Gonadal hormones and early life programming. The reproductive behaviour of adult males and females has long been known to be dependent on the gonadal steroid hormone milieu, which is unique to each sex. Males have higher circulating levels of testosterone derived from the testis, whereas females have a cyclic pattern of oestrogens and progestins arising from the ovary. In the ideal conditions of the laboratory (that is, in the presence of continuous food, water, light and shelter), males are always sexually active, but females will only readily engage in mating at or around the time of ovulation. What was not initially obvious is that the concordance of adult hormonal milieu and mating behaviour actually depends on early life programming, a concept that has been historically referred to as the organizational hypothesis12.

In rodents, this programming is initiated prenatally in males when the fetal testes produce androgens that gain access to the brain, are converted to oestrogens in large amounts and initiate brain masculinization. Both male and female fetuses express high levels of the steroid-binding globulin α-fetoprotein, which sequesters the maternal oestrogens that are present in fetal circulation and thus protects the brain from the influence of maternal steroids. As the female fetal ovary remains quiescent, this sequestration of maternal oestrogens means that only males experience high intracerebral oestrogen levels (as a result of the aromatization of testicular androgens). If α-fetoprotein is ablated, maternal oestradiol penetrates the fetal brain during the critical period, and females are subsequently masculinized13.

The processes of brain masculinization and feminization both occur during a critical period (Fig. 1). In rodents, the critical period begins prenatally at the time of the surge in male fetal androgen production. The end of the critical period is defined as the time at which the female phenotype can no longer be diverted to male by treatment with exogenous steroid hormones, which, in rats and mice, is one week to 10 days after birth. The existence of this critical window provides a tool for studying the process of masculinization. Injecting female rat or mouse pups with testosterone or its aromatized metabolite, oestradiol, for 1 or 2 days immediately after birth initiates many (although not necessarily all) of the same gene expression profiles and cell signalling cascades that occur normally in males in utero (for a review, see Ref. 11). Although this is not a perfect model of masculinization, it provides an excellent means to interrogate both the short-term processes and long-term consequences of brain sexual differentiation by comparing males, females and masculinized females. There is an apparently separate sensitive period for rodent brain feminization that occurs weeks after birth; however, this remains less well characterized than the critical window for masculinization14.

Figure 1: Early life programming of adult sex differences. The role of hormones in the establishment of sex differences in brain and behaviour can be broadly divided into three stages. In the rodent, early life programming occurs during a perinatal sensitive period (the critical window) that is marked by the onset of gonadal steroid production in males. This occurs prenatally and around birth, a period during which there is no corresponding steroidogenesis in females. During this sensitive period, numerous neuroanatomical end points are differentiated in males versus females in a region-specific manner. In some areas, there are sex differences in apoptosis, in others, there are differences in synaptogenesis or neurogenesis. The juvenile hiatus begins shortly after birth and extends until puberty. It is characterized by low to non-existent steroid levels; however, during this time, social play behaviour is more frequent among males than among females, a behavioural sex difference that is organized during early life programming. The adult period is characterized by sex-specific hormonal milieus with males having high and steady levels of testosterone (with some daily and annual variations), whereas females undergo cyclic changes in hormone levels that are associated with ovulation. The steroid milieu of each sex activates the neural circuits of mating that were organized during the perinatal period. In this way, the gonadal phenotype and neural phenotype controlling mating behaviour are synchronized. Non-reproductive behaviours such as stress and anxiety, spatial learning, locomotion and social affiliation are also modulated by steroids in adulthood, and this modulation may or may not depend on earlier programming effects during the sensitive period. PowerPoint slide Full size image

In addition to the adult consequences of the effects of gonadal hormones on brain development (known as 'organizational' effects), many sex differences in adult animals (and probably in humans too) are the result of the markedly different gonadal hormonal milieus that each experiences in adulthood. These are often referred to as 'activational' hormonal effects and may or may not depend on prior organizational hormonal effects.

Chromosome complement: XX versus XY matters too. Not all hormonal effects in adults are preordained developmentally, and not all sex differences are due to hormones. Besides its influence on gonadal differentiation and steroid hormone production, chromosome complement is an additional contributing variable to sex differences in brain and behaviour15,16 (Fig. 2). The potential for direct sex chromosome effects was evident in a comparison of embryonic neural stem cells derived from male and female mouse embryos. When compared before the onset of gonadal steroid production, more than 100 transcripts were differentially expressed between female (XX) and male (XY) stem cells. Intriguingly, stem cells of both sexes were responsive to testosterone treatment but not in the same way: thousands more transcripts varied in response to testosterone in XX stem cells than in XY stem cells17.

Figure 2: Sex chromosomes affect brain development directly and indirectly. The differentiation of the bipotential gonad into a testis versus an ovary depends on the SRY gene, which is located on the Y chromosome. If present, the gonad will become a testis, and, if not, it will become an ovary18. Testosterone and its aromatized end product oestradiol produced by the embryonic testis bind to their cognate receptors, which regulate gene transcription and exert an organizing influence on cells in the developing brain. Ovarian and testicular steroids also activate sex-specific physiology and behaviour in adulthood. Moreover, every cell in the brain is XY in male mammals and XX in female mammals. Genes on the X chromosome may be expressed at different levels in males and females (in cases in which they escape from X inactivation in females)113, whereas genes on the Y chromosome (such as SRY) are expressed only in the brain of males20. In females, the presence of a second X chromosome could create a heterochromatic sink by monopolizing the cellular machinery used for epigenetic regulation and thus alter the expression of other genes in a manner that is distinct from males. Thus, the sex chromosomes can exert both specific and broad influences on the developing brain. ER, oestrogen receptor. PowerPoint slide Full size image

The SRY gene on the Y chromosome is best known for its role as the essential driver of testis development18 but also promotes catecholamine production by dopaminergic neurons of the substantia nigra of the mouse19 and by human NT2 cells differentiated to be dopaminergic. As only males carry the SRY gene, this is a true sex dimorphism and is speculated to contribute to the higher susceptibility of males to dopamine disorders such as Parkinson disease and schizophrenia20.

A genetically modified mouse line in which Sry has been translocated from the Y chromosome to an autosome allows for generation of XX individuals with testis and XY individuals with ovaries, thereby separating sex genotype and gonadal phenotype21. This powerful tool is called the four-core genotype and has shown that multiple sex differences in brain and behaviour can be attributed to chromosome complement rather than gonadal phenotype. These include differences in the incidence of neural tube defects22, pain perception23 and many additional phenotypes (for a review, see Ref. 24).

The four-core genotype was also exploited to dissect the sources of increased vulnerability of females to autoimmune disorders, focusing on multiple sclerosis (which is easily modelled in rodents in a paradigm referred to as experimental autoimmune encephalomyelitis). The severity of symptoms and degree of myelination were greater in XX mice compared with XY, regardless of gonadal phenotype25, suggesting a genetic origin of disease vulnerability. However, it is important to note that the four-core genotype involves every cell in the body, preventing the dissociation of effects in the periphery from those in the CNS. This conundrum was addressed by the use of bone marrow transplantation to generate subjects with an XX nervous system and XY peripheral immune system and vice versa, thereby revealing that an XY nervous system was more sensitive to injury than an XX one26. The opposing effects of sex chromosome complement on the peripheral immune system and CNS, when combined with the sex-specific modulatory effects of gonadal steroids (for a review, see Ref. 27), reveal the enormous complexity of gender bias in disease and the importance of determining the contribution of sex as a biological variable.

Neuroimmunity and brain sex differences. Steroid hormones largely exert their effects by binding to intracellular receptors, which dimerize and dock at hormone response elements on gene promoters to regulate gene transcription28,29,30. Surprisingly, however, relatively few genes have been identified that are directly induced by steroid hormones and mediate masculinization. Instead, over the past decade, immune mediators have been strongly implicated as drivers of the masculinization process.

The brain is considered to be protected from systemic immune responses and pathogens by the blood–brain barrier. However, this does not mean that the brain is immunodeficient. Instead, the brain receives information from the periphery about immune status from various sources, including the vagal nerve, humoral signalling and cytokine transport into the brain, as well as meningeal and epithelial signalling across the blood–brain barrier31,32,33. The brain is also populated by resident immunocompetent cells, known as microglia, that engage in immune surveillance. These cells produce a host of neuroinflammatory mediators that are crucial regulators of brain development and homeostasis, including cytokines, prostanoids, purines and reactive oxygen species. Many of these neuroimmune mediators act in a manner that is indistinguishable from the functions of neuromodulators or neurohormones34,35,36.

Microglia seed the brain early in fetal development, beginning at around 4.5 gestational weeks in humans and embryonic day 8–9.5 in mice and rats37,38,39,40. Microglia derive from yolk-sac progenitor cells, colonize the brain before the blood–brain barrier is formed and then locally proliferate across the remainder of fetal development and into the early postnatal period41. This process differs in males and females. On embryonic day 17, just before the fetal androgen surge in males, male and female rats have the same number of microglia in the brain. One week later, males have significantly more microglia than females in several brain regions, including the parietal cortex, hippocampus, amygdala and paraventricular nucleus of the hypothalamus42. Similarly, in the developing medial preoptic area (mPOA), male rats have significantly more microglia than females in the early postnatal period and twice as many microglia that are in an activated state (based on their ameboid-like morphology)43. Treating newborn females with a masculinizing dose of oestradiol increases both microglia number and activational state to male levels.

In recent years, the functional significance of these sex differences in neuroimmune signalling and microglial number has become apparent (Fig. 3). The mPOA is a crucial brain region for expression of male sexual behaviour. Olfactory cues from sexually receptive females are detected by the male olfactory system and projected to the amygdala, which in turn projects to the mPOA. From there, signals are integrated with the neural circuits of motivation and reward, as well as projected to the hypothalamus and midbrain for execution of mating behaviour44. Dendrites of mPOA neurons are studded with dendritic spines, and, in males, the density of these spines is twofold higher than in females, resulting in greater excitation in response to olfactory input45. A crucial molecular driver of the sex difference in the density of dendritic spine synapses in the rodent mPOA is the prostanoid prostaglandin E 2 (PGE 2 ). The surge in oestradiol levels in the male brain46 during the critical period triggers an upregulation in the expression of cyclooxygenase 2 (COX2), the enzyme that leads to prostaglandin synthesis47,48. Elevated COX2 leads to increased PGE 2 in the mPOA, which is necessary for the induction of both dendritic spine patterning and male typical copulatory behaviour in adulthood48. The spinogenic effect of PGE 2 involves activation of the prostanoid receptors EP2 and EP4 (Ref. 49), which are linked to protein kinase A activation50, resulting in phosphorylation and mobilization of AMPA-type glutamate receptors to the membrane of both neurons and astrocytes51, and glutamate-dependent formation of dendritic spines50,51.

Figure 3: Neuroepigenetic and neuroinflammatory contributions to sex differences in the preoptic area. The rodent preoptic area (POA) is robustly sexually dimorphic and is therefore an ideal model system to understand the role of neuroepigenetics and neuroinflammation in sexual differentiation. The activity of DNA methyltransferase (DNMT) enzymes is higher in the developing POA of female rats than in that of males. This is because it is reduced in males, through unknown mechanisms, by the elevated oestradiol levels that are present owing to the aromatization of testosterone made in the testis. As a result, males have less DNA methylation (in the figure, indicated by 'Me') in POA cells79. Indirect evidence suggests that the lower DNA methylation in males releases immune response genes from epigenetic repression, resulting in elevation of inflammatory signalling molecules such as prostaglandin E 2 (PGE 2 ), histamine and cytokines79. PGE 2 in particular is known to activate a signal transduction cascade that results in the formation of dendritic spine synapses on the dendrites of POA neurons. This process requires the participation of both astrocytes, as a putative source of glutamate, and neighbouring microglia, which provide much of the prostaglandin. In females, immune response genes are epigenetically silenced by higher levels of DNA methylation. PGE 2 levels are therefore low, and both microglia and astrocytes are in a non-reactive state. As a result, females have half the density of dendritic spine synapses in the POA compared with males. These neuroanatomical sex differences undergird sex-specific reproductive behaviours, including male copulatory behaviour (both motivation to copulate and actual sexual performance), social scent marking, female oestrous cycling and maternal behaviour (for reviews, see Refs 44,114). PowerPoint slide Full size image

We now know that microglia are crucial for the PGE 2 -mediated induction of male-typical synaptic patterning in the mPOA of rats and the resultant male-typical copulatory behaviour in adulthood. Treating females with oestradiol increases ameboid microglia numbers to male-typical levels, as well as the number of dendritic spine-like protrusions on mPOA neurons. Oestradiol or PGE 2 treatment will also masculinize POA neurons in a dish, as indicated by an increased density of spine-like processes. However, this response does not occur if the culture is deprived of microglia43. In vivo, oestradiol-induced release of PGE 2 in the mPOA is blunted by minocycline, an antibiotic known to inhibit microglia activity, confirming the requirement for microglia in the inflammatory signalling that is responsible for male-typical synaptic patterning. Furthermore, masculinization of copulatory behaviour in females by neonatal oestradiol treatment can be prevented by co-treatment with minocycline43. Even brief microglial ablation from the neonatal brain of males leads to complete loss of male typical copulatory behaviour in adulthood52, along with other sex-specific effects53.

In the nearby anteroventral periventricular nucleus (AVPV) of the POA, a parallel process of sexual differentiation is underway during the sensitive period. In this region, oestradiol increases cell death in males, significantly reducing the size of the AVPV in comparison to that of females54. Once again, sex differences in inflammatory signalling are responsible for masculinization. An unbiased screen of AVPV gene expression revealed that several members of the tumour necrosis family of cytokines — tumour necrosis factor (TNF), TNF receptor 2 (TNFR2; also known as TNFRSF1B) and nuclear factor-κB (NF-κB) — are constitutively active in neurons of the female AVPV, but this activity is blocked in males by testosterone-induced expression of E3 ubiquitin-protein ligase TRAIP, which allows apoptosis to proceed55,56.

Resident immune cells may not be the only immune players in the sexual differentiation process. Indirect evidence supports a role for immune cells derived from the bone marrow and thymus. T cells from the thymus reside in the meninges, and recent studies have found that T cell-deficient mice have impairments in adult behaviours, including spatial learning and social behaviour57,58. A separate study examined the role of T cells on brain development59. In adulthood, the bed nucleus of the stria terminalis (BNST), a brain region that is contiguous with the POA and is both highly sexually dimorphic and sexually differentiated by steroid hormones during development60, was masculinized, that is, increased in size, in female mice congenitally lacking T cells. The BNST is necessary for sociosexual behaviours, including territoriality and olfactory investigation, as well as mood-related behaviours. The T cell-deficient females showed a decrease in anxiety behaviour that may or may not be a result of the increased volume of the BNST. Social and sexual behaviours have not been investigated in these animals to date but certainly warrant future inquiry. Overall, this study suggests T cells are active participants in brain feminization, but the mechanism by which they influence physiology in the CNS remains unclear.

Mast cells are granulocytes of myeloid lineage derived from the bone marrow and are distributed throughout the body, including the brain61,62. Some mast cells synthesize gonadotropin-releasing hormone63, a peptide crucial for the control of the anterior pituitary and gonadal function. Mast cell numbers increase in specific brain regions in both male and female rodents when exposed to sexually receptive stimuli of the other sex63,64. Furthermore, the transcriptome of peripheral mast cells is markedly different in males and females65. Mast cells have not yet been implicated directly in brain sexual differentiation, but, given their involvement in reproductive responses, the potential for such a role is high.

Neuroepigenetics and brain sex differences. The term 'epigenetics' describes modifications (epigenetic marks) to the DNA or associated histones that do not involve alterations of nucleotide sequences but have an impact on levels of gene expression in the long term. Canonical epigenetic modifications are methylation of the 5′ carbon of cytosines that are proximal to guanines (CpG) and post-translational modifications of histone tails, the most prevalent of which are acetylation and methylation. Epigenetic mechanisms also include additional sites of DNA methylation and other modifications of histones along with the actions of non-coding RNAs and the generation of a constellation of epigenetic marks that inactivate the X chromosome. The observation that epigenetic modifications in the nervous system happen rapidly and are reversible within a lifespan has led to a re-synthesis of the traditional view of cell fate determination and is aptly named neuroepigenetics66.

A neuroepigenetic contribution to sex differences is at once obvious and mysterious. For example, every cell in the female nervous system has one epigenetically inactivated X chromosome, but how that contributes to functional sex differences remains obscure. As mentioned above, it is obvious that sex chromosome composition is a contributing variable to sex differences in the brain16,67, but it is not clear whether X-chromosome genes that escape X inactivation in females drive these differences or whether the effect is due to the heterochromatic 'sink' that is created when large amounts of epigenetic regulatory proteins are required to maintain silencing of the second X chromosome: that is, the reduction in available epigenetic modifiers in cells with two X chromosomes may dilute the capacity for other forms of epigenetic regulation. It is also important not to forget the potential direct effects of Y chromosome genes (see above).

When contemplating an early life programming event that only manifests in adulthood, such as hormone-mediated sexual differentiation, it seems to be obvious that some form of cellular 'memory' must be established. Steroids bind to nuclear transcription factors (which interact directly with DNA), as well as to larger transcriptional complexes that include histone-modifying enzymes68,69, thereby increasing the likelihood that epigenetics is involved. Indeed the early life programming scenario outlined above predicts that masculinization is driven by direct transcriptional regulation of a set of masculinizing genes that are turned on early (and possibly continuously), thereby differentiating the male brain. There are hints that this is true, to at least some degree, in the developing rat amygdala. Male adult rats have more vasopressin mRNA in their amygdala than females, a sex difference that is determined developmentally by higher androgen levels in males70,71. It has been shown that an experimentally induced transient reduction in the levels of the DNA methyl-binding protein methyl-CpG-binding protein 2 (MeCP2) during the sensitive period for sexual differentiation in males can permanently reduce amygdala vasopressin expression to the level of females, suggesting that an epigenetic modification (DNA methylation and the subsequent binding of MeCP2) establishes and maintains the sex difference in amygdala vasopressin levels72 (Fig. 4). The amygdala is a site of multiple neuroanatomical sex differences and is central to the control of multiple behaviours that differ in males and females, including juvenile social play (which is displayed at higher levels by males across a wide range of species)73.

Figure 4: Epigenetic sources of brain sex differences. a | Several crucial epigenetic mediators are X chromosome and Y chromosome linked. These include the histone lysine demethylases KDM6A (also known as UTX) and KDM5C, which escape X inactivation and are thus expressed at higher levels in the female brain115,116. The Y-linked homologue of KDM6A, KDM6C (also known as UTY), is expressed at higher levels in the male brain116. Sex-specific expression of epigenetic modifiers such as these has the potential to establish widespread sex differences in the chromatin landscape and gene expression and thus to drive structural and functional sex differences in the brain. X-linked chromatin-binding proteins, such as methyl-CpG-binding protein 2 (MeCP2), have also been shown to be important for establishment of brain sex differences. b | Several epigenetic mediators are regulated by sex-specific gonadal hormones. For example, male gonadal hormones reduce the expression of MeCP2 in the amygdala72, reduce DNA methyltransferase (DNMT) activity and methylation genome-wide in the preoptic area (POA)79, and alter methylation on specific promoters related to brain masculinization such as the oestrogen and progesterone receptors74,75. Hormonal modulation at the level of histone methylation and acetylation has also been demonstrated in the POA and bed nucleus of the stria terminalis (BNST), potentially mediating both active and repressive chromatin states80,83. HDACs, histone deacetylases. PowerPoint slide Full size image

Epigenetic regulation of vasopressin and MeCP2 expression is presumed to be downstream of steroid hormone action, but what about regulation of the steroid receptors themselves? Direct modulation of these transcription factor genes should provide a wide ranging and enduring regulation of hormonal responsiveness in adulthood. There have been studies of promoter methylation of both isoforms of the oestrogen receptor and the progesterone receptor in rats74,75. However, there is little agreement about the existence, direction and magnitude of any sex differences in promoter methylation76,77. In the few instances in which more than one time point was measured, sex-specific epigenetic patterns established early in life were replaced by different but still sex-specific patterns later in life75. This was most dramatically seen in a genome-wide analysis of genomic areas that are highly methylated. When comparing male mice, female mice and female mice treated with testosterone at birth to induce masculinization, the investigators found relatively few genes for which methylation levels were different in newborn males and females or that were modified by testosterone treatment of females. But, when animals treated the same way were assessed in adulthood, there were hundreds of genes that were differentially methylated by the testosterone treatment78. Among the challenges to epigenetic research is the inability to comprehensively assess DNA methylation and histone modifications at the same time on the same gene, and — even more importantly — to do so retrospectively. Thus, when an epigenetic modification is observed, it is impossible to know if it happened long ago and endured until now or was placed there just yesterday. Instead, we are limited to a single snapshot in time of a single end point. Nevertheless, these studies suggest that the epigenetic impacts of early life hormone exposure are enduring, but dynamic, generating an epigenetic 'echo' that grows and distorts across the lifespan but retains aspects of its original form.

As noted above, steroid-mediated transcription is an obvious means by which males could be differentiated from females. In addition, a recent study79 showed that DNA methyltransferase (DNMT) activity in the POA of female rats was higher than that in males and that treating females with a masculinizing dose of steroid reduced enzymatic activity to that of males. This sex difference lasted for only the first few days after birth. DNA isolated from the female mPOA had more global methylation and a higher number of 100% methylated CpG sites than that isolated from both males and masculinized females, consistent with higher DNMT activity. But is this differential methylation important? RNA-sequencing analyses of the transcriptome from neonatal males and females with and without treatment that reduces DNA methylation was employed to explore this question. As expected, reducing DNA methylation increased the number of genes upregulated in females more than it did in males. More interestingly, when the transcriptome of males and females was directly compared, a surprisingly small number of genes (70) were differentially expressed. Furthermore, about half were expressed at higher levels in males, whereas half were higher in females. This is surprising because a scenario in which steroid receptor-induced transcription differentiates males from females would predict increased gene expression in males. Moreover, most sex differences in differential gene expression were reversed in animals in which DNA methylation was reduced, confirming that epigenetics was the basis for the sex-specific transcriptomes. Further proof was found in the reversal of sex differences in mPOA neuron synaptic density and adult mating behaviour in females subject to reduced DNA methylation as neonates. The mechanism by which steroids reduce DNMT activity is not known, but the capacity is lost by the end of the first week of life, coincident with the closing of the critical period. However, if females are treated with a demethylating agent at the normal end of the critical period, the critical period remains open and females are masculinized. Thus, DNA methylation seems to be essential to both the initiation and maintenance of sexual differentiation of the mPOA.

In contrast to the effects of DNA methylation (which prevent masculinization), histone deacetylation-mediated repression of gene expression is required for masculinization80. This is again surprising, as DNA methylation and histone deacetylation usually work in concert to suppress gene expression. Even more interestingly, there is evidence that these two opposing forces are at work in the same general brain region. As described above, the BNST is larger in males than in females because more cells die in females. This sex difference can be reversed by giving females a masculinizing dose of steroid during the critical period, indicating that steroids support survival60. It takes several days for the pro-survival effects of steroids to manifest, suggesting that an epigenetic change is induced by the steroids that then protects the cells from a subsequent apoptotic programme. Treatment of neonatal mouse pups with a histone deacetylase (HDAC) inhibitor increased histone acetylation and prevented the masculinization of the BNST, suggesting that pro-death genes that are normally suppressed in males were activated81. A functional impact of neonatal HDAC inhibition is evident in the impaired sexual ability of adult males80, an effect that is attributed at least in part to dysregulation of the Cyp19a1 gene, which encodes the enzyme aromatase, responsible for converting testosterone to oestradiol.

There are two approaches to discover sex differences in the epigenome. One is the use of broad based surveys that assay most or all potential targets. The second approach is to focus on specific candidate genes or epigenetic marks. An advantage of broad survey approaches is that intragenic regions, promoters, regulatory elements and reading frames of genes are all included in the analyses. Whole-genome bisulfite sequencing analysis of the neonatal POA demonstrated that most sex differences in DNA methylation levels are in the intergenic region, the functional significance of which remains a mystery79. A more focused view was gained by analysing an epigenetic mark known to cluster at transcription start sites, histone H3 lysine 4 trimethylation (H3K4me3), which is generally permissive of gene expression82. Chromatin immunoprecipiation followed by sequencing (CHIP–seq) established that most genes of males and females exhibited the same number of H3K4me3 marks but that ∼200 genes differed in the number of H3K4me3 marks carried (most of them being higher in females)83. Thirteen genes were confirmed by quantitative polymerase chain reaction to be differentially expressed at the moment of assay, and two of these were located on the X chromosome, where they escape X inactivation. Together, these findings highlight the importance of considering the 'sexome' — the constellation of gene expression changes related to sex — rather than sex differences in individual genes84.

Intersection of neuroimmunity and neuroepigenetics. Given the findings outlined above, it is natural to question whether there is crosstalk between neuroimmune and neuroepigenetic mechanisms of sex differentiation. The X chromosome has the highest concentration of immune-related genes of any chromosome85. Thus, X-chromosome inactivation in females is crucial to keeping overexpression of these immune-related genes repressed, and dysregulated X-chromosome silencing is proposed as a possible reason that females suffer more auto-inflammatory conditions85.

Gene ontology analyses of the genes regulated by DNA methylation in the POA showed an almost fourfold enrichment of immune-related genes in female rat pups treated with a DNMT inhibitor (Fig. 3). These included cytokines, chemokines and their receptors, microglia-specific and/or macrophage-specific genes, genes related to phagocytosis, complement-related proteins and antigen presentation and receptor genes79. There were also many gene isoforms differentially expressed in males and females. Among these were many immune-related genes, including intriguingly a mast cell protease79, implicating this non-neuronal cell type in the process of sexual differentiation.

A complementary possibility is that immunocompetent cells possess sexually differentiated epigenetic tags. Exposure to early life stress via neonatal handling of rat pups induces microglial-specific decreases in methylation of the anti-inflammatory immune gene interleukin-10, resulting in long-term increases in cytokine levels and subsequent resilience to morphine-induced conditioned place preference86. Although no sex differences were reported in this study, future exploration of immune cell-specific sex differences in the methylome or histone modifications would answer the question of whether the epigenetic regulation of these cells contributes to or maintains sexual differentiation of the brain.