests, and greater than chance for the Low LG females in the

final test (Table 5.1).

122

Female Sexual Behavior: Lordosis quotient was the only behavior measured in the
female sexual behavior tests that was altered by maternal LG (Figure 5.2; [F(2,58)=10.2,
p<0.001]). The Low LG females had a significantly lower LQ than did the Mid or High

LG females, which did not differ from each other.

Male Offspring

Partner Preference: No behaviors measured were affected by maternal LG in the male
partner preference tests. However, some measures, regardless of LG treatment, were
affected by the sexual experience received between the partner preference tests. All males
had a longer latency to enter the female chamber during the initial test than during the
final test (X’s: SEM: 29.3 i 4.2 vs 14.2 i 1.5; [F(l,56)=12.1, p=0.001]). They also
increased the time spent in the stimulus female chamber (2? :1: SEM: 556.3 :1: 20.1 vs
723.0 :I: 31.8; [F(l,56)=29.6, p<0.001]) and decreased the time spent in the stimulus male
chamber (2? 3 SEM: 331.0 1 19.5 vs 139.5 :1: 18.0; [F(l,56)=71.6, p<0.001]) from initial
to final partner preference test. The preference score shown by the experimental male
offspring increased from initial to final test (2? i SEM: 225.3 :1: 35.7 vs 583.5 :t 45.9;
[F(l,56)=55.9, p<0.001]). Finally, the male offspring increased the number of mounts (
2? s SEM: 2.4 s 0.5 vs 7.9 :1: 1.0; [F(l,56)=37.9, p<0.001]), intromissions (r :1: SEM: 6.0
:t 1.2 vs 16.2 :1: 1.4; [F(l,56)=48.4, p<0.001]), and ejaculations (X :1: SEM: 0.2 :t 0.1 vs
1.3 :c 0.1; [F(l,56)=56.2, p<0.001]) shown with the stimulus female from initial to final

test.

123

In the initial partner preference test, both the Mid and High LG males had a positive
preference score significantly different from chance, indicating a preference for the
female stimulus animal. In the final partner preference test, all three groups had a positive
preference score significantly different from chance (Table 5.2). Also, the duration of
time spent in the female chamber was significantly greater than chance for all three

groups in both the initial and final tests (Table 5.2).

Male Sexual Behavior: Maternal LG did not affect any behaviors measured in the male

sexual behavior test.

124

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Test
I Initial
I Final
600 - * ‘
3
v 400 -
E
I“
200 -
0.
Low Mid High

LG Level
Figure 5.1. Time female offspring spent in the stimulus male chamber during the partner
preference tests. The Low LG females increased the amount of time spent with the male
from initial to final test. *Significantly different from Low LG initial test and Mid LG

final test.

127

 

 

 

190
80-
?; t
"'5' 60-
a
O
a 40'
3
20-
0.
Low Mid High
LG Level

Figure 5.2. Lordosis quotient of the female offspring during the female sexual behavior
test. Low LG females displayed a lower LQ than did Mid or High LG females.

*Significantly different from Mid and High LG.

128

DISCUSSION

Female Offspring

All females, regardless of level of maternal care they received, showed preference scores
significantly less than expected by chance, indicating a preference for the male in both
initial and final partner preference tests. Therefore, maternal behavior does not seem to
affect the development of partner preference in female offspring. This lack of effect is in
agreement with other work from our laboratory. Cummings et al (2008) did not find a
correlation between amount of LG and female partner preference scores when analyzed

within PCB treatment groups.

However, the level of maternal licking and grooming did affect one measure in the
female offspring partner preference test. Females that received low levels of LG
increased the amount of time spent in the stimulus male chamber from initial to final test,
and during the final test the Low LG females spent more time with the male than did the
Mid LG females. The Low LG females also did not spend more time than was expected
by chance with the stimulus male during the initial test. The Mid and High LG females
did not increase time spent with the male over testing, and spent more time than expected

by chance with the stimulus male in both preference tests.

It seems that the sexual behavior experience during the female sexual behavior tests
affected the amount of time the Low LG females spent with the stimulus male. It is
interesting to note that the Low LG females showed less receptivity with the stimulus

male during the female sexual behavior test (see below), yet the experience led to an

129

increase in the time spent with the male during the final partner preference test. It appears
that even though the LG females were less likely to display lordosis in response to the
stimulus males, the females still found the experience to be rewarding, leading to
increased time spent with the male. Increases in the amount of time spent with a male
after sexual experience has been seen in other studies (Clark et al., 2004; Matuszczyk and
Larsson, 1991), and in the present study the experience received seem to affect the

behavior of the Low LG females.

Maternal licking and grooming also altered female offspring receptivity to a sexually.
active male. Female offspring of Low LG mothers exhibited a lower lordosis quotient
during the female sexual behavior test than did offspring of either Mid or High LG
mothers, who did not differ from each other. In contrast to our findings, Cameron et al
(2008) reported that female offspring from High LG dams displayed lower lordosis
quotients than Low LG female offspring. However, there are significant methodological
differences between the two studies. Cameron et a1 (2008) measure maternal behavior for
five 75-minute daily observations on PND 1 through 6, whereas in the present study,
maternal behavior is observed for last hour of the light phase and the first hour of the
dark phase of the light-dark cycle on PND 1, 2, 4, and 6. Also, we observed female
sexual behavior after ovariectomy and hormone replacement. Cameron et al (2008) tests
for female sexual behavior in intact animals. These methodological differences make

comparisons between two studies difficult.

130

The experience received between the two partner preference tests also affected a number
of measures. Regardless of level of maternal LG received during development, all
females decreased their latency to enter the stimulus female and stimulus male chambers
from initial to final test. The females also received more mounts, intromissions, and
ejaculations from the stimulus male during the final test than during the initial. As
mentioned earlier, sexual experience has been shown in other studies to affect behaviors
seen in partner preference tests (Clark et al., 2004; Matuszczyk and Larsson, 1991). Also,
since the female offspring were sexually naive in the initial partner preference test, it is
apparent that sexual experience for the females was necessary for the display of full

copulatory behavior.

Male Offspring

N o behaviors measured in the male offspring partner preference tests were altered by the
level of maternal licking and grooming during development. Also, in both preference
tests, all three groups spent more time with the stimulus female than was expected by
chance. In the initial test, the Mid and High LG males showed preference scores
significantly higher than chance, indicating a preference for the female, and during the
final test all three groups showed preference for the female. Maternal LG, therefore, does

not appear to play a role in the development of adult partner preference in male offspring.

Maternal LG also did not affect any measures of male sexual behavior. This is in
agreement with other studies that show no effect of maternal LG on male sexual

behaviors (Cameron et al., 2008).

131

However, regardless of level of maternal LG received during development, a number of
measures in the partner preference test were altered by an effect of experience. The male
offspring decreased their latency to enter the female chamber, increased the amount of
time spent with the stimulus female, decreased the time spent with the stimulus male, and
thereby increased their preference scores from initial to final test. The male offspring also
showed an increase in sexual behavior from initial to final partner preference test. The
males displayed more mounts, intromissions, and ejaculations in the final test. As
mentioned in previous chapters, these changes can be explained by the sexual experience
the males received between the two partner preference tests. Sexual experience has been
shown to lead to increased preference for a female over a male stimulus animal
(Matuszczyk and Larsson, 1994). Similar to the females, the male offspring were
sexually nai've during the initial partner preference tests, and sexual experience must have

been necessary for the males to show copulatory behavior.

Overall

Maternal licking and grooming had little effect on the display of partner preference in
female and male offspring. Regardless of level of LG, female offspring continued to
show a preference for the stimulus male, and male offspring continued to show a
preference for the stimulus female. Maternal LG also did not alter any male sexual
behavior measure. However, females that received low levels of LG did show a reduced
lordosis quotient when paired with a stimulus male. Therefore, it appears that maternal
licking and grooming plays no role in the development of adult partner preference in the

laboratory rat.

132

CHAPTER SIX:

GENERAL DISCUSSION

In the studies reported in this dissertation, early postnatal hormone treatments altered
adult partner preference in the laboratory rat. In females, early postnatal exogenous
estradiol masculinized female-typical partner preference and sexual behavior. Females
treated early in development with estradiol preferred to spend more time with an estrous
stimulus female and less time with a stimulus male compared to cholesterol-treated
females. Compared to control females, females exposed to estradiol postnatally also
showed increased male-like sexual behavior when paired with a stimulus female. In
males, treatment with testosterone during the early postnatal period led to an altered
partner preference in which the testosterone-treated males spent more time with a
stimulus male than did cholesterol-treated males. Early postnatal testosterone treatment
also disrupted the display of normal male sexual behavior. The effect of early postnatal
estradiol treatment in males is difficult to analyze due to the impaired health status of the
animals. However, all postnatally estradiol—treated males did show disrupted male sexual
behavior. Finally, these postnatal hormone changes in adult partner preference were not
mediated by maternal behavior changes since altered maternal licking and grooming did
not affect sex-typical adult partner preference expression. These novel findings increase
the significance of the broadening literature on the role of perinatal hormones on the
development of partner preference. As reviewed in Chapter One, perinatal hormones play
a role in the development of adult partner preference in a number of mammalian species

(i.e. rat, ferret, mouse, pig, hamster, dog, human).

133

The development of human sexual orientation is obviously a more complicated process
than that seen in animal models. However, research on laboratory animals can still
provide insight and be applicable to the human. Understanding the endocrine components
and neural factors that play a role in sexual motivation and partner preference across
mammalian species, can also have implications for understanding the basis of human

orientation.

The masculinizing effects of early estradiol on the display of adult partner preference in
the female rat can be associated with the effect of prenatal DES on the increased
incidence of homosexuality and bisexuality in exposed women (Ehrhardt et al., 1985;
Meyer-Bahlburg et al., 1995). However, the organizational effects of estrogen on human
sexual orientation needs further evaluation since XY individuals who carry a
dysfunctional ERa or aromatase gene self report as heterosexual males (for review,
Baum, 2006). The effects of early postnatal testosterone on the expression of partner
preference in the male rat lends further support to the idea that hyper-androgenization
during early development can lead to a male-oriented sexual preference. Having older
brothers increases a male’s chance of being homosexual (Blanchard and Bogaert, 1996)
and also leads to digit length ratios that are more masculine than controls (Williams et al.,
2000). It appears that males with older brothers are exposed to increased levels of

androgen prenatally.

In addition to the notion that hyper-androgenization affects adult sexual orientation, there

are alternative explanations for the fraternal birth order effect that should be discussed.

134

For example, one explanation may be that a younger brother has an increased chance of
being homosexual due to the psychosocial environment in which he grows up. Perhaps
the influence of older brothers leads to a male-oriented preference in a younger brother,
either due to teasing, fighting, or some other measure. However, it has been shown that
the fraternal birth order effect applies only to brothers who share the same mother, i.e. not
brothers with the same father only and not step-brothers (Bogaert, 2006). In addition,
biological brothers with the same mother who are raised separately also show the
fraternal birth order effect on homosexuality (Bogaert, 2006). If an increased probability
of homosexuality in younger brothers was due to a social cause, it should be seen in boys
who have older step-brothers as well and not seen in younger brothers who are raised

apart from older brothers.

Parental age effects have also been suggested as an alternative explanation. Parents are
naturally older when younger sons are born compared to older sons, and homosexuality
may not be associated with birth order but with parental age. However, statistical
analyses indicate that the fraternal birth order effects still remain even after controlling

for both paternal and maternal age (Blanchard and Bogaert, 1996).

One further alternate explanation is the maternal immunization hypothesis. It has been
suggested that a mother carrying a son could have an immune reaction to a Y-linked
minor histocompatibility antigen, known as H-Y antigen (Blanchard, 2001; Blanchard
and Bogaert, 1996). Once the mother is exposed to the male-specific proteins, she

produces antibodies against the H-Y antigen. Subsequent births cause stronger immune

135

reactions, and later sons are exposed (through the placenta) to higher levels of the
maternal antibodies (Blanchard, 2001; Blanchard and Bogaert, 1996). These maternal
antibodies could then bind the fetal H-Y antigens, possibly preventing neural

development in a male-typical fashion (Blanchard, 2001).

The maternal immunization hypothesis would provide an explanation for why only older
brothers, and not older sisters, have an effect on the orientation of younger brothers.
Since female fetuses do not produce any proteins foreign in nature to the mother, no
maternal antibodies would be made. However, the H-Y antigen theory needs to be
examined further as it does not provide an explanation for why homosexual men are
hyper-masculine on some measures such as digit length ratio (Rahman, 2005; Rahman
and Wilson, 2003; Robinson and Manning, 2000), genitalia size (Bogaert and
Hershberger, 1999), and auditory evoked potentials (McFadden and Champlin, 2000).
Much still remains to be determined about the mechanisms underlying the fraternal birth

order effect.

Neural Control

The medial pre0ptic area (MPOA) of the hypothalamus is an essential brain area for the
expression of male sexual behaviors (Hull et al., 2002; Meisel and Sachs, 1994). Lesions
directed to the MPOA lead to deficits in male copulatory behavior in a number of species
(e.g. rat, mice, guinea pig, gerbil, monkey, dog, cat; for review, Hull et al., 2002).
Electrical stimulation of the MPOA, on the other hand, increases the frequency of

ejaculations in a 30 min test, as well as decreases latency to ejaculation, the

136

postejaculatory interval, and the number of intromissions prior to an ejaculation in male

rats (Rodriguez-Manzo et al., 2000).

The MPOA may also be critical for the display of adult partner preference behavior.
Studies in sheep have provided an interesting animal model in which to study neural
control of partner preference because a small percentage (7-9%) of rams are innately
male-oriented, and no hormone manipulations are needed for the males to display a same-
sex preference (Roselli et al., 2004). Male-oriented rams appear to have received levels of
testosterone perinatally equivalent to those of female-oriented rams. In ewes, an estradiol
injection will cause a surge of luteinizing hormone (LH), but in rams, regardless of
orientation, no estradiol-mediated LH surge is seen(Perkins et al., 1995). This implies
that defeminization has occurred in the male-oriented rams. Also, male-oriented rams do
not show any female-like sexual behaviors even after adult castration and treatment with

ovarian hormones (Stormshak et al., 2008).

Studies in sheep may implicate specific areas of the brain in the expression of sex-typical
partner preference, as there are some anatomical and functional difference in neural
circuitry between male- and female-oriented rams. The ovine sexually dimorphic nucleus
(oSDN) of the MPOA is two times larger in female-oriented rams than male—oriented
rams (Roselli et al., 2004). Also, in the oSDN aromatase activity (Resko et al., 1996) and
aromatase mRNA (Roselli et al., 2004) have been shown to be lower in male-oriented

rams than in female—oriented rams. The male-oriented rams, therefore, may experience

137

lower than normal levels of estrogenic exposure during developmental critical periods

compared with female-oriented rams.

In addition to the sheep, the MPOA also seems important for the display of sex-typical
adult partner preference in other animal models. In ferrets, electrolytic lesions of the
male’s MPOA led to female-like approach behavior to stimulus males, and approach
latencies decreased in a dose-dependent manner to increasing levels of adult estradiol
treatment (Cherry and Baum, 1990). Also, lesions in the male ferret MPOA caused males
to prefer to spend time with a stimulus male over a stimulus female after adult estradiol
treatment (Kindon et al., 1996; Paredes and Baum, 1995) and to prefer male odors over

estrous female odors (Baum, 2006).

Similar findings are seen when examining rat behavior. Male rats castrated in adulthood,
treated with either testosterone or estradiol, and given bilateral MPOA lesions prefer to
approach a stimulus male over an estrous female (Paredes et al., 1998). Other studies of
males with MPOA lesions also show a decrease in the preference for a female (Edwards
and Einhom, 1986; Edwards et al., 1996; Paredes et al., 1993). Sham-operated control
males continue to show a male-typical preference for the female. In addition, males
administered lidocaine into the MPOA show a decreased preference for the female
(Hurtazo et al., 2008). Also, in male rats treated neonatally with ATD, SDN-POA volume
correlates positively with male-typical copulatory behavior and partner preference for a
female (Houtsmuller et al., 1994). Further indication that the MPOA is important for the

expression of partner preference can be found in the maternal behavior literature. Prenatal

138

stress leads to a smaller SDN volume in male offspring (Kerchner et al., 1995), and
prenatal stress has also been shown to decrease preference for a stimulus female

compared to that shown by unstressed controls (Wang et al., 2006).

The hypothalamus also may be involved in human sexual orientation, but the data are
slightly more complicated than those found in the animal model literature. The interstitial
nuclei of the anterior hypothalamus region 3 (INAH3) has been found to be sexually
dimorphic with men having a larger volume than women (Allen et al., 1989; Levay,
1991). It was also discovered that homosexual men have a smaller INAH3 than do
heterosexual men (Levay, 1991). Another study (Byne et al., 2001) tried to replicate these
findings, however, and only found a non-significant trend for homosexual men to have a
smaller INAH3 than heterosexual men. One criticism of the Levay (1991) study is that
nearly all of his homosexual subjects had HIV /AIDS, and therefore he could not rule out
the possibility that the disease had an effect on INAH3 size (Baum, 2006). However, in
the follow-up study by Byne (2001), it was determined that HIV/AIDS did not have an
effect on the size of the region in either homosexual or heterosexual subjects. The 1N AHl
region is also sexually dimorphic, but no anatomical differences were seen between
homosexual and heterosexual men in this region (Swaab and Fliers, 1985). However,
other studies were unable to replicate the finding that there exists a sexual dimorphism in

the INAHl (Allen et al., 1989; Levay, 1991).

139

Future Directions

As noted earlier, the MPOA is a critical brain region for the display of male sexual
behavior, and possibly for the expression of male-typical partner preference. However,
the majority of these studies are based on correlations between size of the nucleus or
effect of lesion on behavior. Further research is needed to provide a direct indication that
the MPOA is involved in preference, especially since some data are conflicting. Although
many studies show an alteration in interest in a stimulus female after MPOA lesion, this
is not always the case (Everitt and Stacey, 1987; Hughes et al., 1990). It should also be
noted, however, that MPOA lesions tend to decrease a male’s preference for a female but
not eliminate it completely (Dominguez and Hull, 2005; Everitt, 1990). One possible way
to further link the MPOA to partner preference would be to measure dopamine in the

l

MPOA.

Within the MPOA, the neurotransmitter dopamine (DA) may be critical for the
expression of male sexual behavior, particularly in the rat, and may also facilitate the
display of partner preference. Systemic administration of a DA receptor agonist increases
copulatory behavior in male rats, whereas administration of a DA antagonist has been
shown to decrease mount, intromission and ejaculation frequency (for review,
Dominguez and Hull, 2005). Also, after administration of a DA antagonist, male rats
placed in a bilevel chamber, which can be used to determine sexual motivation, are less
likely to display anticipatory behavior (e. g level changes) prior to the introduction of a

stimulus female (Pfaus and Phillips, 1991).

140

DA agonists administered directly into the MPOA have effects similar to systemic
administration, as do antagonists. Apomorphine, a DA agonist, administered in the
MPOA increases the frequency of ejaculations while decreasing the latency to an
ejaculation and the postejaculatory interval (Hull et al., 1986). DA antagonists
administered directly into the MPOA, however, impair male sexual behavior (Pehek et
al., 1988) and decrease a male’s preference for an estrous female in an X—maze goal box
paradigm (Moses et al., 1995; Warner et al., 1991). DA in the MPOA has also been
shown to increase in vivo when a male is allowed to see, hear, and smell a female (Hull et
al., 1995). This increase in central DA was not seen when the male was exposed to a male
or a running wheel (Hull et al., 1995), indicating DA release occurs in response to only a

female.

I suggest analyzing DA release in the MPOA of early estrogenized females to possibly
provide further support for the idea that the MPOA influences partner preference. As
noted above, a normal male will show an increase in extracellular DA concentration in
the Nfl’OA after being presented with a female (Hull et al., 1995). A normal female,
however, does not appear to show any precopulatory rise in DA in the MPOA
(Matuszewich et al., 2000). The early estrogenized female show a clear partner
preference for a stimulus female. It is likely that the early estrogenized females would
show an increase in DA release in the MPOA when exposed to a receptive female, but
not when exposed to a stimulus male, similar to a control male, indicating their brain and

their preference have been masculinized. If however, there is no change in DA release in

141

the MPOA in these female, that may imply that the MPOA is not a critical brain region

for partner preference.

Based on the assumption that DA concentration would increase in the MPOA of early
estrogenized females after presentation of a stimulus female, further tests could be
undertaken. The hyper-androgenized males did not show a clear preference for either
stimulus animal, so there are a number of possible outcomes for an MPOA DA test. 1)
The males could show DA release in response to both stimulus animals, indicating a
bisexual preference and provide an explanation for why no preference was seen in the
partner preference paradigm. 2) The males could show DA release in response to the
male stimulus animal, but for some reason preference was not clear in the partner
preference paradigm. 3) The males could show DA release in response to the female
stimulus animal, but for some reason preference was not clear in the partner preference
paradigm. 4) The males could show no DA release in response to both stimulus animals.
This outcome could indicate one of two things: a) The males are not interested in either
stimulus animal (asexual), or b) The males may have a feminized MPOA DA system, and
as mentioned above, normal females do not show a pre-copulatory rise in DA when

exposed to a male.

Non-dopamine studies would also be useful to further understand the role of the MPOA
in the display of partner preference. The studies in sheep and ATD-treated rats, imply that
the estrogenic organization of the MPOA during development leads to male-typical

partner preference behavior in adulthood. Male-oriented rams have lower aromatase

142

activity in the oSDN (Resko et al., 1996; Roselli et al., 2004), thereby receiving less

estrogen during early life.

Male—oriented rams also have a smaller oSDN than female-oriented rams (Roselli et al.,
2004). It is acceptable to assume that the estrogenized females would have a larger SDN
than control females given that other studies have shown that neonatal estrogen treatment
increases the size of the SDN in female rats (Tarttelin and Gorski, 1988), and neonatal
estrogen antagonist treatment decreases the size of the SDN in male rats (Dohler et al.,
1986). However, it is not known what super-physiological levels of testosterone may do
to the size of and aromatase activity level in the SDN of hyper-androgenized males. The
effect of testosterone on aromatase activity is not well understood because the data are
conflicting; testosterone has been shown to increase, decrease, or have no effect on
aromatase activity (for review, Negri-Cesi et al., 2008). Environmental contaminants
with steroid hormone~mimicking effects have been shown to alter aromatase in different
ways depending on the dose used (Andrade et al., 2006). Perhaps physiological male-
typical levels of testosterone lead to normal masculine aromatase activity, but super-
physiological levels of testosterone down-regulate activity in the MPOA. Similar to the
male-oriented rams that have lower aromatase activity in the MPOA than female-oriented
rams (Resko et al., 1996; Roselli et al., 2004), perhaps the male—oriented hyper-

androgenized males also have lower aromatase levels than control males.

It should be noted here that Tfm male rats, which do display a masculine partner

preference (Hamson, personal communication), also have a smaller neuronal soma size

143

(Morris et al., 2005) and lower aromatase activity (Roselli et al., 1987) in the SDN than
their wild-type brothers. However, these males have also been shown to have similar
estrogen binding in the hypothalamus to their wild-type brothers (Olsen and Whalen,
1982). Tfm males also show higher levels of circulating estrogen than their wild-type
brothers (Roselli et al., 1987), unlike the sheep (see Roselli et al., 2004) or the hyper-
androgenized rats in the previous chapters, which both show normal levels of circulating
estrogen. It is likely that the Tfm male rats show a male-typical partner preference
because they are exposed to sufficient levels of estrogen during development, whereas

male-oriented rams and hyper-androgenized male rats are exposed to lower levels.

It is likely that the MPOA is not the only brain region involved in regulating partner
preference. However, few other regions have been studied. Further investigation of
differences in the brain circuitry of male- and female-oriented rams may provide useful
information into other areas that control preference. In the rat, lesions in the dorsolateral
tegmentum (DLT) and central tegmental field (CTF) have no effect on the display of
male partner preference, indicating these regions can be excluded from possible sites of
control (Romero—Carbente et al., 2006; Romero-Carbente et al., 2007). The DLT and
CTF regions are reciprocally connected with the MPOA and are implicated in the motor
and sensory function of male sexual behavior, respectively (Hull et al., 2002). Lesions in
the central zona incerta, a region with connections with the DLT that has been shown to
be important for the display of male sexual behavior (Hull et al., 2002), also do not affect
the expression of male partner preference (Edwards and Isaacs, 1991). In addition,

dopamine antagonist administration into the nucleus accumbens, an area involved in the

144

reward pathway, do not affect male partner preference display (Moses et al., 1995). Many
other regions remain to be studied. For example, Bodo and Rissman (2008) suggest that
Tfm male mice may display a female-typical partner preference due to alterations in the
accessory olfactory system, which mediates pheromonal cues. It is also possible that one
brain region is not solely involved in the control of partner preference, and that a full

neural circuit is necessary for the expression of sex-typical preference.

In addition to studies investigating the neural control of partner preference, more
endocrine manipulation studies would also be useful. Studying the effects of a lower dose
of early postnatal estrogen on male partner preference could proved insight into the
effects of environmental contaminants on the sexual behavior of animals in the wild.
Also, attempting to replicate the early postnatal testosterone studies in other species
would lend more support to the partner preference altering effects of hyper-

androgenization seen here.

Overall

A great deal is still unknown about the development of partner preferences in mammalian
species; however, steroid hormone exposure during development is arguably an
important factor. More research is needed both in animals models and humans to tease
apart the neuroendocrine basis of preference. Humans, ultimately, stand separate from
other animal models as more than simply preference needs to be taken into account (e.g.
gender identity), but the importance of understanding the mechanisms of the development

of partner preference in other animal models cannot be understated.

145

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