References 6.b: Causes – Genetic factors – Animal studies

Epigenetically programming gender identity

The presence or absence of a Y chromosome dictates our biological gender. However, the brain is wired to be female unless exposed to testicular steroids (such as androgen and its metabolite estradiol) during a sensitive period shortly before and after birth. The most sexually dimorphic region in the brain is the preoptic area (POA). Nugent et al. found that steroid-induced gender identity in the POA is mediated by an epigenetic mechanism, specifically DNA methylation. The best characterized form of DNA methylation occurs at cytosine residues that are adjacent to guanines (CpG islands). DNA methyltransferase (DNMT) activity and CpG methylation were lower in the POA of male newborn rats than in those of female newborn rats. Subcutaneously injecting newborn female rats with estradiol induced a decrease in DNMT activity and CpG methylation for a few days. DNMT activity did not differ before or after the sensitive perinatal period, with or without estradiol injections. Intracerebral injection of the DNMT inhibitors zebularin or RG108 in female newborn rats mimicked the effect of estradiol on DNMT activity and produced neuronal morphology, protein marker patterns, and adult anxiety-related and sexual behavior more typical of male mice, without affecting the estrous cycle. In addition, adult female mice that lacked Dnmt3a in the POA from birth had anxiety and sexual behaviors more typical of male mice. However, unlike estradiol exposure, the masculinizing effects of knocking out Dnmt3a were not restricted to the sensitive perinatal period, suggesting that enduring DNA methylation maintains the female phenotype in the brain. RNA sequencing of the POA from postnatal male or female rats revealed that a subset of genes derepressed by DNMT inhibition in females was associated with sexual differentiation. The findings further define how neurological gender identity is determined.

Author/-s:       Leslie K. Ferrarelli

Publication:     Science Signaling, 2015

Web link:          http://stke.sciencemag.org/content/8/376/ec120.abstract

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

Web link:          http://www.ncbi.nlm.nih.gov/pubmed/25177264

Early Prenatal Stress Epigenetically Programs Dysmasculinization in Second-Generation Offspring via the Paternal Lineage

Studies have linked sex-biased neurodevelopmental disorders, including autism and schizophrenia, with fetal antecedents such as prenatal stress. Further, these outcomes can persist into subsequent generations, raising the possibility that aspects of heritability in these diseases involve epigenetic mechanisms. Utilizing a mouse model in which we previously identified a period in early gestation when stress results in dysmasculinized and stress-sensitive male offspring, we have examined programming effects in second-generation offspring of prenatally stressed (F2-S) or control (F2-C) sires. Examination of gene expression patterns during the perinatal sensitive period, when organizational gonadal hormones establish the sexually dimorphic brain, confirmed dysmasculinization in F2-S males, where genes important in neurodevelopment showed a female-like pattern. Analyses of the epigenomic miRNA environment detected significant reductions in miR-322, miR-574, and miR-873 in the F2-S male brain, levels that were again more similar to those of control females. Increased expression of a common gene target for these three miRNAs, β-glycan, was confirmed in these males. These developmental effects were associated with the transmission of a stress-sensitive phenotype and shortened anogenital distance in adult F2-S males. As confirmation that the miRNA environment is responsive to organizational testosterone, neonatal males administered the aromatase inhibitor formestane exhibited dramatic changes in brain miRNA patterns, suggesting that miRNAs may serve a previously unappreciated role in organizing the sexually dimorphic brain. Overall, these data support the existence of a sensitive period of early gestation when epigenetic programming of the male germline can occur, permitting transmission of specific phenotypes into subsequent generations.

Author/-s:       Christopher P. Morgan; Tracy L. Bale

Publication:     The Journal of Neuroscience, 2011

Web link:          http://www.jneurosci.org/content/31/33/11748.abstract

Sex differences in brain developing in the presence or absence of gonads

Brain sexual differentiation results from the interaction of genetic and hormonal influences. This study used a unique agonadal mouse model to determine relative contributions of genetic and gonadal hormone influences in the differentiation of selected brain regions. SF-1 knockout (SF-1 KO) mice are born without gonads and adrenal glands and are not exposed to endogenous sex steroids during fetal/neonatal development. Consequently, male and female SF-1 KO mice are born with female external genitalia and if left on their own, die shortly after birth due to adrenal insufficiency. In this study, SF-1 KO mice were rescued by neonatal adrenal transplantation to examine their brain morphology in adult life. To determine potential brain loci that might mediate functional sex differences, we examined the area and distribution of immunoreactive calbindin and neuronal nitric oxide synthase in the preoptic area (POA) and ventromedial nucleus of the hypothalamus, two areas previously reported to be sexually dimorphic in the mammalian brain. A sex difference in the positioning of cells containing immunoreactive calbindin in a group within the POA was clearly gonad dependent based on the elimination of the sex difference in SF-1 KO mice. Several other differences in the area of ventromedial hypothalamus and in POA were maintained in male and female SF-1 KO mice, suggesting gonad-independent genetic influences on sexually dimorphic brain development.

Author/-s:       T. Büdefeld; N. Grgurevic; S. A. Tobet; G. Majdic

Publication:     Developmental neurobiology, 2008

Web link:          http://www.ncbi.nlm.nih.gov/pubmed/18418875

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

Web link:          http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2706155/?tool=pubmed

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

Web link:          http://europepmc.org/abstract/MED/17307903

Sex differences in brain and behavior: hormones versus genes.

Sex determination is the commitment of an organism toward male or female development. Traditionally, in mammals, sex determination is considered equivalent to gonadal determination. Since the presence or the absence of the testes ultimately determines the phenotype of the external genitalia, sex determination is typically seen as equivalent to testis determination. But what exactly does sex determine? The endpoint of sex determination is almost invariably seen as the reproductive structures, which represent the most obvious phenotypic difference between the sexes. One could argue that the most striking differences between males and females are not the anatomy of the genitals, but the size of the gametes-considerably larger in females than males. In fact, there could be many different endpoints to sex determination, leading to differences between the sexes: brain sexual differences, behavioral differences, and susceptibility to disease. The central dogma of sexual differentiation, stemming initially from the gonad-transfer experiments of Alfred Jost, is that sexual dimorphisms of all somatic tissues are dependent on the testicular secretion of the developing fetus. In this chapter, we will take the example of sex differences in brain and behavior as an endpoint of sex determination. We will argue that genetic factors play a role in sexually dimorphic traits such as the number of dopaminergic cells in the mesencephalon, aggression, and sexual orientation, independently from gonadal hormones.

Author/-s:       S. Bocklandt; E. Vilain

Publication:     Advances in genetics, 2007

Web link:          http://www.ncbi.nlm.nih.gov/pubmed/17888801

Direct regulation of adult brain function by the male-specific factor SRY

The central dogma of mammalian brain sexual differentiation has contended that sex steroids of gonadal origin organize the neural circuits of the developing brain. Recent evidence has begun to challenge this idea and has suggested that, independent of the masculinizing effects of gonadal secretions, XY and XX brain cells have different patterns of gene expression that influence their differentiation and function. We have previously shown that specific differences in gene expression exist between male and female developing brains and that these differences precede the influences of gonadal hormones. Here we demonstrate that the Y chromosome-linked, male-determining gene Sry is specifically expressed in the substantia nigra of the adult male rodent in tyrosine hydroxylase-expressing neurons. Furthermore, using antisense oligodeoxynucleotides, we show that Sry downregulation in the substantia nigra causes a statistically significant decrease in tyrosine hydroxylase expression with no overall effect on neuronal numbers and that this decrease leads to motor deficits in male rats. Our studies suggest that Sry directly affects the biochemical properties of the dopaminergic neurons of the nigrostriatal system and the specific motor behaviors they control. These results demonstrate a direct male-specific effect on the brain by a gene encoded only in the male genome, without any mediation by gonadal hormones.

Author/-s:       P. Dewing; C. W. Chiang; K. Sinchak; H. Sim; P. O. Fernagut; S. Kelly; M. F. Chesselet; P. E. Micevych; K. H. Albrecht; V. R. Harley; E. Vilain

Publication:     Current biology, 2006

Web link:          http://www.ncbi.nlm.nih.gov/pubmed/16488877

Sex chromosome complement and gonadal sex influence aggressive and parental behaviors in mice

Across human cultures and mammalian species, sex differences can be found in the expression of aggression and parental nurturing behaviors: males are typically more aggressive and less parental than females. These sex differences are primarily attributed to steroid hormone differences during development and/or adulthood, especially the higher levels of androgens experienced by males, which are caused ultimately by the presence of the testis-determining gene Sry on the Y chromosome. The potential for sex differences arising from the different complements of sex-linked genes in male and female cells has received little research attention. To directly test the hypothesis that social behaviors are influenced by differences in sex chromosome complement other than Sry, we used a transgenic mouse model in which gonadal sex and sex chromosome complement are uncoupled. We find that latency to exhibit aggression and one form of parental behavior, pup retrieval, can be influenced by both gonadal sex and sex chromosome complement. For both behaviors, females but not males with XX sex chromosomes differ from XY. We also measured vasopressin immunoreactivity in the lateral septum, which was higher in gonadal males than females, but also differed according to sex chromosome complement. These results imply that a gene(s) on the sex chromosomes (other than Sry) affects sex differences in brain and behavior. Identifying the specific X and/or Y genes involved will increase our understanding of normal and abnormal aggression and parental behavior, including behavioral abnormalities associated with mental illness.

Author/-s:       J. D. Gatewood; A. Wills; S. Shetty; J. Xu; A. P. Arnold; P. S. Burgoyne; E. F. Rissman

Publication:     The Journal of neuroscience, 2006

Web link:          http://www.ncbi.nlm.nih.gov/pubmed/16495461

Tissue-specific expression and regulation of sexually dimorphic genes in mice

We report a comprehensive analysis of gene expression differences between sexes in multiple somatic tissues of 334 mice derived from an intercross between inbred mouse strains C57BL/6J and C3H/HeJ. The analysis of a large number of individuals provided the power to detect relatively small differences in expression between sexes, and the use of an intercross allowed analysis of the genetic control of sexually dimorphic gene expression. Microarray analysis of 23 574 transcripts revealed that the extent of sexual dimorphism in gene expression was much greater than previously recognized. Thus, thousands of genes showed sexual dimorphism in liver, adipose, and muscle, and hundreds of genes were sexually dimorphic in brain. These genes exhibited highly tissue-specific patterns of expression and were enriched for distinct pathways represented in the Gene Ontology database. They also showed evidence of chromosomal enrichment, not only on the sex chromosomes, but also on several autosomes. Genetic analyses provided evidence of the global regulation of subsets of the sexually dimorphic genes, as the transcript levels of a large number of these genes were controlled by several expression quantitative trait loci (eQTL) hotspots that exhibited tissue-specific control. Moreover, many tissue-specific transcription factor binding sites were found to be enriched in the sexually dimorphic genes.

Author/-s:       Xia Yang; Eric E. Schadt; Susanna Wang; Hui Wang; Arthur P. Arnold; Leslie Ingram-Drake; Thomas A. Drake; Aldons J. Lusis

Publication:     Genome Research, 2006

Web link:          http://genome.cshlp.org/content/16/8/995

Partial demasculinization of several brain regions in adult male (XY) rats with a dysfunctional androgen receptor gene

The adult rat posterodorsal medial amygdala (MePD) is sexually dimorphic in regional volume and neuronal soma size, both of which are larger in males than in females. This sexual dimorphism is entirely dependent on adult circulating levels of testicular androgens, and both androgen and estrogen treatment can masculinize MePD structure. We examined male rats that are rendered androgen-insensitive by the testicular feminization mutation (tfm) of the androgen receptor (AR) gene to determine how a dysfunctional AR affects this and other brain sexual dimorphisms. In adult wild-type rats, the MePD in males had a greater regional volume, rostrocaudal extent, and soma size than in females. In genetic males, defective ARs affected some but not all of these indices: MePD volume and soma size in tfm males were intermediate between those of wild-type males and females, but the rostrocaudal extent of the MePD was unaffected by the mutation, being as great in tfm males as in wild-type males. Regional volume and soma size in the suprachiasmatic nucleus was reduced in tfm males compared with wild-type males, suggesting that AR normally affects this region in male rats. Interestingly, whereas volume of the sexually dimorphic nucleus of the preoptic area was unaffected by the tfm allele, soma size in this region was reduced in tfm males compared with wild-type males. Although estrogen receptor activation has been shown to be vital for masculinization of the rodent brain, our results indicate that ARs also contribute to this process in several brain regions.

Author/-s:       John A. Morris; Cynthia L. Jordan; Brittany N. Dugger; S. Marc Breedlove

Publication:     The Journal of Comparative Neurology, 2005

Web link:          http://onlinelibrary.wiley.com/doi/10.1002/cne.20558/abstract

Are XX and XY brain cells intrinsically different?

In mammals and birds, the sex of the gonads is determined by genes on the sex chromosomes. For example, the mammalian Y-linked gene Sry causes testis differentiation. The testes then secrete testosterone, which acts on the brain (often after conversion to estradiol) to cause masculine patterns of development. If this were the only reason for sex differences in neural development, then XX and XY brain cells would have to be deemed otherwise equivalent. This equivalence is doubtful because of recent experimental results demonstrating that some XX and XY tissues, including the brain, are sexually dimorphic even when they develop in a similar endocrine environment. Although X and Y genes probably influence brain phenotype in a sex-specific manner, much more information is needed to identify the magnitude and character of these effects.

Author/-s:       Arthur P. Arnold; P. S. Burgoyne

Publication:     Trends in endocrinology and metabolism, 2004

Web link:          http://www.ncbi.nlm.nih.gov/pubmed/14693420

Sexually dimorphic gene expression in mouse brain precedes gonadal differentiation

The classic view of brain sexual differentiation and behavior is that gonadal steroid hormones act directly to promote sex differences in neural and behavioral development. In particular, the actions of testosterone and its metabolites induce a masculine pattern of brain development, while inhibiting feminine neural and behavioral patterns of differentiation. However, recent evidence indicates that gonadal hormones may not solely be responsible for sex differences in brain development and behavior between males and females. Here we examine an alternative hypothesis that genes, by directly inducing sexually dimorphic patterns of neural development, can influence the sexual differences between male and female brains. Using microarrays and RT-PCR, we have detected over 50 candidate genes for differential sex expression, and confirmed at least seven murine genes which show differential expression between the developing brains of male and female mice at stage 10.5 days post coitum (dpc), before any gonadal hormone influence. The identification of genes differentially expressed between male and female brains prior to gonadal formation suggests that genetic factors may have roles in influencing brain sexual differentiation.

Author/-s:       Phoebe Dewing; Tao Shi; Steve Horvath; Eric Vilain

Publication:     Molecular Brain Research, 2003

Web link:          http://www.ncbi.nlm.nih.gov/pubmed/14559357

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

Web link:          http://www.ncbi.nlm.nih.gov/pubmed/12388607

Sex chromosome genes directly affect brain sexual differentiation

Sex differences in the brain are caused by differences in gonadal secretions: higher levels of testosterone during fetal and neonatal life cause the male brain to develop differently than the female brain. In contrast, genes encoded on the sex chromosomes are not thought to contribute directly to sex differences in brain development, even though male (XY) cells express Y-chromosome genes that are not present in female (XX) cells, and XX cells may have a higher dose of some X-chromosome genes. Using mice in which the genetic sex of the brain (XX versus XY) was independent of gonadal phenotype (testes versus ovaries), we found that XY and XX brain cells differed in phenotype, indicating that a brain cell's complement of sex chromosomes may contribute to its sexual differentiation.

Author/-s:       L. L. Carruth; I. Reisert; Arthur P. Arnold

Publication:     Nature neuroscience, 2002

Web link:          http://www.ncbi.nlm.nih.gov/pubmed/12244322

Genetically triggered sexual differentiation of brain and behavior

The dominant theory of sexual differentiation of the brain holds that sex differences in brain anatomy and function arise because of the action of gonadal steroids during embryonic and neonatal life. In mammals, testicular steroids trigger masculine patterns of neural development, and feminine patterns of neural development occur in the absence of such testicular secretions. In contrast, gonadal differentiation in mammals is not initiated by hormonal mechanisms, but is regulated by the action of gene products such as SRY, a testis-determining gene on the Y chromosome. This paper argues that such genetic, nonhormonal signals may also trigger specific examples of sexual differentiation of the brain. This thesis is supported by two arguments. The first is that "direct genetic" (i.e., nonhormonal) control of sexual differentiation may be as likely to evolve as hormonal control. The second line of argument is that neural and nonneural dimorphisms have already been described that are not well explained by classical theories of steroid-dependent sexual differentiation and for which other factors need to be invoked.

Author/-s:       Arthur P. Arnold

Publication:     Hormones and behavior, 1996

Web link:          http://www.ncbi.nlm.nih.gov/pubmed/9047274