Evidence for distinct biodevelopmental influences on male sexual orientation.

Several biological mechanisms have been proposed to influence male sexual orientation, but the extent to which these mechanisms cooccur is unclear. Putative markers of biological processes are often used to evaluate the biological basis of male sexual orientation, including fraternal birth order, handedness, and familiality of same-sex sexual orientation; these biomarkers are proxies for immunological, endocrine, and genetic mechanisms. Here, we used latent profile analysis (LPA) to assess whether these biomarkers cluster within the same individuals or are present in different subgroups of nonheterosexual men. LPA defined four profiles of men based on these biomarkers: 1) A subgroup who did not have these biomarkers, 2) fraternal birth order, 3) handedness, and 4) familiality. While the majority of both heterosexual and nonheterosexual men were grouped in the profile that did not have any biomarker, the three profiles associated with a biomarker were composed primarily of nonheterosexual men. We then evaluated whether these subgroups differed on measures of gender nonconformity and personality that reliably show male sexual orientation differences. The subgroup without biomarkers was the most gender-conforming whereas the fraternal birth order subgroup was the most female-typical and agreeable, compared with the other profiles. Together, these findings suggest there are multiple distinct biodevelopmental pathways influencing same-sex sexual orientation in men.

Overexpression of Androgen Receptors Masculinizes 2D:4D Digit Ratios in Mice.

Studying the role of the prenatal endocrine environment in humans is challenging due to the ethical and practical considerations of measuring hormone levels of the developing fetus. Because it has been difficult to ascertain whether prenatal androgens contribute to the brain and behavior in humans as it does in non-human species, retrospective markers of prenatal androgens, such as the second-to-fourth finger digit ratio (2D:4D), are of interest to the studying of human behavioral endocrinology. To assess the validity of such markers, laboratory animals have been studied. Some strains of mice have been reported to show a sex difference in 2D:4D, and pharmacological and genetic manipulation of the androgen and estrogen receptors (AR and ER) has implicated a role for prenatal androgens in mediating this sex difference, although there have been conflicting reports. Here, we compared mice with global AR overexpression to mice with wildtype (WT) littermates and mice with neural-specific AR overexpression. We found a sex difference in the right hind paw, such that males had larger digit ratios than females. Regardless of sex, mice with global AR overexpression showed an increase in the right hind 2D:4D ratio compared with both WT and neural-specific AR overexpression mice. These results support a role for non-neural AR in the development of 2D:4D and suggest that increased sensitivity to androgens via increased AR is sufficient to increase the masculinization of digit ratios. Future directions for confirming the validity of 2D:4D as a marker for prenatal androgen exposure are discussed.

Through a glass, darkly: Human digit ratios reflect prenatal androgens, imperfectly.

On average, the length of the index finger (digit 2) divided by the length of the ring finger (digit 4) on the right hand, is greater in women than in men. Converging evidence makes it clear that prenatal androgens affect the development of digit ratios in humans and so are likely responsible for this sex difference. Thus, differences in 2D:4D between groups within a sex may be due to average differences between those groups in prenatal androgen exposure. There have been many reports that lesbians, on average, have a smaller (more masculine) digit ratio than straight women, which has been confirmed by metaanalysis. These findings indicate that lesbians were, on average, exposed to greater prenatal androgen than straight women, which further indicates that greater levels of prenatal androgen predispose humans to be attracted to women in adulthood. Nevertheless, these results only apply to group differences between straight women and lesbians; digit ratios cannot be used to classify individual women as gay or straight.

RFamide-related peptide-3 (RFRP-3) suppresses sexual maturation in a eusocial mammal.

Neuroendocrine mechanisms underlying social inhibition of puberty are not well understood. Here, we use a model exhibiting the most profound case of pubertal suppression among mammals to explore a role for RFamide-related peptide-3 [RFRP-3; mammalian ortholog to gonadotropin-inhibitory hormone (GnIH)] in neuroendocrine control of reproductive development. Naked mole rats (NMRs) live in sizable colonies where breeding is monopolized by two to four dominant animals, and no other members exhibit signs of puberty throughout their lives unless they are removed from the colony. Because of its inhibitory action on the reproductive axis in other vertebrates, we investigated the role of RFRP-3 in social reproductive suppression in NMRs. We report that RFRP-3 immunofluorescence expression patterns and RFRP-3/GnRH cross-talk are largely conserved in the NMR brain, with the exception of the unique presence of RFRP-3 cell bodies in the arcuate nucleus (Arc). Immunofluorescence comparisons revealed that central expression of RFRP-3 is altered by reproductive status, with RFRP-3 immunoreactivity enhanced in the paraventricular nucleus, dorsomedial nucleus, and Arc of reproductively quiescent NMRs. We further observed that exogenous RFRP-3 suppresses gonadal steroidogenesis and mating behavior in NMRs given the opportunity to undergo puberty. Together, our findings establish a role for RFRP-3 in preserving reproductive immaturity, and challenge the view that stimulatory peptides are the ultimate gatekeepers of puberty.

Individual differences in the biological basis of androphilia in mice and men.

For nearly 60 years since the seminal paper from W.C Young and colleagues (Phoenix et al., 1959), the principles of sexual differentiation of the brain and behavior have maintained that female-typical sexual behaviors (e.g., lordosis) and sexual preferences (e.g., attraction to males) are the result of low androgen levels during development, whereas higher androgen levels promote male-typical sexual behaviors (e.g., mounting and thrusting) and preferences (e.g., attraction to females). However, recent reports suggest that the relationship between androgens and male-typical behaviors is not always linear - when androgen signaling is increased in male rodents, via exogenous androgen exposure or androgen receptor overexpression, males continue to exhibit male-typical sexual behaviors, but their sexual preferences are altered such that their interest in same-sex partners is increased. Analogous to this rodent literature, recent findings indicate that high level androgen exposure may contribute to the sexual orientation of a subset of gay men who prefer insertive anal sex and report more male-typical gender traits, whereas gay men who prefer receptive anal sex, and who on average report more gender nonconformity, present with biomarkers suggestive of low androgen exposure. Together, the evidence indicates that for both mice and men there is an inverted-U curvilinear relationship between androgens and sexual preferences, such that low and high androgen exposure increases androphilic sexual attraction, whereas relative mid-range androgen exposure leads to gynephilic attraction. Future directions for studying how individual differences in biological development mediate sexual behavior and sexual preferences in both mice and humans are discussed.

Gender Nonconformity and Birth Order in Relation to Anal Sex Role Among Gay Men.

Androphilia is associated with an elevated number of older brothers among natal males. This association, termed the fraternal birth order effect, has been observed among gay men who exhibit marked gender nonconformity. Gender nonconformity has been linked to gay men's preferred anal sex role. The present study investigated whether these two lines of research intersect by addressing whether the fraternal birth order effect was associated with both gender nonconformity and a receptive anal sex role (243 gay men, 91 heterosexual men). Consistent with previous research, we identified the fraternal birth order effect in our sample of gay men. Also, gay men were significantly more gender-nonconforming on adulthood and recalled childhood measures compared to heterosexual men. When gay men were compared based on anal sex role (i.e., top, versatile, bottom), all groups showed significantly greater recalled childhood and adult male gender nonconformity than heterosexual men, but bottoms were most nonconforming. Only gay men with a bottom anal sex role showed evidence of a fraternal birth order effect. A sororal birth order effect was found in our sample of gay men, driven by versatiles. No significant associations were found between fraternal birth order and gender nonconformity measures. These results suggest that the fraternal birth order effect may apply to a subset of gay men who have a bottom anal sex role preference and that this subgroup is more gender-nonconforming. However, there were no significant associations between fraternal birth order and gender nonconformity at the individual level. As such, based on the present study, whether processes underpinning the fraternal birth order effect influence gender nonconformity is equivocal.

Distinct Etiological Roles for Myocytes and Motor Neurons in a Mouse Model of Kennedy's Disease/Spinobulbar Muscular Atrophy.

Polyglutamine (polyQ) expansion of the androgen receptor (AR) causes Kennedy's disease/spinobulbar muscular atrophy (KD/SBMA) through poorly defined cellular mechanisms. Although KD/SBMA has been thought of as a motor neuron disease, recent evidence indicates a key role for skeletal muscle. To resolve which early aspects of the disease can be caused by neurogenic or myogenic mechanisms, we made use of the tet-On and Cre-loxP genetic systems to selectively and acutely express polyQ AR in either motor neurons (NeuroAR) or myocytes (MyoAR) of transgenic mice. After 4 weeks of transgene induction in adulthood, deficits in gross motor function were seen in NeuroAR mice, but not MyoAR mice. Conversely, reduced size of fast glycolytic fibers and alterations in expression of candidate genes were observed only in MyoAR mice. Both NeuroAR and MyoAR mice exhibited reduced oxidative capacity in skeletal muscles, as well as a shift in fast fibers from oxidative to glycolytic. Markers of oxidative stress were increased in the muscle of NeuroAR mice and were reduced in motor neurons of both NeuroAR and MyoAR mice. Despite secondary pathology in skeletal muscle and behavioral deficits, no pathological signs were observed in motor neurons of NeuroAR mice, possibly due to relatively low levels of polyQ AR expression. These results indicate that polyQ AR in motor neurons can produce secondary pathology in muscle. Results also support both neurogenic and myogenic contributions of polyQ AR to several acute aspects of pathology and provide further evidence for disordered cellular respiration in KD/SBMA skeletal muscle.

Contrasting effects of opposite- versus same-sex housing on hormones, behavior and neurogenesis in a eusocial mammal.

Competitive interactions can have striking and enduring effects on behavior, but the mechanisms underlying this experience-induced plasticity are unclear, particularly in females. Naked mole-rat (NMR) colonies are characterized by the strictest social and reproductive hierarchy among mammals, and represent an ideal system for studies of social competition. In large matriarchal colonies, breeding is monopolized by one female and 1-3 males, with other colony members being socially subordinate and reproductively suppressed. To date, competition for breeding status has been examined in-colony, with female, but not male, aggression observed following the death/removal of established queens. To determine whether this sex difference extends to colony-founding contexts, and clarify neural and endocrine mechanisms underlying behavioral change in females competing for status, we examined neurogenesis and steroid hormone concentrations in colony-housed subordinates, and NMRs given the opportunity to transition status via pair-housing. To this end, Ki-67 and doublecortin immunoreactivity were compared in the hippocampal dentate gyrus (DG) and basolateral amygdala (BLA) of colony-housed subordinates, and subordinates housed with a same-sex (SS) or opposite-sex (OS) conspecific. Results suggest that OS pairing in eusocial mammals promotes cooperation and enhances hippocampal plasticity, while SS pairing is stressful, resulting in enhanced HPA activation and muted hippocampal neurogenesis relative to OS pairs. Data further indicate that competition for status is confined to females, with female-female housing exerting contrasting effects on hippocampal and amygdalar neurogenesis. These findings advance understanding of social stress effects on neuroplasticity and behavior, and highlight the importance of including female-dominated species in research on aggression and intrasexual competition.

Cross-sex cecal microbiota transfer alters depressive-like behaviours in mice.

Major depressive disorder (MDD) is a leading cause of non-fatal global disease burden, with females being two-fold more likely than males to be diagnosed with the disorder. Despite this sex-linked disparity of diagnosis, it is unclear what underlies the sex bias in MDD. Recent findings suggest a role for the gut in mediating affective disorders through the gut-brain-axis (GBA). However, few studies have included sex as a biological variable. For this study, cross-sex transfer of cecal microbiota was performed between male and female C57Bl/6 mice to elucidate the effects of sex and the gut microbiome on a standard battery of tests measuring depressive-like behaviours. Specifically, regardless of sex, recipients of male cecal content had a greater sucrose preference than controls and recipients of female cecum, although recipients of male cecal transfer also showed a trend towards increased passive coping on the force swim test. Conversely, in the splash test, recipients of female cecum displayed a decrease in grooming behaviour compared to both controls and recipients of male cecum, suggestive of an increase in depressive-like behaviour. These results support a role for female-specific gut microbes in contributing to female vulnerability to depression, particularly on self-care measures, while male-specific gut microbes may protect in part against an anhedonia-like phenotype, but increase passive coping.

Antibiotic-induced socio-sexual behavioral deficits are reversed via cecal microbiota transplantation but not androgen treatment.

Recent evidence has demonstrated a sex-specific role of the gut microbiome on social behavior such as anxiety, possibly driven by a reciprocal relationship between the gut microbiome and gonadal hormones. For instance, gonadal hormones drive sex differences in gut microbiota composition, and certain gut bacteria can produce androgens from glucocorticoids. We thus asked whether the gut microbiome can influence androgen-dependent socio-sexual behaviors. We first treated C57BL/6 mice with broad-spectrum antibiotics (ABX) in drinking water to deplete the gut microbiota either transiently during early development (embryonic day 16-postnatal day [PND] 21) or in adulthood (PND 60-85). We hypothesized that if ABX interferes with androgens, then early ABX would interfere with critical periods for sexual differentiation of brain and thus lead to long-term decreases in males' socio-sexual behavior, while adult ABX would interfere with androgens' activational effects on behavior. We found that in males but not females, early and adult ABX treatment decreased territorial aggression, and adult ABX also decreased sexual odor preference. We then assessed whether testosterone and/or cecal microbiota transplantation (CMT) via oral gavage could prevent ABX-induced socio-sexual behavioral deficits in adult ABX-treated males. Mice were treated with same- or other-sex control cecum contents or with testosterone for two weeks. While testosterone was not effective in rescuing any behavior, we found that male CMT restored both olfactory preference and aggression in adult ABX male mice, while female CMT restored olfactory preference but not aggression. These results suggest sex-specific effects of the gut microbiome on socio-sexual behaviors, independent of androgens.

Evidence that ovarian hormones, but not diet and exercise, contribute to the sex disparity in post-traumatic stress disorder.

Females are twice as likely as males to receive a diagnosis of post-traumatic stress disorder (PTSD). One hypothesis for this sex disparity is that ovarian hormones, including estrogen and progesterone, contribute to PTSD risk. Alternatively, sex differences in lifestyle factors, such as diet and exercise, may play a role in PTSD risk. Using data from the Atlantic Partnership for Tomorrow's Health (PATH) cohort (n = 16,899), the relationship between endogenous hormone fluctuations (e.g., menarche, pregnancy, and menopause), exogenous hormone use (e.g., hormonal contraception and hormone replacement therapy (HRT)) and lifestyle variables (diet and exercise habits, as measured by the Mediterranean Diet Adherence Screener, Healthy Eating Index, and International Physical Activity Questionnaire) with PTSD diagnosis and treatment were analyzed. While several hormonal variables, including contraceptive use, higher total number of pregnancies, younger menarche age, and having undergone menopause increased the risk of PTSD, no lifestyle variables contributed to an increased risk of PTSD diagnosis. These findings support the theory that ovarian hormones contribute to the sex-linked disparity in PTSD diagnosis.

Evidence for Perinatal Steroid Influence on Human Sexual Orientation and Gendered Behavior.

In laboratory animals, exposure to gonadal steroid hormones before and immediately after birth can exert permanent effects on many behaviors, particularly reproductive behaviors. The extent to which such effects occur in humans remains an open question, but several lines of evidence indicate that perinatal levels of both androgens and estrogens may affect adult human psychology and behavior, including sexual orientation and gender nonconformity. Some putative indicators of prenatal androgen exposure, including the ratio of the length of the index finger to that of the ring finger (2D:4D), have repeatedly indicated that lesbians, on average, were exposed to more prenatal androgens than straight women, suggesting that sufficient fetal androgen exposure predisposes a fetus to gynephilia (attraction to women) at maturity. The digit ratios of gay men do not differ from those of straight men, suggesting that prenatal androgen levels are not responsible for their androphilia (attraction to men). However, evidence that gay men who prefer an insertive anal sex role (ASR) have more masculine digit ratios than those preferring a receptive ASR suggests that early androgens influence some sexual preferences in men. Furthermore, digit ratios among gay men have been found to correlate with recalled childhood gender nonconformity (CGN). People with isolated gonadotropin-releasing hormone (GnRH) deficiency (IGD) offer further insight into the effects of perinatal gonadal steroid exposure. In people with IGD, gonadal hormone production is low or absent after the first trimester of gestation. However, because placental gonadotropins drive gonadal hormone secretion during the first trimester when genitalia sexually differentiate, individuals with IGD are unambiguously male or female at birth, consistent with their chromosomal and gonadal sex. Men with IGD report greater CGN, again suggesting that perinatal androgen exposure contributes to male-typical behavioral patterns in humans. Interestingly, women with IGD report less androphilia and more bisexuality than control women, suggesting that perinatal ovarian steroids in females typically augment androphilia in adulthood. Taken together, these findings indicate that the perinatal hormonal milieu influences human sexual orientation and gender conformity.

Recalled Childhood Separation Anxiety Differs by Anal Sex Role among Gay Men.

Male androphilia (i.e., male sexual attraction to adult males) is considered an evolutionary paradox because it is partially influenced by genes and associated with decreased reproduction. Traits associated with attachment to genetic relatives (i.e., kin) could prompt increased kin-directed altruism, thereby offsetting decreased reproduction by helping kin reproduce. These traits include childhood separation anxiety and adulthood neuroticism, which have been associated with feminine gender expression. In prior research, gay men with a receptive (Bottom or Versatile) anal sex role (ASR) reported greater childhood gender nonconformity (GNC) than those with an insertive (Top) ASR. We examined whether ASR groups also differed on recalled childhood separation anxiety and adulthood neuroticism. The Separation Anxiety Scale-Revised and Big-Five Personality Inventory - short form were completed by 350 gay and 146 heterosexual men. For neuroticism, ASR preference groups differed from heterosexual men but not from one another. Gay men who preferred a Bottom or Versatile ASR reported higher recalled childhood separation anxiety than Tops and heterosexual men. Recalled childhood GNC mediated ASR group differences with heterosexual men on childhood separation anxiety. These results indicate that subgroups of gay men delineated by ASR differ on an evolutionarily relevant developmental trait, childhood separation anxiety.

Differences in digit ratios between gay men who prefer receptive versus insertive sex roles indicate a role for prenatal androgen.

Among non-human mammals, exposure to androgens during critical periods of development leads to gynephilia (attraction to females), whereas the absence or low levels of prenatal androgens leads to androphilia (attraction to males). However, in humans, retrospective markers of prenatal androgens have only been associated with gynephilia among women, but not with androphilia among men. Here, we asked whether an indirect indication of prenatal androgen exposure, 2D:4D, differs between subsets of gay men delineated by anal sex role (ASR). ASR was used as a proxy for subgroups because ASR groups tend to differ in other measures affected by brain sexual differentiation, such as gender conformity. First, we replicated the finding that gay men with a receptive ASR preference (bottoms) report greater gender nonconformity (GNC) compared to gay men with an insertive ASR preference (tops). We then found that Tops have a lower (male-typical) average right-hand digit ratio than Bottoms, and that among all gay men the right-hand 2D:4D correlated with GNC, indicating that a higher (female-typical) 2D:4D is associated with increased GNC. Differences were found between non-exclusive and exclusive same-sex attraction and GNC, and ASR group differences on digit ratios do not reach significance when all non-heterosexual men are included in the analyses, suggesting greater heterogeneity in the development of non-exclusive same-sex sexual orientations. Overall, results support a role for prenatal androgens, as approximated by digit ratios, in influencing the sexual orientation and GNC of a subset of gay men.

Puberty is a Critical Period for Vomeronasal Organ Mediation of Socio-sexual Behavior in Mice.

Genetic disruption of the vomeronasal organ (VNO), an organ responsible for pheromone processing, drastically alters socio-sexual behavior in mice. However, it is not known whether the VNO has a role during the pubertal organizational period when sex-typical socio-sexual behaviors emerge, or if disruption of the organ in adulthood is sufficient to alter socio-sexual behavior. To bypass the lifelong VNO disruption of genetic knockout models, we surgically ablated the VNO of male and female mice either during the peripubertal period [postnatal day (PND) 28-30] or adulthood (PND 58-60), with sham controls at both ages. We ruled out anosmia via the buried food test and assessed sexual odor preferences by simultaneously exposing mice to same- and opposite-sex soiled-bedding. We then measured territorial aggression with the resident-intruder paradigm and assessed sexual behavior in response to an encounter with an estrus-induced female. Neural activity approximated by FOS-immunoreactivity along the VNO-accessory olfactory pathway was measured in response to opposite-sex odors. We found that peripubertal VNO ablation decreased sexual odor preferences and neural activity in response to opposite-sex odors, and drastically reduced territorial aggression in male mice. Conversely, adult VNO ablation resulted in subtle differences in sexual odor preferences compared with sham controls. Regardless of the VNO condition, mice displayed sex-typical copulatory behaviors. Together, these results suggest that puberty is a critical period in development whereby the VNO contributes to the sexual differentiation of behavior and neural response to conspecific odors.

Androgen-Dependent Excitability of Mouse Ventral Hippocampal Afferents to Nucleus Accumbens Underlies Sex-Specific Susceptibility to Stress.

BACKGROUND: Depression affects women nearly twice as often as men, but the neurobiological underpinnings of this discrepancy are unclear. Preclinical studies in male mice suggest that activity of ventral hippocampus (vHPC) neurons projecting to the nucleus accumbens (NAc) regulates mood-related behavioral responses to stress. We sought to characterize this circuit in both sexes and to investigate its role in potential sex differences in models of depression. METHODS: We used male and female adult C57BL/6J mice in the subchronic variable stress model to precipitate female-specific reduction in sucrose preference and performed gonadectomies to test the contributions of gonadal hormones to this stress response. In addition, ex vivo slice electrophysiology of transgenic Cre-inducible Rosa-eGFP-L10a mice in combination with retrograde viral tracing to identify circuits was used to test contributions of gonadal hormones to sex differences in vHPC afferents. Finally, we used an intersecting viral DREADD (designer receptor exclusively activated by designer drugs) strategy to manipulate vHPC-NAc excitability directly in awake behaving mice. RESULTS: We show a testosterone-dependent lower excitability in male versus female vHPC-NAc neurons and corresponding testosterone-dependent male resilience to reduced sucrose preference after subchronic variable stress. Importantly, we show that long-term DREADD stimulation of vHPC-NAc neurons causes decreased sucrose preference in male mice after subchronic variable stress, whereas DREADD inhibition of this circuit prevents this effect in female mice. CONCLUSIONS: We demonstrate a circuit-specific sex difference in vHPC-NAc neurons that is dependent on testosterone and causes susceptibility to stress in female mice. These data provide a substantive mechanism linking gonadal hormones to cellular excitability and anhedonia-a key feature in depressive states.

Both neural and global androgen receptor overexpression affect sexual dimorphism in the mouse brain.

Testosterone is the main endocrine mechanism mediating sexual differentiation of the mammalian brain, although testosterone signalling is complex and important mechanistic questions remain. Notably, the extent to which testosterone acts via androgen receptors (AR) in this process remains unknown and it is also not clear where testosterone acts in the body to produce sexual dimorphisms in neuroanatomy. To address these questions, we used a transgenic mouse model of Cre/loxP-driven AR overexpression in which AR was induced selectively in neural tissue (Nestin-cre) or in all tissues (CMV-cre). We then studied sexually dimorphic features of several well-characterised sexual dimorphisms: calbindin-immunoreactive neurones in the medial preoptic area (CALB-SDN), tyrosine hydroxylase neurones in the anteroventral periventricular nucleus, and vasopressin-immunoreactive neurones originating in the bed nucleus of the stria terminalis and their projections in the lateral septum. We additionally evaluated oestrogen receptor α immunoreactivity in these nuclei. Briefly, we found that global but not neural overexpression of AR resulted in masculinisation of CALB-SDN nucleus volume, cell number and cell size in transgenic females. Furthermore, neural AR overexpression resulted in increased oestrogen receptor α staining in females compared to males in the medial preoptic area. AR overexpression did not affect other measures. Overall, the results of the present study provide support for the hypothesis that androgenic mechanisms external to the nervous system can affect sexual differentiation of the brain.