Hormone-dependent sexual responses of female mice in response to manual genital stimulation.

Although copulatory behavior is thought to have a strong innate basis in mice, there is also clear evidence that sexual experience shapes its expression. Reinforcement of behavior through rewarding genital tactile stimulation is a primary candidate mechanism for this modification. In rats, manual tactile clitoral stimulation is rewarding only when it is temporally distributed, which is hypothesized to result from an innate preference for species-typical copulatory patterning. Here we test this hypothesis using mice, which have a temporal copulatory pattern which is distinctly less temporally distributed than that of rats. Female mice received manual clitoral stimulation which was either temporally continuous every second, or stimulation which was temporally distributed, occurring every 5 s, This pattern of stimulation was paired with environmental cues in a conditioned place preference apparatus to assess reward. Neural activation in response to this stimulation was evaluated by measuring FOS immunoreactivity. Results indicated that both temporal patterns of clitoral stimulation were rewarding, but that continuous stimulation better reproduced brain activation associated with sexual reward. Furthermore, continuous, but not distributed stimulation elicited a lordosis response in some females, and this response increased within and across days. Sexual reward, neural activation and lordosis resulting from tactile genital stimulation were eliminated by ovariectomy and restored with combined 17ß-estradiol and progesterone treatment but not 17ß-estradiol treatment alone. These observations are consistent with the hypothesis that sexual reward resulting from species-typical genital tactile stimulation has a permissive effect on copulatory behavior of female mice.

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.

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.

Homeostatic control of nuclear-encoded mitochondrial gene expression by the histone variant H2A.Z is essential for neuronal survival.

Histone variants are crucial regulators of chromatin structure and gene transcription, yet their functions within the brain remain largely unexplored. Here, we show that the H2A histone variant H2A.Z is essential for neuronal survival. Mice lacking H2A.Z in GABAergic neurons or Purkinje cells (PCs) present with a progressive cerebellar ataxia accompanied by widespread degeneration of PCs. Ablation of H2A.Z in other neuronal subtypes also triggers cell death. H2A.Z binds to the promoters of key nuclear-encoded mitochondrial genes to regulate their expression and promote organelle function. Bolstering mitochondrial activity genetically or by organelle transplant enhances the survival of H2A.Z-ablated neurons. Changes in bioenergetic status alter H2A.Z occupancy at the promoters of nuclear-encoded mitochondrial genes, an adaptive response essential for cell survival. Our results highlight that H2A.Z fulfills a key, conserved role in neuronal survival by acting as a transcriptional rheostat to regulate the expression of genes critical to mitochondrial function.

Sex-specific effects of the histone variant H2A.Z on fear memory, stress-enhanced fear learning and hypersensitivity to pain.

Emerging evidence suggests that histone variants are novel epigenetic regulators of memory, whereby histone H2A.Z suppresses fear memory. However, it is not clear if altered fear memory can also modify risk for PTSD, and whether these effects differ in males and females. Using conditional-inducible H2A.Z knockout (cKO) mice, we showed that H2A.Z binding is higher in females and that H2A.Z cKO enhanced fear memory only in males. However, H2A.Z cKO improved memory on the non-aversive object-in-place task in both sexes, suggesting that H2A.Z suppresses non-stressful memory irrespective of sex. Given that risk for fear-related disorders, such as PTSD, is biased toward females, we examined whether H2A.Z cKO also has sex-specific effects on fear sensitization in the stress-enhanced fear learning (SEFL) model of PTSD, as well as associated changes in pain sensitivity. We found that H2A.Z cKO reduced stress-induced sensitization of fear learning and pain responses preferentially in female mice, indicating that the effects of H2A.Z depend on sex and the type of task, and are influenced by history of stress. These data suggest that H2A.Z may be a sex-specific epigenetic risk factor for PTSD susceptibility, with implications for developing sex-specific therapeutic interventions.

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.

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.

Androgen receptor is a negative regulator of contextual fear memory in male mice.

Although sex-hormones have a well-documented role in memory formation, most literature has focused on estrogens, whereas the role of androgens and their receptor (the androgen receptor; AR) in fear memory is relatively unexplored. To address this gap, we used a transgenic mouse model of AR overexpression (CMV-AR) to determine if AR regulates fear memory, and if this effect can be reversed either by the removal of circulating androgens via gonadectomy, or by antagonising AR activity with flutamide. We found that AR overexpression results in reduced freezing in response to foot shock, and that this difference is reversed with both gonadectomy and flutamide treatment. Differences between genotypes were reinstated by testosterone replacement in gonadectomized mice, suggesting that reduced fear memory in mutants results from AR activation by testosterone and is not secondary to group differences in circulating testosterone. Potential transcriptional mechanisms by which CMV-AR exerts its effects on fear memory were assessed by quantitating the expression of memory-related genes in area CA1 of the hippocampus. Several genes that are altered with AR inhibition and activation, including genes that encode for the histone variant H2A.Z, cholinergic receptors, glutamate receptors, and brain-derived neurotrophic factor. Overall, our findings suggest that AR is a negative regulator of fear memory and identify potential gene targets through which AR may mediate this effect.

Muscle, a conduit to brain for hormonal control of behavior.

SBN Elsevier Lecture Investigation into mechanisms whereby hormones control behavior often starts with actions on central nervous system (CNS) motivation and motor systems and is followed by assessment of CNS drive of coordinated striated muscle contractions. Here we turn this perspective on its head by discussing ways in which hormones might first act on muscle that then secondarily drive upstream the evolution and function of the CNS. While there is a lengthy history for consideration of this perspective, newly discovered properties of muscle signaling reveal novel mechanisms that may well be captured by endocrine systems and thus of interest to behavioral endocrinologists.

Sex- and brain region-specific patterns of gene expression associated with socially-mediated puberty in a eusocial mammal.

The social environment can alter pubertal timing through neuroendocrine mechanisms that are not fully understood; it is thought that stress hormones (e.g., glucocorticoids or corticotropin-releasing hormone) influence the hypothalamic-pituitary-gonadal axis to inhibit puberty. Here, we use the eusocial naked mole-rat, a unique species in which social interactions in a colony (i.e. dominance of a breeding female) suppress puberty in subordinate animals. Removing subordinate naked mole-rats from this social context initiates puberty, allowing for experimental control of pubertal timing. The present study quantified gene expression for reproduction- and stress-relevant genes acting upstream of gonadotropin-releasing hormone in brain regions with reproductive and social functions in pre-pubertal, post-pubertal, and opposite sex-paired animals (which are in various stages of pubertal transition). Results indicate sex differences in patterns of neural gene expression. Known functions of genes in brain suggest stress as a key contributing factor in regulating male pubertal delay. Network analysis implicates neurokinin B (Tac3) in the arcuate nucleus of the hypothalamus as a key node in this pathway. Results also suggest an unappreciated role for the nucleus accumbens in regulating puberty.

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.

Androgen receptors and muscle: a key mechanism underlying life history trade-offs.

Sexual dimorphism in skeletal muscle is prominent in mammals, with males typically having larger and stronger muscles than females. Furthermore, neuromuscular systems with sexual functions are remarkably sexually dimorphic in a wide variety of vertebrates. Endocrine mechanisms are of central importance for sexual differentiation of these traits, and anabolic actions of gonadal testosterone have been intensively studied. Here we review the relationship between androgen receptor (AR) and sexual differentiation of neuromuscular systems. We focus our review on the hypotheses that sexual dimorphism and androgen responsiveness of neuromuscular systems is a function of the amount of AR expressed by muscle and that AR in muscle is a key mechanism on which evolution acts to shape individual and species differences in reproductive behavior.

Handedness is a biomarker of variation in anal sex role behavior and Recalled Childhood Gender Nonconformity among gay men.

Developmental theories of the biological basis of sexual orientation suggest that sexually differentiated psychological and behavioural traits should be linked with sexual orientation. Subgroups of gay men delineated by anal sex roles differ according to at least one such trait: gender expression. The present study assessed the hypothesis that handedness, a biologically determined sexually differentiated trait, corresponds to differences in subgroups of gay men based on anal sex role. Furthermore, it assessed whether handedness mediates the association between gender nonconformity and male sexual orientation. Straight and gay men (N = 333) completed the Edinburgh Inventory of Handedness and the Recalled Childhood Gender Nonconformity Scale. Gay men also completed measures of anal sex role preference. As in previous studies, gay men showed greater non-right-handedness and gender nonconformity than straight men. Also, among gay men, bottoms/versatiles (i.e., gay men who take a receptive anal sex role, or who take on both a receptive and insertive anal sex role) were more gender-nonconforming than tops (i.e., gay men who take an insertive anal sex role). In support of the hypothesis, bottoms/versatiles were more non-right-handed than tops and handedness mediated the male sexual orientation and anal sex role differences in Recalled Childhood Gender Nonconformity. Together, these findings suggest that developmental processes linked to handedness underpin variation among men in sexual orientation and gender nonconformity as well as variation among subgroups of gay men that are delineated by anal sex roles.

Nonneural Androgen Receptors Affect Sexual Differentiation of Brain and Behavior.

Testosterone, acting via estrogenic and androgenic pathways, is the major endocrine mechanism promoting sexual differentiation of the mammalian nervous system and behavior, but we have an incomplete knowledge of which cells and tissues mediate these effects. To distinguish between neural and nonneural actions of androgens in sexual differentiation of brain and behavior, we generated a loxP-based transgenic mouse, which overexpresses androgen receptors (ARs) when activated by Cre. We used this transgene to overexpress AR globally in all tissues using a cytomegalovirus (CMV)-Cre driver (CMV-AR), and we used a Nestin-Cre driver to overexpress AR only in neural tissue (Nes-AR). We then examined whether neural or global AR overexpression can affect socio-sexual behaviors using a resident-intruder paradigm. We found that both neural and global AR overexpression resulted in decreased aggressive behaviors and increased thrusting during mounting of intruders, consistent with a neural site of action. Global, but not neural, AR overexpression in males led to an increase in same-sex anogenital investigation. Together, these results suggest novel roles for nonneural AR in sexual differentiation of mice, and indicate that excess AR can lead to a paradoxical reduction of male-typical behavior.

Removal of reproductive suppression reveals latent sex differences in brain steroid hormone receptors in naked mole-rats, Heterocephalus glaber.

BACKGROUND: Naked mole-rats are eusocial mammals, living in large colonies with a single breeding female and 1-3 breeding males. Breeders are socially dominant, and only the breeders exhibit traditional sex differences in circulating gonadal steroid hormones and reproductive behaviors. Non-reproductive subordinates also fail to show sex differences in overall body size, external genital morphology, and non-reproductive behaviors. However, subordinates can transition to breeding status if removed from their colony and housed with an opposite-sex conspecific, suggesting the presence of latent sex differences. Here, we assessed the expression of steroid hormone receptor and aromatase messenger RNA (mRNA) in the brains of males and females as they transitioned in social and reproductive status. METHODS: We compared in-colony subordinates to opposite-sex subordinate pairs that were removed from their colony for either 1 day, 1 week, 1 month, or until they became breeders (i.e., produced a litter). Diencephalic tissue was collected and mRNA of androgen receptor (Ar), estrogen receptor alpha (Esr1), progesterone receptor (Pgr), and aromatase (Cyp19a1) was measured using qPCR. Testosterone, 17ß-estradiol, and progesterone from serum were also measured. RESULTS: As early as 1 week post-removal, males exhibited increased diencephalic Ar mRNA and circulating testosterone, whereas females had increased Cyp19a1 mRNA in the diencephalon. At 1 month post-removal, females exhibited increased 17ß-estradiol and progesterone. The largest changes in steroid hormone receptors were observed in breeders. Breeding females had a threefold increase in Cyp19a1 and fivefold increases in Esr1 and Pgr, whereas breeding males had reduced Pgr and increased Ar. CONCLUSIONS: These data demonstrate that sex differences in circulating gonadal steroids and hypothalamic gene expression emerge weeks to months after subordinate animals are removed from reproductive suppression in their home colony.

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.

Androgen receptor expression in satellite cells of the neonatal levator ani of the rat.

Androgens are thought to mediate sexual differentiation of spinal nucleus of the bulbocavernosus (SNB) motoneurons via actions on androgen receptors (ARs) within their target muscles bulbocavernosus and levator ani (LA). However, the cells within these muscles which mediate masculinization of the SNB remain undefined. Until recently, myocytes were thought to be the most likely candidate cell type. However, genetic tests of AR function in myocytes have failed to support a sufficient role for these cells in producing masculine SNB morphology, suggesting the involvement of other cell types. To identify other candidate cell types in the LA, we evaluated whether satellite cells or fibroblasts express AR. Fluorescent immunohistochemistry and confocal microscopy were used to evaluate whether satellite cells and fibroblasts express AR in neonatal male and female rats in the LA and an adjacent sexually monomorphic control muscle (CM). We found that a small proportion of satellite cells in the LA express AR and that this proportion is significantly greater in the LA compared to the CM. No sex differences were found between the proportions of satellite cells expressing AR in either muscle. Less colocalization of satellite cells and AR was seen in postnatal day 3 muscle than in postnatal day 1 muscle. In contrast, only negligible amounts of fibroblasts labeled with S100A4 express AR in either the LA or the CM. Together, findings support satellite cells, but not fibroblasts, as a candidate cell type involved in the sexual differentiation of the SNB neuromuscular system.

Turning sex inside-out: Peripheral contributions to sexual differentiation of the central nervous system.

Sexual differentiation of the nervous system occurs via the interplay of genetics, endocrinology and social experience through development. Much of the research into mechanisms of sexual differentiation has been driven by an implicit theoretical framework in which these causal factors act primarily and directly on sexually dimorphic neural populations within the central nervous system. This review will examine an alternative explanation by describing what is known about the role of peripheral structures and mechanisms (both neural and non-neural) in producing sex differences in the central nervous system. The focus of the review will be on experimental evidence obtained from studies of androgenic masculinization of the spinal nucleus of the bulbocavernosus, but other systems will also be considered.

Maintenance of the spinal nucleus of the bulbocavernosus neuromuscular system is not influenced by physiological levels of glucocorticoids.

The spinal nucleus of the bulbocavernosus (SNB) neuromuscular system mediates sexual reflexes, and is highly sexually dimorphic in rats. While maintenance of this system in adulthood is mainly dependent on androgens, there is also evidence to suggest that glucocorticoids may have a catabolic effect. We conducted a series of studies to fully examine the influence of basal glucocorticoids on the size of the SNB motoneurons and the associated bulbocavernosus (BC) and levator ani (LA) muscles. Specifically, we examined whether the muscles and motoneurons of the SNB neuromuscular system are affected by: (1) blockade of endogenous glucocorticoids via delivery of the antagonist RU-486 at doses ranging from low to high, (2) removal of endogenous glucocorticoids via adrenalectomy, or (3) restoration of physiological corticosterone levels via implants following adrenalectomy. In each study, we found that muscle and motoneuron size were unaffected by glucocorticoid manipulation. In contrast to previous results with supraphysiological levels of glucocorticoids, our results indicate that basal, nonstress levels of glucocorticoids do not influence the size of the BC/LA muscles or their associated SNB motoneurons.