References 5.b: Causes – Hormone exposure – Animal studies

Gene Expression and Sequence Analysis of the Neonatal Female Mouse Brain after Administration of Testosterone Propionate

Gender Dysphoria (GD), previously known as gender identity disorder, is characterized by dissociation between psychological gender identity and physical sex. This condition causes clinically significant distress or impairment in social, occupational, or other important areas of functioning. Clinical care decisions for peripubertal adolescents with GD should be made carefully. Furthermore, the identification of biomarkers is very important for rapid and accurate diagnosis of GD in young people.

Studies of the biological mechanisms in GD have been reported continually. However, in terms of steroidogenic enzymes and sex hormone receptors, there are no reports of genetic variants that are strongly associated with GD. It is inadequate to consider that only steroidogenic enzymes and sex hormone receptors drive determination of gender identity because these genes are partly responsible for disorders of sexual development.

Thus, we present a novel hypothesis that genes directly related to GD are target genes of sex hormones and/or downstream genes of sex hormone signaling pathways as well as steroidogenic enzymes or sex hormone receptors. However, the specific gene regulation and molecular mechanisms during sexual differentiation of the mammalian brain are still unclear. The aim of this study was to investigate gene expression profiles during masculinization of the neonatal female mouse brain by testosterone and to identify biomarkers related to GD.

To identify GD-related genes as biomarkers for diagnosis, microarray analysis was performed using RNAs extracted from the brains of neonatal mice treated by intraperitoneal injection of testosterone propionate during the sexual determination period. Sequence motif enrichment analysis for sex hormone receptor responsive elements was performed for the flanking regions of genes that showed significant expression changes following administration of testosterone propionate.

We identified 334 genes that showed differential expression in the masculinized neonatal female brain after testosterone propionate treatment. Interestingly, most of these genes are not reported to be expressed in a sexually dimorphic manner. Moreover, sequence motif enrichment analysis suggested that masculinization of the neonatal female brain by testosterone was controlled more by estrogen receptors than androgen receptors.

Differences in genes that are expressed differentially following administration of testosterone injection from known sexually dimorphic genes suggest that many GD-related genes are upregulated during female brain masculinization. The gene set identified in this study provides a basis to better understand the mechanisms of GD and delineate its associated biomarkers.

Author/-s:       Yutaka Nakachi; Mioko Iseki; Tomotaka Yokoo; Yosuke Mizuno; Yasushi Okazaki

Publication:     Endocrine Society's 97th Annual Meeting and Expo, 2015

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Gene Expression Profile of the Neonatal Female Mouse Brain After Administration of Testosterone Propionate

Introduction: Clinical care decisions for peripubertal adolescents with gender dysphoria (GD) should be made carefully. Furthermore, the identification of biomarkers is very important for rapid and accurate diagnosis of GD in young people.

Aim: The aim of this study was to investigate gene expression profiles during masculinization of the neonatal female mouse brain by testosterone and to identify biomarkers related to GD.

Methods: Microarray analysis was performed using RNAs extracted from the brains of neonatal mice treated by intraperitoneal injection of testosterone propionate during the sexual determination period. Sequence motif enrichment analysis for sex hormone receptor responsive elements was performed for the flanking regions of genes that showed significant expression changes following administration of testosterone propionate.

Main Outcome Measures: We revealed a gene set with marked changes in expression during brain masculinization of neonatal female mice following administration of testosterone propionate.

Results: We identified 334 genes that showed differential expression in the masculinized neonatal female brain after testosterone propionate treatment. Interestingly, most of these genes are not reported to be expressed in a sexually dimorphic manner. Moreover, sequence motif enrichment analysis suggested that masculinization of the neonatal female brain by testosterone was controlled more by estrogen receptors than androgen receptors.

Conclusions: Differences in genes that are expressed differentially following administration of testosterone injection from known sexually dimorphic genes suggest that many GD-related genes are upregulated during female brain masculinization. The gene set identified in this study provides a basis to better understand the mechanisms of GD and delineate its associated biomarkers.

Author/-s:       Yutaka Nakachi; Mioko Iseki; Tomotaka Yokoo; Yosuke Mizuno; Yasushi Okazaki

Publication:     The Journal of sexual medicine, 2015

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Perinatal Administration of Aromatase Inhibitors in Rodents as Animal Models of Human Male Homosexuality: Similarities and Differences

In this chapter we briefly review the evidence supporting the existence of biological influences on sexual orientation. We focus on basic research studies that have affected the estrogen synthesis during the critical periods of brain sexual differentiation in male rat offspring with the use of aromatase inhibitors, such as 1,4,6-androstatriene-3,17 (ATD) and letrozole. The results after prenatal and/or postnatal treatment with ATD reveal that these animals, when adults, show female sexual responses, such as lordosis or proceptive behaviors, but retain their ability to display male sexual activity with a receptive female. Interestingly, the preference and sexual behavior of these rats vary depending upon the circadian rhythm.

Recently, we have established that the treatment with low doses of letrozole during the second half of pregnancy produces male rat offspring, that when adults spend more time in the company of a sexually active male than with a receptive female in a preference test. In addition, they display female sexual behavior when forced to interact with a sexually experienced male and some typical male sexual behavior when faced with a sexually receptive female. Interestingly, these males displayed both sexual behavior patterns spontaneously, i.e., in absence of exogenous steroid hormone treatment. Most of these features correspond with those found in human male homosexuals; however, the “bisexual” behavior shown by the letrozole-treated rats may be related to a particular human population. All these data, taken together, permit to propose letrozole prenatal treatment as a suitable animal model to study human male homosexuality and reinforce the hypothesis that human sexual orientation is underlied by changes in the endocrine milieu during early development.

Author/-s:       Sandra Olvera-Hernández; Alonso Fernández-Guasti

Publication:     Advances in Neurobiology: Perinatal Programming of Neurodevelopment, 2014

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Sexual Differentiation of the Brain Requires Perinatal Kisspeptin-GnRH Neuron Signaling

Sex differences in brain function underlie robust differences between males and females in both normal and disease states. Although alternative mechanisms exist, sexual differentiation of the male mammalian brain is initiated predominantly by testosterone secreted by the testes during the perinatal period. Despite considerable advances in understanding how testosterone and its metabolite estradiol sexually differentiate the brain, little is known about the mechanism that generates the male-specific perinatal testosterone surge. In mice, we show that a male-specific activation of GnRH neurons occurs 0–2 h following birth and that this correlates with the male-specific surge of testosterone occurring up to 5 h after birth. The necessity of GnRH signaling for the sexually differentiating effects of the perinatal testosterone surge was demonstrated by the persistence of female-like brain characteristics in adult male, GnRH receptor knock-out mice. Kisspeptin neurons have recently been identified to be potent, direct activators of GnRH neurons. We demonstrate that a population of kisspeptin neurons appears in the preoptic area of only the male between E19 and P1. The importance of kisspeptin inputs to GnRH neurons for the process of sexual differentiation was demonstrated by the lack of a normal neonatal testosterone surge, and disordered brain sexual differentiation of male mice in which the kisspeptin receptor was deleted selectively from GnRH neurons. These observations demonstrate the necessity of perinatal GnRH signaling for driving brain sexual differentiation and indicate that kisspeptin inputs to GnRH neurons are essential for this process to occur.

Author/-s:       Jenny Clarkson; Ellen R. Busby; Milen Kirilov; Günther Schütz; Nancy M. Sherwood; Allan E. Herbison

Publication:     The Journal of Neuroscience, 2014

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The mechanisms underlying sexual differentiation of behavior and physiology in mammals and birds: relative contributions of sex steroids and sex chromosomes.

From a classical viewpoint, sex-specific behavior and physiological functions as well as the brain structures of mammals such as rats and mice, have been thought to be influenced by perinatal sex steroids secreted by the gonads. Sex steroids have also been thought to affect the differentiation of the sex-typical behavior of a few members of the avian order Galliformes, including the Japanese quail and chickens, during their development in ovo. However, recent mammalian studies that focused on the artificial shuffling or knockout of the sex-determining gene, Sry, have revealed that sex chromosomal effects may be associated with particular types of sex-linked differences such as aggression levels, social interaction, and autoimmune diseases, independently of sex steroid-mediated effects. In addition, studies on naturally occurring, rare phenomena such as gynandromorphic birds and experimentally constructed chimeras in which the composition of sex chromosomes in the brain differs from that in the other parts of the body, indicated that sex chromosomes play certain direct roles in the sex-specific differentiation of the gonads and the brain. In this article, we review the relative contributions of sex steroids and sex chromosomes in the determination of brain functions related to sexual behavior and reproductive physiology in mammals and birds.

Author/-s:       F. Maekawa; S. Tsukahara; T. Kawashima; K. Nohara; H. Ohki-Hamazaki

Publication:     Frontiers in neuroscience, 2014

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Testosterone exposure during the critical period decreases corticotropin-releasing hormone-immunoreactive neurons in the bed nucleus of the stria terminalis of female rats

We previously described sex differences in the number of corticotropin-releasing hormone-immunoreactive (CRH-ir) neurons in the dorsolateral division of the bed nucleus of the stria terminalis (BSTLD). Female rats were found to have more CRH neurons than male rats. We hypothesized that testosterone exposure during the critical period of sexual differentiation of the brain decreased the number of CRH-ir neurons in the hypothalamus, including the BSTLD and preoptic area. In the present study we confirm that testosterone exposure during the neonatal period results in changes to a variety of typical aspects of the female reproductive system, including estrous cyclicity as shown by virginal smear, the positive feedback effects of estrogen alone or combined with progesterone, luteinizing hormone secretions, and estrogen and progesterone-induced Fos expression in gonadotropin-releasing hormone neurons. The number of CRH-ir neurons in the preoptic area did not change, whereas CRH-ir neurons in the BSTLD significantly decreased in estrogen-primed ovariectomized rats exposed to testosterone during the neonatal period. These results suggest that the sexual differentiation of CRH neurons in the BSTLD is a result of testosterone exposure during the critical period and the BSTLD is more fragile than the preoptic area during sexual differentiation. Furthermore, sex differences in CRH in the preoptic area may not be caused by testosterone during this period.

Author/-s:       Atsushi Fukushima; Miyako Furuta; Fukuko Kimura; Tatsuo Akema; Toshiya Funabashi

Publication:     Neuroscience Letters, 2013

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Effects of prenatal androgens on rhesus monkeys: a model system to explore the organizational hypothesis in primates

After proposing the organizational hypothesis from research in prenatally androgenized guinea pigs (Phoenix, C.H., Goy, R.W., Gerall, A.A., Young, W.C., 1959. Organizational action of prenatally administered testosterone propionate on the tissues mediating mating behavior in the female guinea pig. Endocrinology 65, 369-382.), the same authors almost immediately extended the hypothesis to a nonhuman primate model, the rhesus monkey. Studies over the last 50 years have verified that prenatal androgens have permanent effects in rhesus monkeys on the neural circuits that underlie sexually dimorphic behaviors. These behaviors include both sexual and social behaviors, all of which are also influenced by social experience. Many juvenile behaviors such as play, mounting, and vocal behaviors are masculinized and/or defeminized, and aspects of adult sexual behavior are both masculinized (e.g. approaches, sex contacts, and mounts) and defeminized (e.g. sexual solicits). Different behavioral endpoints have different periods of maximal susceptibility to the organizing actions of prenatal androgens. Aromatization is not important, as both testosterone and dihydrotestosterone are equally effective in rhesus monkeys. Although the full story of the effects of prenatal androgens on sexual and social behaviors in the rhesus monkey has not yet completely unfolded, much progress has been made. Amazingly, a large number of the inferences drawn from the original 1959 study have proved applicable to this nonhuman primate model.

Author/-s:       J. Thornton; J. L. Zehr; M. D. Loose

Publication:     Hormones and Behavior, 2009

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Juvenile Rank Can Predict Male-Typical Adult Mating Behavior in Female Sheep Treated Prenatally with Testosterone

Previous research with female sheep indicates that exposure to excess testosterone for 60 days (from Gestational Days 30–90 of the 147-day gestation) leads to virilized genitalia, severe neuroendocrine deficits, as well as masculinization and defeminization of sexual behavior (T60 females). In contrast, 30 days of testosterone exposure (Gestational Days 60–90) produce animals with female-typical genitalia, less severe neuroendocrine alterations, and variable gender patterns of sexual behavior (T30 females). Variation in adult sexual behavior of male ungulates is influenced by early social experience, but this has never been tested in females. Here we investigate the influence of rank in the dominance hierarchy on the expression of adult sexual behavior in females. Specifically, we hypothesized that juvenile rank would predict the amount of male- and female-typical mating behavior exhibited by adult female sheep. This hypothesis was tested in two treatment groups and their controls (group 1: T60 females; group 2: T30 females). Dominance hierarchies were determined by observing competition over resources. Both groups of prenatal testosterone-treated females were higher ranking than controls (T60: P = 0.05; T30: P < 0.01). During the breeding season, both T60 and T30 females exhibited more male-typical mating behavior than did controls; however, the T30 animals also exhibited female-typical behavior. For the T60 group, prenatal treatment, not juvenile rank, best predicted male-typical sex behavior (P = 0.007), while juvenile rank better predicted male mating behavior for the T30 group (P = 0.006). Rank did not predict female mating behavior in the hormone-treated or control ewes. We conclude that the effect of prenatal testosterone exposure on adult male-specific but not female-specific mating behavior is modulated by juvenile social experiences.

Author/-s:       Eila K. Roberts; Jonathan N. Flak; Wen Ye; Vasantha Padmanabhan; Theresa M. Lee

Publication:     Biology of reproduction, 2009

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Sexual Differentiation of Behaviour in Monkeys: Role of Prenatal Hormones

The theoretical debate over the relative contributions of nature and nurture to the sexual differentiation of behaviour has increasingly moved towards an interactionist explanation that requires both influences. In practice, however, nature and nurture have often been seen as separable, influencing human clinical sex assignment decisions, sometimes with disastrous consequences. Decisions about the sex assignment of children born with intersex conditions have been based almost exclusively on the appearance of the genitals and how other’s reactions to the gender role of the assigned sex affect individual gender socialisation. Effects of the social environment and gender expectations in human cultures are ubiquitous, overshadowing the potential underlying biological contributions in favour of the more observable social influences. Recent work in nonhuman primates showing behavioural sex differences paralleling human sex differences, including toy preferences, suggests that less easily observed biological factors also influence behavioural sexual differentiation in both monkeys and humans. We review research, including Robert W. Goy’s pioneering work with rhesus monkeys, which manipulated prenatal hormones at different gestation times and demonstrated that genital anatomy and specific behaviours are independently sexually differentiated. Such studies demonstrate that, for a variety of behaviours, including juvenile mounting and rough play, individuals can have the genitals of one sex but show the behaviour more typical of the other sex. We describe another case, infant distress vocalisations, where maternal responsiveness is best accounted for by the mother’s response to the genital appearance of her offspring. Taken together, these studies demonstrate that sexual differentiation arises from complex interactions where anatomical and behavioural biases, produced by hormonal and other biological processes, are shaped by social experience into the behavioural sex differences that distinguish males and females.

Author/-s:       K. Wallen; J. M. Hassett

Publication:     Journal of Neuroendocrinology, 2009

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Testosterone Programs Adult Social Behavior before and during, But Not after, Adolescence

Whereas the adolescent brain is a major target for gonadal hormones, our understanding of hormonal influences on adolescent neural and behavioral development remains limited. These experiments investigated how variations in the timing of testosterone (T) exposure, relative to adolescence, alters the strength of steroid-sensitive neural circuits underlying social behavior in male Syrian hamsters. Experiment 1 simulated early, on-time, and late pubertal development by gonadectomizing males on postnatal d 10 and treating with SILASTIC brand T implants for 19 d before, during, or after adolescence. T treatment before or during, but not after, adolescence facilitated mating behavior in adulthood. In addition, preadolescent T treatments most effectively increased mating behavior overall, indicating that the timing of exposure to pubertal hormones contributes to individual differences in adult behavior. Experiment 2 examined the effects of preadolescent T treatment on behavior and brain regional volumes within the mating neural circuit of juvenile males (i.e. still preadolescent). Although preadolescent T treatment did not induce reproductive behavior in juvenile males, it did increase volumes of the bed nucleus of the stria terminalis, sexually dimorphic nucleus, posterodorsal medial amygdala, and posteroventral medial amygdala to adult-typical size. In contrast, juvenile anterodorsal medial amygdala and ventromedial hypothalamus volumes were not changed by preadolescent T treatment yet differed significantly in volume from adult controls, suggesting that further maturation of these brain regions during adolescence is required for the expression of male reproductive behavior. Thus, adolescent maturation of social behavior may involve both steroid-independent and -dependent processes, and adolescence marks the end of a postnatal period of sensitivity to steroid-dependent organization of the brain.

The brain is a key target of gonadal hormones such as estradiol and testosterone (T), and numerous animal studies document the powerful effects of these hormones on a variety of behaviors ranging from sexual interest to cognitive abilities. We have known for almost 50 years that exposure to gonadal hormones during early neural development permanently alters sex-specific behavioral responses to gonadal hormones in adulthood, a seminal finding that spawned the idea that hormones organize behavioral circuits during sensitive periods of development. Despite this understanding, only a handful of studies have investigated the contribution of pubertal hormones to adolescent brain development and whether hormone action in the brain during this postnatal developmental period results in enduring changes in adult behavior.

The scarcity of animal research investigating pubertal hormone influences on adolescent brain development is particularly notable considering that deviations in pubertal timing are associated with adolescent-emerging psychopathologies such as depression, anxiety, disordered eating, and conduct disorder. The effects of variation in pubertal timing on psychopathology have largely been attributed to psychosocial factors that come into play with the intense changes in experience that accompany sexual maturation. However, gonadal hormones secreted at pubertal onset also have wide-ranging effects on the brain. Thus, mistimed, direct hormonal influences on the brain may also skew the course of adolescent brain development toward increased risk for psychopathology. If so, then the timely diagnosis and treatment of disorders of pubertal timing have repercussions that extend beyond normalization of reproductive function to more global influences on social behavior.

Animal models have immense potential for elucidating the mechanisms by which gonadal hormones directly impact adolescent neural and behavioral development. For example, depriving Syrian hamsters of hormones during adolescence compromises social behaviors, even after hormones are replaced in adulthood. This suggests that exposure of the adolescent brain to gonadal hormones organizes behavioral circuits and programs long-lasting behavioral responses. Furthermore, these data imply that a window of sensitivity to organizational effects of gonadal steroid hormones may close after adolescence, a possibility we tested herein.

The current studies sought to define the temporal parameters spanning the pre- and postadolescent periods within which neural circuits mediating social behavior are sensitive to the organizational effects of gonadal steroid hormones. Although previous work has established that perinatal exposure to gonadal steroid hormones masculinizes and defeminizes behavioral neural circuits, more recent work suggests that a second window of sensitivity may open at adolescence. For example, steroid hormones fail to elicit maximal expression of male reproductive behavior immediately before adolescence but readily activate high levels of reproductive behavior after adolescence, raising the possibility that adolescence is a second sensitive period for the organizational effects of gonadal steroid hormones on male reproductive behavior. Experiment 1 tested the hypothesis that brain sensitivity to hormonal influences fluctuates over the course of postnatal development. After gonadectomy (GDX) on postnatal d 10, we simulated early, on-time, or delayed onset of pubertal hormone secretion by administering SILASTIC brand T capsules (Dow Corning Corp., Midland, MI) before, during, or after adolescent development and examined the potential for sexual behavior in adulthood.

We also sought to extend the previous finding that 1 week of preadolescent T-treatment does not facilitate mating behavior in juveniles in Exp. 2 by 1) determining whether a longer duration of preadolescent T-treatment facilitates mating behavior in juveniles, 2) determining what behaviors are displayed by juveniles in the presence of estrous females, and 3) identifying which brain regions underlying social behaviors are capable of steroid-dependent organization before adolescence, and which regions undergo additional maturation during adolescence. Identifying the brain regions capable of steroid-dependent change before adolescence and those that require further maturation during adolescence is an essential first step in understanding the brain regions underlying the adolescent maturation of social behaviors.

Author/-s:       Kalynn M. Schulz; Julia L. Zehr; Kaliris Y. Salas-Ramirez; Cheryl L. Sisk

Publication:     Endocrinology, 2009

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Sex differences in rhesus monkey toy preferences parallel those of children

Sex differences in toy preferences in children are marked, with boys expressing stronger and more rigid toy preferences than girls, whose preferences are more flexible. Socialization processes, parents, or peers encouraging play with gender-specific toys are thought to be the primary force shaping sex differences in toy preference. A contrast in view is that toy preferences reflect biologically-determined preferences for specific activities facilitated by specific toys. Sex differences in juvenile activities, such as rough-and-tumble play, peer preferences, and infant interest, share similarities in humans and monkeys. Thus if activity preferences shape toy preferences, male and female monkeys may show toy preferences similar to those seen in boys and girls. We compared the interactions of 34 rhesus monkeys, living within a 135 monkey troop, with human wheeled toys and plush toys. Male monkeys, like boys, showed consistent and strong preferences for wheeled toys, while female monkeys, like girls, showed greater variability in preferences. Thus, the magnitude of preference for wheeled over plush toys differed significantly between males and females. The similarities to human findings demonstrate that such preferences can develop without explicit gendered socialization. We offer the hypothesis that toy preferences reflect hormonally influenced behavioral and cognitive biases which are sculpted by social processes into the sex differences seen in monkeys and humans.

Author/-s:       J. M. Hassett; E. R. Siebert; K. Wallen

Publication:     Hormones and Behavior, 2008

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The Role of Androgen Receptors in the Masculinization of Brain and Behavior: What we’ve learned from the Testicular Feminization Mutation

Many studies demonstrate that exposure to testicular steroids such as testosterone early in life masculinizes the developing brain, leading to permanent changes in behavior. Traditionally, masculinization of the rodent brain is believed to depend on estrogen receptors (ERs) and not androgen receptors (ARs). According to the aromatization hypothesis, circulating testosterone from the testes is converted locally in the brain by aromatase to estrogens, which then activate ERs to masculinize the brain. However, an emerging body of evidence indicates that the aromatization hypothesis cannot fully account for sex differences in brain morphology and behavior, and that androgens acting on ARs also play a role. The testicular feminization mutation (Tfm) in rodents, which produces a nonfunctional AR protein, provides an excellent model to probe the role of ARs in the development of brain and behavior. Tfm rodent models indicate that ARs are normally involved in the masculinization of many sexually dimorphic brain regions and a variety of behaviors, including sexual behaviors, stress response and cognitive processing. We review the role of ARs in the development of the brain and behavior, with an emphasis on what has been learned from Tfm rodents as well as from related mutations in humans causing complete androgen insensitivity.

Author/-s:       Damian G. Zuloaga; David A. Puts; Cynthia L. Jordan; S. Marc Breedlove

Publication:     Hormones and Behavior, 2008

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The control of sexual differentiation of the reproductive system and brain

This review summarizes current knowledge of the genetic and hormonal control of sexual differentiation of the reproductive system, brain and brain function. While the chromosomal regulation of sexual differentiation has been understood for over 60 years, the genes involved and their actions on the reproductive system and brain are still under investigation. In 1990, the predicted testicular determining factor was shown to be the SRY gene. However, this discovery has not been followed up by elucidation of the actions of SRY, which may either stimulate a cascade of downstream genes, or inhibit a suppressor gene. The number of other genes known to be involved in sexual differentiation is increasing and the way in which they may interact is discussed. The hormonal control of sexual differentiation is well-established in rodents, in which prenatal androgens masculinize the reproductive tract and perinatal oestradiol (derived from testosterone) masculinizes the brain. In humans, genetic mutations have revealed that it is probably prenatal testosterone that masculinizes both the reproductive system and the brain. Sexual differentiation of brain structures and the way in which steroids induce this differentiation, is an active research area. The multiplicity of steroid actions, which may be specific to individual cell types, demonstrates how a single hormonal regulator, e.g. oestradiol, can exert different and even opposite actions at different sites. This complexity is enhanced by the involvement of neurotransmitters as mediators of steroid hormone actions. In view of current environmental concerns, a brief summary of the effects of endocrine disruptors on sexual differentiation is presented.

Author/-s:       C. A. Wilson; D. C. Davies

Publication:     Reproduction, 2007

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Hormonal influences on sexually differentiated behavior in nonhuman primates

Sexually dimorphic behavior in nonhuman primates results from behavioral predispositions organized by prenatal androgens. The rhesus monkey has been the primary primate model for understanding the hormonal organization of sexually dimorphic behavior. Historically, female fetuses have received high prenatal androgen doses to investigate the masculinizing and defeminizing effects of androgens. Such treatments masculinized juvenile and adult copulatory behavior and defeminized female-typical sexual initiation to adult estrogen treatment. Testosterone and the nonaromatizable androgen, 5alpha-dihydrotestosterone, produced similar effects suggesting that estrogenic metabolites of androgens are not critical for masculinization and defeminization in rhesus monkeys. Long duration androgen treatments masculinized both behavior and genitalia suggesting that socializing responses to the females' male-like appearance may have produced the behavioral changes. Treatments limited to 35 days early or late in gestation differentially affected behavioral and genital masculinization demonstrating direct organizing actions of prenatal androgens. Recent studies exposed fetal females to smaller doses of androgens and interfered with endogenous androgens using the anti-androgen flutamide. Low dose androgen treatment only significantly masculinized infant vocalizations and produced no behavioral defeminization. Females receiving late gestation flutamide showed masculinized infant vocalizations and defeminized interest in infants. Both late androgen and flutamide treatment hypermasculinized some male juvenile behaviors. Early flutamide treatment blocked full male genital masculinization, but did not alter their juvenile or adult behavior. The role of neuroendocrine feedback mechanisms in the flutamide effects is discussed. Sexually differentiated behavior ultimately reflects both hormonally organized behavioral predispositions and the social experience that converts these predispositions into behavior.

Author/-s:       K. Wallen

Publication:     Frontiers in Neuroendocrinology, 2005

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Perinatal Exposure to Low Levels of the Environmental Antiandrogen Vinclozolin Alters Sex-Differentiated Social Play and Sexual Behaviors in the Rat

In this study we examined the effects of exposure to the antiandrogenic fungicide vinclozolin (Vz) on the development of two sex-differentiated behaviors that are organized by the perinatal actions of androgens. Pregnant Long-Evans rats were administered a daily oral dose of 0, 1.5, 3, 6, or 12 mg/kg Vz from the 14th day of gestation through postnatal day (PND)3. The social play behavior of juvenile offspring was examined on PND22 and again on PND34 during play sessions with a same-sex littermate. After they reached adulthood, the male offspring were examined with the ex copula penile reflex procedure to assess erectile function. Vz did not produce any gross maternal or neonatal toxicity, nor did it reduce the anogenital distance in male pups. We observed no effects of Vz on play behavior on PND22. However, the 12-mg/kg Vz dose significantly increased play behavior in the male offspring on PND34 compared with controls. The most dramatic increases were seen with the nape contact and pounce behavior components of play. The Vz effect was more pronounced in male than in female offspring. As adults, male offspring showed a significant reduction of erections at all dose levels during the ex copula penile reflex tests. The 12-mg/kg dose was also associated with an increase in seminal emissions. These effects demonstrate that perinatal Vz disrupts the development of androgen-mediated behavioral functions at exposure levels that do not produce obvious structural changes or weight reductions in androgen-sensitive reproductive organs.

Author/-s:       Nathan K. W. Colbert; Nicole C. Pelletier; Joyce M. Cote; John B. Concannon; Nicole A. Jurdak; Sara B. Minott; Vincent P. Markowski

Publication:     Environmental Health Perspectives, 2005

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Sex steroid-related genes and male-to-female transsexualism

Transsexualism is characterised by lifelong discomfort with the assigned sex and a strong identification with the opposite sex. The cause of transsexualism is unknown, but it has been suggested that an aberration in the early sexual differentiation of various brain structures may be involved. Animal experiments have revealed that the sexual differentiation of the brain is mainly due to an influence of testosterone, acting both via androgen receptors (ARs) and—after aromatase-catalyzed conversion to estradiol—via estrogen receptors (ERs). The present study examined the possible importance of three polymorphisms and their pairwise interactions for the development of male-to-female transsexualism: a CAG repeat sequence in the first exon of the AR gene, a tetra nucleotide repeat polymorphism in intron 4 of the aromatase gene, and a CA repeat polymorphism in intron 5 of the ERβ gene. Subjects were 29 Caucasian male-to-female transsexuals and 229 healthy male controls. Transsexuals differed from controls with respect to the mean length of the ERβ repeat polymorphism, but not with respect to the length of the other two studied polymorphisms. However, binary logistic regression analysis revealed significant partial effects for all three polymorphisms, as well as for the interaction between the AR and aromatase gene polymorphisms, on the risk of developing transsexualism. Given the small number of transsexuals in the study, the results should be interpreted with the utmost caution. Further study of the putative role of these and other sex steroid-related genes for the development of transsexualism may, however, be worthwhile.

Author/-s:       Susanne Henningsson; Lars Westberg; Staffan Nilsson; Bengt Lundström; Lisa Ekselius; Owe Bodlund; Eva Lindström; Monika Hellstrand; Roland Rosmond; Elias Eriksson; Mikael Landén

Publication:     Psychoneuroendocrinology, 2005

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Induction of PGE2 by estradiol mediates developmental masculinization of sex behaviour

Adult male sexual behavior in mammals requires the neuronal organizing effects of gonadal steroids during a sensitive perinatal period. During development, estradiol differentiates the rat preoptic area (POA), an essential brain region in the male copulatory circuit. Here we report that increases in prostaglandin-E2 (PGE2), resulting from changes in cyclooxygenase-2 (COX-2) regulation induced by perinatal exposure to estradiol, are necessary and sufficient to organize the crucial neural substrate that mediates male sexual behavior. Briefly preventing prostaglandin synthesis in newborn males with the COX inhibitor indomethacin permanently downregulates markers of dendritic spines in the POA and severely impairs male sexual behavior. Developmental exposure to the COX inhibitor aspirin results in mild impairment of sexual behavior. Conversely, administration of PGE2 to newborn females masculinizes the POA and leads to male sex behavior in adults, thereby highlighting the pathway of steroid-independent brain masculinization. Our findings show that PGE2 functions as a downstream effector of estradiol to permanently masculinize the brain.

Author/-s:       Stuart K. Amateau; Margaret M. McCarthy

Publication:     Nature Neuroscience, 2004

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A model system for study of sex chromosome effects on sexually dimorphic neural and behavioral traits

We tested the hypothesis that genes encoded on the sex chromosomes play a direct role in sexual differentiation of brain and behavior. We used mice in which the testis-determining gene (Sry) was moved from the Y chromosome to an autosome (by deletion of Sry from the Y and subsequent insertion of an Sry transgene onto an autosome), so that the determination of testis development occurred independently of the complement of X or Y chromosomes. We compared XX and XY mice with ovaries (females) and XX and XY mice with testes (males). These comparisons allowed us to assess the effect of sex chromosome complement (XX vs XY) independent of gonadal status (testes vs ovaries) on sexually dimorphic neural and behavioral phenotypes. The phenotypes included measures of male copulatory behavior, social exploration behavior, and sexually dimorphic neuroanatomical structures in the septum, hypothalamus, and lumbar spinal cord. Most of the sexually dimorphic phenotypes correlated with the presence of ovaries or testes and therefore reflect the hormonal output of the gonads. We found, however, that both male and female mice with XY sex chromosomes were more masculine than XX mice in the density of vasopressin-immunoreactive fibers in the lateral septum. Moreover, two male groups differing only in the form of their Sry gene showed differences in behavior. The results show that sex chromosome genes contribute directly to the development of a sex difference in the brain.

Author/-s:       Geert J. de Vries; Emilie F. Rissman; Richard B. Simerly; Liang-Yo Yang; Elka M. Scordalakes; Catherine J. Auger; Amanda Swain; Robin Lovell-Badge; Paul S. Burgoyne; Arthur P. Arnold

Publication:     The Journal of Neuroscience, 2002

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Androgen imprinting of the brain in animal models and humans with intersex disorders: review and recommendations.

Purpose: Psychosexual development, gender assignment and surgical treatment in patients with intersex are controversial issues in the medical literature. Some groups are of the opinion that gender identity and sexual orientation are determined prenatally secondary to the fetal hormonal environment causing irreversible development of the nervous system. We reviewed the evidence in animal and human studies to determine the possible role of early postnatal androgen production in gender development.

Materials and methods: An extensive literature review was performed of data from animal experiments and human studies.

Results: Many animal studies show that adding or removing hormonal stimulus in early postnatal life can profoundly alter gender behavior of the adult animal. Human case studies show that late intervention is unable to reverse gender orientation from male to female. Most studies have not permitted testing of whether early gender assignment and treatment as female with suppression/ablation of postnatal androgen production leads to improved concordance of the gender identity and sex of rearing.

Conclusions: Animal studies support a role for postnatal androgens in brain/behavior development with human studies neither completely supportive nor antagonistic. Therefore, gender assignment in infants with intersex should be made with the possibility in mind that postnatal testicular hormones at ages 1 to 6 months may affect gender identity. A case-control study is required to test the hypothesis that postnatal androgen exposure may convert ambisexual brain functions to committed male behavior patterns.

Author/-s:       Z. Hrabovszky; J. M. Hutson

Publication:     The Journal of urology, 2002

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Sex differences in response to children's toys in nonhuman primates (Cercopithecus aethiops sabaeus)

Sex differences in children's toy preferences are thought by many to arise from gender socialization. However, evidence from patients with endocrine disorders suggests that biological factors during early development (e.g., levels of androgens) are influential. In this study, we found that vervet monkeys (Cercopithecus aethiops sabaeus) show sex differences in toy preferences similar to those documented previously in children. The percent of contact time with toys typically preferred by boys (a car and a ball) was greater in male vervets (n=33) than in female vervets (n=30) (P<.05), whereas the percent of contact time with toys typically preferred by girls (a doll and a pot) was greater in female vervets than in male vervets (P<.01). In contrast, contact time with toys preferred equally by boys and girls (a picture book and a stuffed dog) was comparable in male and female vervets. The results suggest that sexually differentiated object preferences arose early in human evolution, prior to the emergence of a distinct hominid lineage. This implies that sexually dimorphic preferences for features (e.g., color, shape, movement) may have evolved from differential selection pressures based on the different behavioral roles of males and females, and that evolved object feature preferences may contribute to present day sexually dimorphic toy preferences in children.

Author/-s:       Gerianne M. Alexander; Melissa Hines

Publication:     Evolution & Human Behavior, 2002

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Wired for reproduction: Organization and Development of Sexually Dimorphic Circuits in the Mammalian Forebrain

Mammalian reproduction depends on the coordinated expression of behavior with precisely timed physiological events that are fundamentally different in males and females. An improved understanding of the neuroanatomical relationships between sexually dimorphic parts of the forebrain has contributed to a significant paradigm shift in how functional neural systems are approached experimentally. This review focuses on the organization of interconnected limbic-hypothalamic pathways that participate in the neural control of reproduction and summarizes what is known about the developmental neurobiology of these pathways. Sex steroid hormones such as estrogen and testosterone have much in common with neurotrophins and regulate cell death, neuronal migration, neurogenesis, and neurotransmitter plasticity. In addition, these hormones direct formation of sexually dimorphic circuits by influencing axonal guidance and synaptogenesis. The signaling events underlying the developmental activities of sex steroids involve interactions between nuclear hormone receptors and other transcriptional regulators, as well as interactions at multiple levels with neurotrophin and neurotransmitter signal transduction pathways.

Author/-s:       Richard B. Simerly

Publication:     Annual Review of Neuroscience, 2002

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Changes in sexual behavior of adult male and female rats neonatally treated with vitamin D3

1: Neonatal treatment of rats with vitamin D3 resulted in a change of sexual behavior in adulthood.

2: 2.5 mg vitamin D3 completely inhibited the ejaculation of males without any apparent influence on sexual desire. 250 mg vitamin D3 influenced both the desire and ejaculation.

3: Sexual activity of females was depressed by both doses.

4: The experiments demonstrate that vitamin D3, a steroid in structure, given in the critical period of hormonal imprinting may influence steroid hormone-receptor commanded events for life, in a way similar to the effects exhibited by synthetic steroid hormone analogues and benzpyrene in earlier studies.

Author/-s:       G. Csaba; O. Dobozy; Cs. Karabélyos; S. Mirzahosseini

Publication:     Human & Experimental Toxicology, 1996

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The pre- and postnatal influence of hormones and neurotransmitters on sexual differentiation of the mammalian hypothalamus

A number of brain structures and a great number of brain functions have been shown to be sexually dimorphic. It has also been shown that development and differentiation of these structures and functions proceeds during a critical pre- and postnatal period of increased susceptibility, and is controlled by gonadal steroids and neurotransmitter substances. The brain of male and female mammals seems to be still undifferentiated before the period of increased susceptibility to gonadal steroids and neurotransmitters starts. Feminization of brain structure and functions, e.g., establishment of the cyclic LH-surge mechanism and the expression of lordosis behavior, seems to depend on the moderate interaction of estrogens with the developing nervous system. Defeminization and masculinization of brain functions seem to be established during interaction of the developing nervous system with androgens, which have to be converted, at least in part, into estrogens. Structural differentiation of the male brain, e.g., the sexually dimorphic nucleus of the preoptic area (SDN-POA), seems to be exclusively estrogen-dependent, during differentiation of male brain functions, however, estrogens may be supportive, rather than directive, to the primary action of androgens. The molecular mechanisms of sexual differentiation of the brain are not yet fully understood. It seems, however, that the priming action of gonadal steroids during the period of increased susceptibility is either mediated by neurotransmitters, or neurotransmitters modulate the priming action of gonadal steroids. In particular, the adrenergic, the serotoninergic, the cholinergic, and possibly the dopaminergic system were shown to have strong influences on sexual differentiation of brain structure and functions. In contrast to the great number of available studies on the influence of gonadal steroids on sexual differentiation of the brain, there are rather few studies available concerning the influence of neurotransmitter systems. The available results are partly contradictory, so that an interpretation must be done with caution and will leave plenty of room for speculation. Postnatal application of compounds which stimulate or inhibit adrenergic activity mainly affected the neural control of gonadotropin secretion, and had only minor influences on differentiation of behavior patterns. It seems, however, that adrenergic participation in the differentiation of the center for cyclic gonadotropin release is very complex and stimulatory and inhibitory components may operate simultaneously. Activation or inhibition of beta-adrenergic receptors during postnatal development was shown to impair the responsiveness of the center for cyclic gonadotropin release to gonadal steroids, and impairs the expression of ejaculatory behavior in male rats.

Author/-s:       K. D. Döhler

Publication:     International review of cytology, 1991

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Behavioral masculinization is independent of genital masculinization in prenatally androgenized female rhesus macaques

Genetic female fetuses were exposed transplacentally to testosterone propionate injected into their mothers either early (Days 40 through 64) or late (Days 115 through 139) in gestation. Early and late androgenized females (EAFs and LAFs, respectively) were raised with normal males and females that served as criteria for evaluating degree of behavioral masculinization induced by the prenatal androgen. EAFs were genitally virilized and LAFs were not. Males and untreated females differed reliably on five behavioral measures: males showed more mother-mounting, more peer-mounting, more rough play with peers, a preference for initiating play with male partners, and less grooming of mothers. Neither type of prenatally androgenized female showed masculinization of all five types of behavior. Compared with females, EAFs showed more mother-mounting, more peer-mounting, less mother-grooming, did not differ from females in rough play, and did not manifest a preference for male partners. LAFs, like females, groomed but did not mount their mothers, and did not show a preference for male partners; but unlike females they showed elevated rough play and mounting with peers. EAFs showed a statistically significant delay in puberty onset (menarche), but LAFs did not. Mothers inspected genitalia of their offspring more often if they were males than if they were females. Mothers of EAFs inspected their offspring's genitalia as often as mothers of males, but mothers of LAFs did not. No aspect of maternal behavior was associated with either the amount or kind of masculine behavior shown toward peers. We interpret the results to mean that genital virilization is independent of, and largely irrelevant to, the expression of those behavioral traits that characterize the juvenile male social role. Moreover, the individual behavior traits that are components of the juvenile male role are independently regulated by the organizing actions of androgen and have separable critical periods. Of the two major traits, mounting peers and rough play with peers, the latter has a greater requirement for androgenic stimulation late in prenatal life.

Author/-s:       R. W. Goy; F. B. Bercovitch; M. C. McBrair

Publication:     Hormones and behaviour, 1988

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Social play soliciting by male and female juvenile rats: effects of neonatal androgenization and sex of cagemates

Male and female juvenile rats were individually exposed to nonplayful juvenile social stimuli in a novel test of play-soliciting behavior to examine hormonal and experiential determinants of sex differences. In Experiment 1, neonatally androgenized females engaged in play soliciting at a level equal to that of male controls and greater than that of nonandrogenized female controls. In Experiment 2, males and females were reared in unisexual and bisexual groups in order to compare long-term sex-related social experience effects on juvenile play soliciting. Males exposed only to other young males engaged in greater play soliciting than males exposed to both sexes; females, in contrast, were unaffected by sex of cagemates. Within rearing conditions, however, males engaged in greater play soliciting than females. The combined results suggest that perinatal gonadal androgen exposure effects on social play are prepotent and contribute essentially to sex differences in the initiation of social play behavior.

Author/-s:       W. R. Holloway Junior; D. H. Thor

Publication:     Behavioral neuroscience, 1986

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Testosterone implants into the amygdala during the neonatal period masculinize the social play of juvenile female rats

The masculinization of social play behavior in the rat is dependent upon the actions of androgens during the neonatal period. The amygdala, a major androgen-target region in the rat limbic brain, appears to be a critical site for this androgenic effect. We tested this hypothesis by implanting testosterone-bearing cannulae into the amygdala of female rat pups on Day 1 of life; the implants were removed on Day 8 of life. The animals were then observed daily between Days 26 and 40 of life and the frequency of play-fighting was recorded. Testosterone-implanted females, like normal males, engaged in significantly more play-fighting than did control females (implanted with cholesterol-bearing cannulae). We have also presented data indicating that the testosterone diffusion from the cannulae was, for the most part, restricted to the amygdala. Thus, testosterone implanted into the amygdala mimicked the effects previously reported for systemic testosterone injections, supporting the idea that the amygdala is a critical region for the actions of androgens on the sexual differentiation of social play behavior in the rat.

Author/-s:       B. S. McEwen; Michael J. Meaney

Publication:     Brain Research; 1986

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