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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Pflugers Arch. Author manuscript; available in PMC 2014 May 1.
Published in final edited form as:
PMCID: PMC3654067
NIHMSID: NIHMS456818

Gender Differences in Neurodevelopment and Epigenetics

Summary

The concept that the brain differs in make-up between males and females is not new. For example, it is well-established that anatomists in the nineteenth century found sex differences in human brain weight. The importance of sex differences in the organization of the brain cannot be overstated as they may directly affect cognitive functions, such as verbal skills and visio-spatial tasks in a sex-dependent fashion. Moreover, the incidence of neurological and psychiatric diseases is also highly dependent on sex. These clinical observations reiterate the importance that gender must be taken into account as a relevant possible contributing factor in order to understand the pathogenesis of neurological and psychiatric disorders. Gender-dependent differentiation of the brain has been detected at every levels of organization: morphological, neurochemical, and functional, and have been shown to be primarily controlled by sex differences in gonadal steroid hormone levels during perinatal development. In this review, we discuss how the gonadal steroid hormone testosterone and its metabolites, affect downstream signaling cascades, including gonadal steroid receptor activation, and epigenetic events in order to differentiate the brain in a gender-dependent fashion.

Introduction

The importance of gonadal steroid hormones for behavioral regulation was shown early on in Berthold’s experiments conducted in 1849 [23], who showed that male-typical behaviors in roosters, such as crowing, aggression, and male sexual behavior, disappeared after castration, whereas replacement of the missing gonads restored the male-typical behaviors. However, it was not until Phoenix and colleagues (1959) who demonstrated that testosterone administration in pregnant guinea pigs caused the female offspring to display male sexual behavior as adults, that the idea of gonadal steroid hormone-dependent sexual differentiation of the brain itself was put forward [133]. More importantly, these studies led to the pivotal hypothesis that gonadal steroid hormones actions on the mammalian brain can be categorized as organizational versus activational. In general, organizational effects of gonadal steroid hormones during perinatal development are thought to be permanent, whereas activational effects are transient, and mainly restricted to adulthood.

In the early 1970’s it was finally confirmed that the central nervous system (CNS) itself contains specific regions that differ between males and females at the neuronal and synaptic level [32,136]. A further landmark discovery was that the medial preoptic nucleus (MPN) is 2–6 larger in males than in females [64]. These studies also confirmed the organizational versus activational hypothesis at the level of the brain, as changes in gonadal steroid hormone levels had no effect on the size of the adult rat MPN [64]. In contrast, perinatal castration of male rat pups resulted in a female-sized MPN, whereas neonatal female rats injected with testosterone showed a malesized MPN in adulthood [64]. These initial reports and others solidified the idea that the vertebrate brain is organized in a sex-dependent fashion under the control of perinatal gonadal steroid hormones (i.e., testosterone) [10,114,21]. In this review, we will discuss the role(s) of the gonadal steroid hormone system, and its interaction with epigenetic events to cause brain sexual differentiation.

Sex Determination

The fundamental fact is that brain sexual differentiation cannot begin without the initiation of normal sex determination of the fetal gonads under influence of genetic sex. In early fetal development, the gonads (i.e., primary source of plasma gonadal steroid hormones) do not differ between males and females, and have therefore been called bi-potential gonads. Differentiation of the male fetal gonads into testes is in essence controlled by the sex determining region-Y chromosome (SRY) protein, which is encoded by the sex determining region-Y chromosome (Sry) gene located on the short arm of the Y chromosome [153,85,22,92]. The SRY protein, a member of the SOX protein family of DNA transcription factors, differentiates pre-Sertoli cells to Sertoli cells in the testis cords [134]. In turn, Sertoli cells secrete Desert Hedgehog protein, which causes the differentiation and expansion of the nearby located testosterone-synthesizing Leydig cells [177]. In the absence of Sry, as is the case in the female fetus, the bi-potential gonads differentiate into ovaries.

Inherent to testes formation, overall testosterone levels are higher in males than in females during mammalian fetal and perinatal development [42,52,171]. Interestingly, circulating levels of testosterone are markedly increased in males at specific time points in development. In male perinatal rats, circulating testosterone levels peak around embryonic day 18 and 19, which is followed by a smaller peak in testosterone levels just hours after birth [41,42,171]. Studies in rats showed that this sex difference in circulating levels of testosterone only has a small developmental window of opportunity to cause the organizational (permanent) sex-dependent changes in mammalian brain morphology and function. In rats, this so-called “critical period”, in which testosterone can program permanent sex-dependent central changes to the morphology and neurochemical phenotype of the brain has been pinpointed to start between embryonic day 18 and approximately end 10 days after birth, which coincides with the perinatal sex differences in circulating levels of testosterone in the rat [45]. In humans, circulating testosterone levels in the male fetus are also much higher than in the female fetus. Specifically, testosterone production in the male human fetus start and rises during the second month of the first trimester and reach its highest levels in the second trimester, which are maintained until late gestation (i.e., third trimester) when testosterone are only slightly higher in males than in females at the time of birth. In the first neonatal year, a second surge in testosterone plasma levels has been observed, which subsides until the onset of puberty [1,41]. Therefore, the sex difference in testosterone levels is, as in rodents, the primary signal that initiates human brain sexual differentiation.

Observations of Morphological and Neurochemical Brain Sex Differences

The concept that the brain differs in make-up between males and females is, of course, not new. For example, it is well-established that anatomists in the nineteenth century found sex differences in human brain weight [157]. These early weight measures are in line with more recent studies showing that the total volume and number of neurons of the human neocortex is about 10–15% larger in men than in women [129].

The importance of sex differences in the organization of the brain cannot be overstated as they may directly affect cognitive functions, such as verbal skills and visio-spatial tasks in a gender-dependent fashion see for reviews [21,155,159]. Moreover, the incidence of human neurological and psychiatric disorders is highly dependent on gender. For instance, the incidence of anorexia nervosa and bulimia is much higher in women than in men, whereas the opposite is true for dyslexia, sleep apnea and Gilles de la Tourette [159]. The incidence of gender identity disorders depends on sex as well. For instance, in the Netherlands, there are about three times fewer male-to-female transsexuals than female-to-male transsexuals [166]. These clinical observations reiterate the importance that sex must be taken into account as an important and relevant contributing factor when considering the possible mechanisms that result in the onset of neurological and psychiatric disorders.

Sex differences in the CNS have been found at every level of brain organization: brain area volume, cell number, cell cytoarchitecture, cell activity, synaptic connectivity and neurochemical content, and in a large number of organisms, such as fish, lizards, songbirds, rodents and primates including humans [15,21,19]. For example, the thoracolumbar intermediolateral nucleus of the spinal cord in male cats was shown to contain more sympathetic motoneurons than in the female cat [32]. In the same year, Raisman and Field (1971) reported that the preoptic area in rats contained more synapses from non-amygdaloid origin in females than in males in adulthood [136]. This was of particular interest, because they also demonstrated that a single injection with testosterone in newborn female rats decreased the number of synaptic contacts, whereas neonatal castration increased the number of synaptic contacts [136,137]. Similar sex differences in synaptic wiring were also found in other hypothalamic and limbic regions of the rodent brain [120,106]. Soon after the findings of Raisman and Field, much more dramatic gonadal steroid-responsive sex differences were found in other vertebrate species, such as in song birds. For instance, song-regulating brain areas in canaries and zebra finches are 6 times larger in males than in females [122]. In the next sections, we will highlight the most prominent sexually dimorphic areas that have been discovered to date in the preoptic/hypothalamic and limbic brain regions.

Medial Preoptic Nucleus

The rat sexually dimorphic medial preoptic nucleus (MPN) is anatomically larger and contains more cells in males than in females [150,64,125,30], and has been found in the preoptic area of the ferret, gerbil, guinea pig and hamster brain [27,40,72,161]. The divergence of MPN between males and females becomes measurable around birth and is completed within the first 10 days after birth [45,64,82,39]. Lesion studies in rats showed that the MPN may be involved in the regulation of sexual behavior [8,46]. The first study to report that the human preoptic/hypothalamic region also contains a sexually dimorphic nucleus was published by Swaab and Fliers in 1985, who showed that the human interstitial nucleus of the anterior hypothalamus 1 (INAH-1) is about two times larger, and contains more cells in young adult men than in young adult women [156,158,74]. Since then, other studies reported additional sexually dimorphic nuclei in the human preoptic/hypothalamic region. Notably, INAH-2 and INAH-3 were shown to be also larger in men than in women [5,99,31].

The MPN is also sexually dimorphic in terms of neurochemistry. For example, the male rodent MPN contains more galanin expressing cells and is more heavily innervated with serotonin fibers than the female MPN [28,150]. On the over hand, the female’s rat MPN is heavily interconnected with brain areas that are involved in gonadotropin release, and female copulatory behavior, such as the anteroventral periventricular nucleus (AVPv), bed nucleus of the stria terminalis (BST), amygdaloid nucleus and ventral medial hypothalamus (VMH). Therefore, it may also contribute significantly to the regulation of female sexual behavior [66,77,50]. In humans, it is at present not known whether the MPN is involved in similar behaviors. The presence of morphological and neurochemical sex differences in the MPN is indicative for the possibility of sexual differences in brain functions. On the other hand, it has been proposed that brain sexual differentiation may be an adaptation in order for the male and female brain to function similarly [50].

Bed Nucleus of the Stria Terminalis

This limbic forebrain region is part of a continuum of columns of sublenticular cell groups traversing the basal forebrain and cell groups that accompany the stria terminalis, which is developmentally closely related to the amygdaloid nucleus [3,144]. The rodent principal nucleus of the BST (BSTp) is about two times larger and contains more cells in males than in females [72,51,67,88,39], whereas the opposite is true for the lateral anterior BST and medial anterior BST [51, 67]. Similar to the MPN, the sexual differentiation of the BSTp occurs during the first 10 days after birth [39,51,67]. The human BST also contains several subdivisions that differ in volume between men and women. Both the darkly staining posteromedial component of the BST and the central nucleus of the BST (BSTc) are larger in men than in women in adulthood [180,4,5,36], both of which have been shown to be observable after ten years of age or in adulthood only, respectively [36,4]. These data demonstrate that the period of organizational sexual differentiation of the human brain occur over a much more protracted period of time than found in the rodent brain. Consequently, illustrating the importance that data from animal studies must complement with human brain studies in order to understand the sexual differentiation of the human brain itself.

The rodent and human BST also exhibits prominent sex differences in neurochemistry. For example, the number of vasopressin, substance P, and cholecystokinin producing cells in the rat BST is larger in male than in female rats [47,48,50,113]. Relatively, recent studies showed that the human male BSTc in contains more vasoactive intestinal polypeptide fibers and somatostatin neuronal staining than in the female BSTc [94,180,36]. These data have helped us to better understand the inherent functions of this region in the human brain. Functionally, the BST has been shown to be involved in the regulation of a number of behaviors, such as reproduction, aggression, addictions, parental behavior and stress [59,57,2,69,101,163, 169, 20]. In humans, the BSTc has been shown to develop differentially in people with a gender identity disorder called transsexuality, in which subjects express the strong feeling of being born in the wrong body. Indeed, these studies showed that the size of the BSTc in male-to-female transsexuals is similar to that found in control women, whereas in the only female-to-male transsexual studied so far the BSTc size was similar to that found in men [94,180]. Therefore, it might be that the human BSTc has a role in human gender identity, however, it must be noted that this is merely correlational, and require far more studies to clearly elucidate the functions of the human BSTc.

Anteroventral Periventricular Nucleus

Some brain regions are larger in females than in males. For example, the rat, mouse, hamster and gerbil AVPv was shown to be larger in females than in males [27,121], and is heavily interconnected with other brain areas, such as the BST, organum vasculosum laminae terminalis (OVLT) and arcuate nucleus [77].

The rat AVPv also contains neurochemical sex differences that are biased in a female direction. For instance, more dopaminergic cells are found in the female AVPv than in the male AVPv [66]. Interestingly, the AVPv is more heavily innervated by the BST and MPN in males than in females [77]. The ascending AVPv projections terminate, in part, close to the OVLT where gonadotropin-releasing hormone containing cells have been observed. Because descending AVPv fibers terminate in the periventricular nucleus and arcuate nucleus, it has been suggested that the AVPv may function as a nodal point in the regulation of gonadotropin secretion [174,56]. At present, it is not known whether the human brain contains a similar analogous AVPv brain region.

Ventromedial Hypothalamic Nucleus

The VMH in the rat brain is another prominent region that is larger in males than in females, which is first detectable ten days after birth [105,36]. Further analysis showed that the number of synaptic contacts is higher in males than in females [106,135]. Tracing experiments showed that the VMH projects to many sexually dimorphic and non-sexually dimorphic brain areas, such as the MPN, lateral septum, BST and paraventricular nucleus [33]. Presently, it is unknown whether the VMH in the human brain is sexually dimorphic in volume. However, studies showed that metabolic activity in the human VMH seemed to be higher in young women than in young men. As the metabolic activity of the VMH appears to increase with age in men, it has been proposed that androgens may inhibit metabolic activity in the VMH [80].

The VMH has been implicated in the regulation of feeding behavior, and also plays a central role in the regulation of male and female reproductive behavior [131,132,123]. For instance, neurons residing in the lateral ventral portion of the VMH have been implicated in the regulation of lordosis after appropriate priming with estradiol and progesterone [26,11]. The VMH in the human brain may be involved in the sexually dimorphic integration of pheromonal input. Positron emission tomography scan studies in humans showed that an androgen-like compound activated the female hypothalamus centering on the VMH, while in males the activation of the hypothalamus by an estrogen-like substance was centered on the PVN and dorsomedial hypothalamus [143].

Testosterone Metabolites Facilitate Neuronal Sexual Differentiation

Evidence from studies investigating the development of sex differences in animals showed that testosterone’s effect on brain sexual differentiation are mediated by its metabolite estradiol, a conversion process facilitated by neuronal aromatase [110,102]. In general, rodent studies showed that perinatal estradiol treatment can reproduce the masculinizing effects of testosterone on brain organization and behavior in gonadectomized animals [19,15,38]. For example, although the neonatal MPN showed the ability to bind testosterone or its metabolites estradiol and dihydrotestosterone (DHT), it was only estradiol treatment in female and castrated male rat pups that was able to increase the size of the MPN [64,81]. Moreover, newborn brain areas that contain anatomical and neurochemical sex differences in adulthood, such as the preoptic area, hypothalamus and limbic system contain high aromatase expression [86,118,142]. Masculinizing effects of testosterone brain development and function were also effectively blocked by estrogen antagonists and aromatase inhibitors [109,110,168,1618].

On the other hand, perinatal treatment with the non-aromatizable androgenic metabolite of testosterone, DHT, showed little to no ability to affect the MPN in a testosterone-like manner [15,21,19]. However, DHT’s role in sexual differentiation cannot be fully discounted. Indeed, the sexually dimorphic vasopressinergic system in the BST (m > f) requires the presence of both estradiol and DHT to be fully maintained [47,48,128]. The ability of DHT to partially maintain this particular sexual dimorphism may be due to its intracellular conversion to its metabolite 3β-diol, which has a relatively high affinity for estrogen receptors (ER) [128]. Although DHT’s role cannot be discounted completely during brain sexual differentiation, it has been shown beyond doubt that the testosterone causes sex-dependent brain development, primarily through its intracellular aromatase-dependent conversion to estradiol.

Estrogen Receptors

Actions of gonadal steroid hormones are classically described as being mediated through their specific receptors, which are part of a large family of nuclear steroid hormone receptors [60,141]. Despite the large diversity in steroid hormone receptors, they are highly conserved transcription factors, which have variations of a general basic modular organization. They contain a ligand binding domain, DNA binding domain and transactivation domain. The ligand - and DNA binding domain of the steroid receptor conveys specificity for ligands and binding to hormone response elements on DNA, respectively. Classical activation of steroid hormone receptors through a specific ligand(s) causes homo- or heterodimerization and translocation of the ligand-receptor complex to the nucleus, which in turn will interact through the DNA binding domain with its specific hormone response element on DNA in order to regulate gene transcription. Classical activation of steroid receptors is not the solitary mode of cellular signaling, but may include the activation of so-called non-genomic second messenger pathways. Moreover, several studies showed that many of these steroid hormone receptors, such as ER’s are expressed as mRNA splicing variants or membrane receptors throughout the body, including the brain, and has specific transcriptional and epigenetic consequences [37,116,147,172,173,58, 68, 53, 79].

Estrogen receptors were the first nuclear receptors to be discovered [162]. Several laboratories have found two subtypes of ERs in the mouse, rat, and human brain [95,115,164], which have been referred to as ERα and ERβ. The two receptors are encoded by separate genes that are located on different chromosomes [95]. ERα and ERβ expression in the rat brain shows considerable overlap in brain areas, such as the MPN, BST, amygdala, lateral habenula, and midbrain regions [149,146]. On the other hand, the VMH and subfornical organ contain almost exclusively ERα, while neurons of the olfactory bulb, supraoptic nucleus, PVN, suprachiasmatic nucleus, zona incerta, ventral tegmental area, cerebellum, and pineal gland among other areas are exclusively ERβ positive [146].

ERα and ERβ distribution has also been studied in the human brain, and is largely consistent with the results from animal studies. Examples of human brain areas that contain ERα and ERβ expressing cells are the diagonal band of Broca, nucleus basalis of Meynert, interstitial nucleus of the anterior hypothalamus, BST, amygdala, PVN, supraoptic nucleus, arcuate nucleus, hippocampus, and cerebral cortex [54,63,127,70]. Postmortem human brain studies further showed that that both ERα and ERβ differ in expression in the INAH-1 and BST between men and women in adulthood [93].

Expression of ERs in rodents and primates is regulated by circulating levels of gonadal steroid hormones. Indeed, removal of circulating levels of testosterone or its metabolite estradiol increases expression of ERα in the rat sexually differentiated brain region, such as the AVPv, MPN, BST, and VMH [151,100,55]. ERβ expression in the PVN of the rat brain was shown to be decreased by estradiol, while estradiol had no effect on ERβ expression in the MPN or BST [130,154]. On the other hand, estradiol seems to up-regulate ERβ expression in the arcuate nucleus of the rat brain [126]. In humans, ERβ expression may be affected by circulating levels of gonadal steroid hormones. For example, ERβ expression was higher in the SON of young women as compared to postmenopausal women, while ERα expression was lower in young women than in postmenopausal women [78]. Together these data are indicative of a gonadal steroid hormone system that is dynamic in nature and responsive to ever changing central and peripheral cues.

How Do Gonadal Steroid Hormones Cause Sexual Differentiation?

Gonadal steroid hormones acting through their specific receptors regulate sexual differentiation of the mammalian brain by affecting one or more of four major developmental processes: neurogenesis, neuronal migration, apoptosis and/or differentiation of cell phenotype. To date no studies have found strong evidence for the direct involvement of gonadal steroid hormone-dependent neurogenesis or neuronal migration in the sexually dimorphic organization of the MPN or BST [83,84,82].

Apoptosis is a highly regulated distinct form of cell death that histologically is characterized by shrinkage of cell cytoplasm, condensation of chromatin, blebbing of cell membrane and formation of membrane bound apoptotic bodies containing intact organelles and condensed chromatin [89]. Biochemical analysis indicated that DNA fragmentation during apoptosis occurs in multiples of 180–200 base pairs [9]. Many studies showed that apoptosis is a widespread phenomenon during early brain development, which is required to remove brain cells that do not migrate, differentiate and/or form appropriate neuronal circuits in a given developmental time period [43,124]. Examples of apoptotic cell death during brain development have been described early on, for instance in the rodent striatum, hippocampus, amygdala and cerebellum [111,87]. The presence of apoptotic cell death has also been documented in a number of studies investigating fetal human brain [35,152,138].

Apoptosis has been observed during sexual differentiation of a number of regions in the preoptic area in the rat brain. For example, the incidence of apoptosis in the perinatal AVPv was higher in males than in females, while the incidence of apoptosis in the postnatal MPN was higher in females than in males [6,7,44,39]. Moreover, these studies showed that testosterone or its metabolite estradiol increased the incidence of apoptosis in the perinatal rat AVPv, whereas testosterone or its metabolite estradiol decreased the incidence of apoptosis in the developing rat MPN [39,44]. These studies suggest that gonadal steroid hormones control the sexual differentiation of the vertebrate brain through the context-dependent induction or prevention of apoptotic cell death.

The importance of apoptosis during sexual differentiation of the BST was inferred from earlier studies. One the most prominent cytoarchitectural sex difference in the rat is the BSTp, which is larger and contains more cells in males than in females [51,67,71,39]. Sexual differentiation of the BSTp is controlled by the sex difference in early circulating levels of testosterone [51,67,39]. Our studies showed that the incidence of apoptosis in the BSTp during the first postnatal week was much higher in females than in males. Moreover, the size of the BSTp became larger in males than in females only after the sex difference in apoptosis [39]. In addition, the incidence of apoptosis was higher in animals devoid of testosterone than in animals with testosterone [39]. These results strongly suggest that sex differences in developmental apoptosis are prerequisite for the gonadal steroid hormone-dependent sexual differentiation of the BSTp in the rat brain.

Gonadal Steroid Hormone Regulation of Apoptosis

Although testosterone was shown to be effective in protecting BST and MPN cells against apoptosis; this effect is facilitated by its estrogenic metabolite acting on ERs. Estrogen-bound ERs form homodimers or heterodimers and translocate to the cell nucleus to associate with specific estrogen response elements (EREs) on DNA [91,148,49], which are palindromic enhancer sequences located in promoter regions to modulate the transcriptional activity of genes involved during poptosis. For example, members of the Bcl-2 gene family, including the anti apoptotic Bcl-2 and Bcl-XL have the putative EREs in their promoter regions supporting the idea that the presence of testosterone-derived estradiol may directly modulate the transcriptional activity of genes that favor cell survival [49,175]. Indeed, estradiol increased Bcl-2 and Bcl-XL expression in neuronal cell lines [175,49,108], while decreasing the expression of Bad mRNA, a proapoptotic Bcl-2 family member [107]. Estrogens also decrease the expression of cellular factors, such as Bnip-2 mRNA which in turn down-regulate Bcl-2 expression [76]. Conversely, estradiol removal increased mRNA expression of two proteolytic so-called initiator Caspases (i.e., 1 and 2) in chick oviduct studies, while at the same time activating the executioner proenzymes, caspase-3 and caspase-6 [75]. More recently, the sex difference in BSTp apoptosis was shown to be dependent on Bax function [39,104]. Interestingly, testosterone’s ability to prevent BSTp apoptosis could be recapitulated with ER and ER selective agonists [73]. Together these studies suggest that estrogen-bound ERs oppose apoptosis by genomically acting on the molecular mechanisms that control cell survival.

Gonadal steroid hormones may regulate cell survival by acting on the transcription level of neurotrophic factors. Indeed, the gene encoding for brain-derived neurotrophic factor (BDNF) contains a putative ERE. Moreover, estrogen increased mRNA levels of BDNF in the rat cerebral cortex and olfactory bulb [147]. Similarly, androgens rescue motoneurons in the spinal nucleus of the bulbocavernosus androgens is facilitated by ciliary neurotrophic factor (CNTF) expressed in the perineal muscles, which act on CNTF receptors located on the motoneurons [173]. These studies indicate that gonadal steroid hormone regulate cell survival not only directly, by targeting the expression of specific components of the apoptotic cell death mechanisms, but also indirectly by modulating the expression of neurotrophic factors.

In vitro studies strongly suggest that gonadal steroid hormones can also regulate apoptosis through non-genomic pathways. In particular, estrogen-bound ERs interact directly with phosphatidylinositisol-3 kinase (PI3K) through protein:protein binding, which in turn phosphorylates the downstream effector AKT to rescue cortical neurons [75,76]. Estrogen-dependent rescue through the PI-3K/AKT pathway was prevented by ICI 182,780, a selective ER antagonist or LY 294002, a selective PI3K inhibitor [76]. In vitro studies also showed that PI3K/AKT phosphorylation of the AR in similar prostate cancer cells may inhibit apoptosis [145]. In addition, PI3K/AKT was shown to increase AR mRNA expression [104]. These studies suggest that apoptosis-dependent sexual differentiation of the vertebrate brain may be mediated by the PI3K/AKT signaling pathway.

Epigenetics: A Further Layer of Complexity

Research from the last few decades has uncovered a wealth of information about the vertebrate brain sexual differentiation. More recent studies have focused on the field of epigenetics, which has been defined in a multitude ways, ranging from those that include a heritable component in gene function to those simply state that epigenetic changes are molecular events that remodel chromatin without altering the underlying DNA code [24]. Much of our fundamental knowledge is derived from studies examining the epigenetic regulation of steroid hormone receptors, in particular glucocorticoid receptors and ER’s [170,34,97]. In this review, we minimally defined epigenetics as a change to chromatin without changing the underlying DNA code, similar to Adrian Bird’s definition [24]. Therefore, the heritability of epigenetic remodeling is irrelevant for this definition. Furthermore, it should be noted that an epigenetic change does not always result in immediate alteration of gene transcription, but it may alter the genes response to future signaling events. This inherently complicates our task to relate epigenetic remodeling of DNA to disease and mental health, because epigenetic changes of a particular gene(s) may directly participate in neurological pathogenesis, or merely alter the probability or severity of a disorder in response to additional gene or environmental challenges.

Steroid Receptor Co-Regulators

Most of what we know about early gonadal steroid hormone action in the brain is due to its activational effects (i.e., increase) on gene transcription, which generally involves the recruitment of coactivator complexes, such as steroid receptor coactivator-1 (SRC-1) and cAMP response element binding protein-binding protein (CPB) [160]. These coactivator complexes can directly alter histone acetylation through their own histone acetyltransferase (HAT) activity or indirectly through their association with other complexes exhibiting HAT activity. The primary consequence of histone acetylation is to reduce the affinity between histones, resulting in the unwinding of chromatin structure, and consequently in heightened gene transcription efficiency.

Both SRC-1 and CBP have been shown to be critical for brain sexual differentiation. Indeed, reduced SRC-1 expression in the developing hypothalamus prevented sexual differentiation of adult sexual behavior in males, and the androgenization of sexual behavior in females [13]. These data indicate that gonadal steroid hormone actions in the developing brain require the increase of histone acetylation and unwinding of the chromatin structure [12]. This idea is supported by recent studies showing that males have higher levels of acetylated histone H3 as compared to females during neonatal brain development [165].

Recent findings indicate that some molecules associated with gene repression are also important in mediating brain sexual differentiation. For instance, DNA methylation can initiate a cascade of events leading to gene repression, and occurs when a methyl group is attached to cytosine within a 5’-CpN-3’ dinucleotide, an enzymatic reaction catalyzed by DNA cytosine-5-methyltransferases (DNMTs) primarily at CpG sites [65,139]. The act of CpG site methylation in itself typically does not have a direct impact on gene transcription rates. Rather inhibition of gene transcription occurs when methyl-CpG-binding proteins (MBPs) are bound bind to the methylated DNA, which recruits co-repressor complexes as opposed to coactivators. The histones deacylatase (HDAC) activity from these co-repressor complexes will deacetylate histones resulting in chromatin condensation, and consequently gene repression [179,25,90].

An very exciting line of research showed that relatively subtle changes in maternal care resulted in the significant modification of the DNA methylation patterns of steroid hormone receptor genes, such as ER and glucorticoid receptors, within the developing offspring brain [170,34]. These data illustrate the plasticity of DNA methylation patterning within the developing postnatal brain. This is further reiterated in our recent assessment of the tactile components of maternal care on ERα expression, and its promoter methylation by simulating maternal grooming (SMG).

During brain development and in adulthood ERα expression within the rodent MPN is higher in males than in females [55,176,178,29,98,103]. In line with these observations, we found that the 5’ flanking region of ERα exon 1b promoter region in males had higher levels of CpG methylation than females, which may be the underlying cause of reduced ERα expression [97]. Moreover, in our studies we were able to use SMG to not only decrease ERα expression, but also increase ERα promoter methylation in females to male-like levels [97]. These data indicate that programming of a critical signaling pathway for brain sexual differentiation, ERα expression, during brain development is not only influenced by internal changes in gonadal steroid hormone environment, but also neonatal social (i.e., external) cues. Interestingly, the programming of ERα expression appears to be associated with gene repression in males. Also intriguing, is that estradiol treatment caused increased ERα promoter methylation in females. Together, these data suggest that sexual differentiation of the brain may require the interplay of both activation and repression of gene expression.

As stated above, DNA methylation does not directly alter gene transcription rates; rather it is methyl-binding proteins binding to methylated CpG sites that lead to gene repression. Therefore, we transiently reduced the expression of methyl CpG binding protein 2 (MeCP2) during amygdala development, and assessed whether this intervention could impact the sexual differentiation of juvenile social play behavior. Remarkably, our studies showed that a relatively subtle reduction in neonatal amygdaloid MeCP2 expression was able to block the masculinization of juvenile social play behavior in males [96,61] without disrupting juvenile sociability. Together these data suggest that the differentiation of the male brain can necessitate increased methylation of some genes during development, and consequently require methyl-binding protein function (i.e. MeCP2) to fully differentiate the male brain. These data further support the idea that some genes need to be repressed for appropriate masculinization of the brain. Further evidence for this concept comes from experiments that disrupt HDAC function during early brain development. Specifically, treatment of neonatal males with valproic acid (HDAC inhibitor) disrupted BST masculinization [117], and was shown to increase histone H3 acetylation. A likely interpretation of these data is that masculinization of the brain requires the inhibition of some genes. Of course, one could then hypothesize that these genes may be involved in feminizing the brain, and therefore need to be turned OFF for masculinization of the male brain to proceed properly.

Epigenetics is a further regulatory layer that can explain how cells with the same genetic material produce different phenotypes [119]. The general consensus is that methylation of cytosine into 5-methylcytosine is relatively stable, and is maintained through DNA replication. However, it was also suggested over 30 years ago that there may be plasticity (i.e."shuffling”) in DNA methylation patterns throughout cellular differentiation [140]. This was recently confirmed in a study that showed that methylation patterns of the estrogen-responsive pS2/TFF1 gene can undergo cyclical changes within minutes to hours [112]. Similar findings have been reported to occur in regulation of the gonadal steroid hormone-dependent vasopressinergic system in the rat BST [14]. Based on these dynamic experiments, it has been conjectured that cyclic pattern of methylation is required for gene transcription, and disrupting this cycle may ultimately block gene transcription. These findings challenge and seriously question the validity of the concept that methylation patterns are always stable, and support the idea that there are both stable pattern and highly dynamic methylation patterns. Previous research has hypothesized that there are gradual shifts in human DNA methylation patterns during ageing. Sometimes these inappropriate shifts in DNA methylation patterns can be observed as aberrant methylation patterns in some cancer cells or in some neurological diseases later in life [167]. Moreover, studies in twins revealed their DNA methylation patterns to be very similar during early childhood, but go on to be very difference in adulthood [62]. These epigenetic studies are in line with this reviews common theme that the cellular and molecular mechanisms of brain sexual differentiation are far from being static, but show a high level of temporal and spatial plasticity.

In Conclusion

The wealth of information that has been generated over the last several decades has revealed that brain sexual differentiation is a highly regulated “event” that occurs throughout life, and has been shown to include a multitude of molecular, cellular and epigenetic mechanisms, which can even be detected through gross measures, such as brain area size or cell number. However, there are still many important uestions that remain to be studied. For instance, how does testosterone cause male-biased cell death in the AVPv, while this is female-biased in the MPN and BST? In other words, what are the molecular mechanisms that underlie and specify region-specific sexual differentiation? Similarly, can sex-dependent epigenetic changes on the DNA during early brain development be reversed in adulthood, and would this cause changes in established behaviors? Notwithstanding the importance of these immediate questions, there is need to include gender in the analysis of the pathogenesis of neurological and psychiatric disorders, especially given the fact many of these disorders are distributed in a gender-dependent fashion. However, what does this mean? Does it mean that gender increases the vulnerability and predisposes the brain to a particular neurological or psychiatric disorder? Or could it be that the cause(s) of a disorder is similar in males and females, but that the pathogenesis process is more or less ‘effective’ in males versus females. To better understand this concept, further analyzes are necessary to examine the true relationship between the sexual differentiation process of human brain and the gender-dependent incidence of neurological and psychiatric disorders.

Footnotes

Disclosure Summary: The authors have nothing to disclose.

References

1. Abramovich DR, Rowe P. Foetal plasma testosterone levels at mid-pregnancy and at term: relationship to foetal sex. The Journal of endocrinology. 1973;56(3):621–622. [PubMed]
2. Albert DJ, Petrovic DM, Walsh ML, Jonik RH. Medial accumbens lesions attenuate testosterone-dependent aggression in male rats. Physiology & behavior. 1989;46(4):625–631. [PubMed]
3. Alheid GF, Beltramino CA, De Olmos JS, Forbes MS, Swanson DJ, Heimer L. The neuronal organization of the supracapsular part of the stria terminalis in the rat: the dorsal component of the extended amygdala. Neuroscience. 1998;84(4):967–996. [PubMed]
4. Allen LS, Gorski RA. Sex difference in the bed nucleus of the stria terminalis of the human brain. The Journal of comparative neurology. 1990;302(4):697–706. [PubMed]
5. Allen LS, Hines M, Shryne JE, Gorski RA. Two sexually dimorphic cell groups in the human brain. The Journal of neuroscience : the official journal of the Society for Neuroscience. 1989;9(2):497–506. [PubMed]
6. Arai Y, Murakami S, Nishizuka M. Androgen enhances neuronal degeneration in the developing preoptic area: apoptosis in the anteroventral periventricular nucleus (AVPvNPOA) Hormones and behavior. 1994;28(4):313–319. [PubMed]
7. Arai Y, Sekine Y, Murakami S. Estrogen and apoptosis in the developing sexually dimorphic preoptic area in female rats. Neuroscience research. 1996;25(4):403–407. [PubMed]
8. Arendash GW, Gorski RA. Effects of discrete lesions of the sexually dimorphic nucleus of the preoptic area or other medial preoptic regions on the sexual behavior of male rats. Brain research bulletin. 1983;10(1):147–154. [PubMed]
9. Arends MJ, Morris RG, Wyllie AH. Apoptosis. The role of the endonuclease. The American journal of pathology. 1990;136(3):593–608. [PubMed]
10. Arnold AP, Gorski RA. Gonadal steroid induction of structural sex differences in the central nervous system. Annual review of neuroscience. 1984;7:413–442. [PubMed]
11. Auger AP, Blaustein JD. Progesterone enhances an estradiol-induced increase in Fos immunoreactivity in localized regions of female rat forebrain. The Journal of neuroscience : the official journal of the Society for Neuroscience. 1995;15(3 Pt 2):2272–2279. [PubMed]
12. Auger AP, Perrot-Sinal TS, Auger CJ, Ekas LA, Tetel MJ, McCarthy MM. Expression of the nuclear receptor coactivator, cAMP response element-binding protein, is sexually dimorphic and modulates sexual differentiation of neonatal rat brain. Endocrinology. 2002;143(8):3009–3016. [PMC free article] [PubMed]
13. Auger AP, Tetel MJ, McCarthy MM. Steroid receptor coactivator-1 (SRC-1) mediates the development of sex-specific brain morphology and behavior. ProcNatlAcadSciUSA. 2000;97(13):7551–7555. [PubMed]
14. Auger CJ, Coss D, Auger AP, Forbes-Lorman RM. Epigenetic control of vasopressin expression is maintained by steroid hormones in the adult male rat brain. Proceedings of the National Academy of Sciences of the United States of America. 2011;108(10):4242–4247. [PubMed]
15. Bakker J, Baum MJ. Role for estradiol in female-typical brain and behavioral sexual differentiation. Frontiers in neuroendocrinology. 2008;29(1):1–16. [PMC free article] [PubMed]
16. Bakker J, Brand T, van Ophemert J, Slob AK. Hormonal regulation of adult partner preference behavior in neonatally ATD-treated male rats. Behavioral neuroscience. 1993;107(3):480–487. [PubMed]
17. Bakker J, van Ophemert J, Slob AK. Postweaning housing conditions and partner preference and sexual behavior of neonatally ATD-treated male rats. Psychoneuroendocrinology. 1995;20(3):299–310. [PubMed]
18. Bakker J, van Ophemert J, Timmerman MA, de Jong FH, Slob AK. Endogenous reproductive hormones and nocturnal rhythms in partner preference and sexual behavior of ATD-treated male rats. Neuroendocrinology. 1995;62(4):396–405. [PubMed]
19. Balthazart J, Ball GF. New insights into the regulation and function of brain estrogen synthase (aromatase) Trends in neurosciences. 1998;21(6):243–249. [PubMed]
20. Bamshad M, Novak MA, De Vries GJ. Sex and species differences in the vasopressin innervation of sexually naive and parental prairie voles, Microtus ochrogaster and meadow voles, Microtus pennsylvanicus. Journal of neuroendocrinology. 1993;5(3):247–255. [PubMed]
21. Bao AM, Swaab DF. Sexual differentiation of the human brain: relation to gender identity, sexual orientation and neuropsychiatric disorders. Frontiers in neuroendocrinology. 2011;32(2):214–226. [PubMed]
22. Berta P, Hawkins JR, Sinclair AH, Taylor A, Griffiths BL, Goodfellow PN, Fellous M. Genetic evidence equating SRY and the testis-determining factor. Nature. 1990;348(6300):448–450. [PubMed]
23. Berthold AA. Transplantion der Hoden. Arch Anat Physiol Wissensch. 1849
24. Bird A. Perceptions of epigenetics. Nature. 2007;447(7143):396–398. [PubMed]
25. Bird AP, Wolffe AP. Methylation-induced repression--belts, braces, and chromatin. Cell. 1999;99(5):451–454. [PubMed]
26. Blaustein JD, Turcotte JC. Estradiol-induced progestin receptor immunoreactivity is found only in estrogen receptor-immunoreactive cells in guinea pig brain. Neuroendocrinology. 1989;49(5):454–461. [PubMed]
27. Bleier R, Byne W, Siggelkow I. Cytoarchitectonic sexual dimorphisms of the medial preoptic and anterior hypothalamic areas in guinea pig, rat, hamster, and mouse. The Journal of comparative neurology. 1982;212(2):118–130. [PubMed]
28. Bloch GJ, Eckersell C, Mills R. Distribution of galanin-immunoreactive cells within sexually dimorphic components of the medial preoptic area of the male and female rat. Brain research. 1993;620(2):259–268. [PubMed]
29. Brown TJ, Hochberg RB, Zielinski JE, MacLusky NJ. Regional sex differences in cell nuclear estrogen-binding capacity in the rat hypothalamus and preoptic area. Endocrinology. 1988;123(4):1761–1770. [PubMed]
30. Budefeld T, Grgurevic N, Tobet SA, Majdic G. Sex differences in brain developing in the presence or absence of gonads. Developmental neurobiology. 2008;68(7):981–995. [PMC free article] [PubMed]
31. Byne W, Lasco MS, Kemether E, Shinwari A, Edgar MA, Morgello S, Jones LB, Tobet S. The interstitial nuclei of the human anterior hypothalamus: an investigation of sexual variation in volume and cell size, number and density. Brain research. 2000;856(1–2):254–258. [PubMed]
32. Calaresu FR, Henry JL. Sex difference in the number of the sympathetic neurons in the spinal cord of the cat. Science (New York, NY) 1971;173(3994):343–344. [PubMed]
33. Canteras NS, Simerly RB, Swanson LW. Organization of projections from the ventromedial nucleus of the hypothalamus: a Phaseolus vulgaris-leucoagglutinin study in the rat. The Journal of comparative neurology. 1994;348(1):41–79. [PubMed]
34. Champagne FA, Weaver IC, Diorio J, Dymov S, Szyf M, Meaney MJ. Maternal care associated with methylation of the estrogen receptor-alpha1b promoter and estrogen receptor-alpha expression in the medial preoptic area of female offspring. Endocrinology. 2006;147(6):2909–2915. [PubMed]
35. Chan WY, Yew DT. Apoptosis and Bcl-2 oncoprotein expression in the human fetal central nervous system. The Anatomical record. 1998;252(2):165–175. [PubMed]
36. Chung WC, De Vries GJ, Swaab DF. Sexual differentiation of the bed nucleus of the stria terminalis in humans may extend into adulthood. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2002;22(3):1027–1033. [PubMed]
37. Chung WC, Pak TR, Suzuki S, Pouliot WA, Andersen ME, Handa RJ. Detection and localization of an estrogen receptor beta splice variant protein (ERbeta2) in the adult female rat forebrain and midbrain regions. The Journal of comparative neurology. 2007;505(3):249–267. [PubMed]
38. Chung WC, Pak TR, Weiser MJ, Hinds LR, Andersen ME, Handa RJ. Progestin receptor expression in the developing rat brain depends upon activation of estrogen receptor alpha and not estrogen receptor beta. Brain research. 2006;1082(1):50–60. [PubMed]
39. Chung WC, Swaab DF, De Vries GJ. Apoptosis during sexual differentiation of the bed nucleus of the stria terminalis in the rat brain. Journal of neurobiology. 2000;43(3):234–243. [PubMed]
40. Commins D, Yahr P. Acetylcholinesterase activity in the sexually dimorphic area of the gerbil brain: sex differences and influences of adult gonadal steroids. The Journal of comparative neurology. 1984;224(1):123–131. [PubMed]
41. Corbier P, Dehennin L, Castanier M, Mebazaa A, Edwards DA, Roffi J. Sex differences in serum luteinizing hormone and testosterone in the human neonate during the first few hours after birth. The Journal of clinical endocrinology and metabolism. 1990;71(5):1344–1348. [PubMed]
42. Corbier P, Kerdelhue B, Picon R, Roffi J. Changes in testicular weight and serum gonadotropin and testosterone levels before, during, and after birth in the perinatal rat. Endocrinology. 1978;103(6):1985–1991. [PubMed]
43. Davies AM. Neurotrophic factors. Switching neurotrophin dependence. Current biology : CB. 1994;4(3):273–276. [PubMed]
44. Davis EC, Popper P, Gorski RA. The role of apoptosis in sexual differentiation of the rat sexually dimorphic nucleus of the preoptic area. Brain research. 1996;734(1–2):10–18. [PubMed]
45. Davis EC, Shryne JE, Gorski RA. A revised critical period for the sexual differentiation of the sexually dimorphic nucleus of the preoptic area in the rat. Neuroendocrinology. 1995;62(6):579–585. [PubMed]
46. De Jonge FH, Louwerse AL, Ooms MP, Evers P, Endert E, van de Poll NE. Lesions of the SDN-POA inhibit sexual behavior of male Wistar rats. Brain research bulletin. 1989;23(6):483–492. [PubMed]
47. De Vries GJ, Best W, Sluiter AA. The influence of androgens on the development of a sex difference in the vasopressinergic innervation of the rat lateral septum. Brain research. 1983;284(2–3):377–380. [PubMed]
48. de Vries GJ, Buijs RM, Swaab DF. Ontogeny of the vasopressinergic neurons of the suprachiasmatic nucleus and their extrahypothalamic projections in the rat brain-- presence of a sex difference in the lateral septum. Brain research. 1981;218(1–2):67–78. [PubMed]
49. De Vries GJ, Rissman EF, Simerly RB, Yang LY, Scordalakes EM, Auger CJ, Swain A, Lovell-Badge R, Burgoyne PS, Arnold AP. A model system for study of sex chromosome effects on sexually dimorphic neural and behavioral traits. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2002;22(20):9005–9014. [PubMed]
50. De Vries GJ, Rissman EF, Simerly RB, Yang LY, Scordalakes EM, Auger CJ, Swain A, Lovell-Badge R, Burgoyne PS, Arnold AP. A model system for study of sex chromosome effects on sexually dimorphic neural and behavioral traits. JNeurosci. 2002;22(20):9005–9014. [PubMed]
51. del Abril A, Segovia S, Guillamon A. The bed nucleus of the stria terminalis in the rat: regional sex differences controlled by gonadal steroids early after birth. Brain research. 1987;429(2):295–300. [PubMed]
52. Dohler KD, Wuttke W. Changes with age in levels of serum gonadotropins, prolactin and gonadal steroids in prepubertal male and female rats. Endocrinology. 1975;97(4):898–907. [PubMed]
53. Dominguez R, Micevych P. Estradiol rapidly regulates membrane estrogen receptor alpha levels in hypothalamic neurons. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2010;30(38):12589–12596. [PMC free article] [PubMed]
54. Donahue JE, Stopa EG, Chorsky RL, King JC, Schipper HM, Tobet SA, Blaustein JD, Reichlin S. Cells containing immunoreactive estrogen receptor-alpha in the human basal forebrain. Brain research. 2000;856(1–2):142–151. [PubMed]
55. DonCarlos LL, Handa RJ. Developmental profile of estrogen receptor mRNA in the preoptic area of male and female neonatal rats. Brain ResDevBrain Res. 1994;79(2):283–289. [PubMed]
56. Ducret E, Gaidamaka G, Herbison AE. Electrical and morphological characteristics of anteroventral periventricular nucleus kisspeptin and other neurons in the female mouse. Endocrinology. 2010;151(5):2223–2232. [PubMed]
57. Dunn JD. Plasma corticosterone responses to electrical stimulation of the bed nucleus of the stria terminalis. Brain research. 1987;407(2):327–331. [PubMed]
58. Edelmann MN, Auger AP. Epigenetic impact of simulated maternal grooming on estrogen receptor alpha within the developing amygdala. Brain, behavior, and immunity. 2011;25(7):1299–1304. [PMC free article] [PubMed]
59. Emery DE, Sachs BD. Copulatory behavior in male rats with lesions in the bed nucleus of the stria terminalis. Physiology & behavior. 1976;17(5):803–806. [PubMed]
60. Evans RM. The steroid and thyroid hormone receptor superfamily. Science (New York, NY) 1988;240(4854):889–895. [PubMed]
61. Forbes-Lorman RM, Rautio JJ, Kurian JR, Auger AP, Auger CJ. Neonatal MeCP2 is important for the organization of sex differences in vasopressin expression. Epigenetics : official journal of the DNA Methylation Society. 2012;7(3):230–238. [PMC free article] [PubMed]
62. Fraga MF, Ballestar E, Paz MF, Ropero S, Setien F, Ballestar ML, Heine-Suner D, Cigudosa JC, Urioste M, Benitez J, Boix-Chornet M, Sanchez-Aguilera A, Ling C, Carlsson E, Poulsen P, Vaag A, Stephan Z, Spector TD, Wu YZ, Plass C, Esteller M. Epigenetic differences arise during the lifetime of monozygotic twins. ProcNatlAcadSciUSA. 2005;102(30):10604–10609. [PubMed]
63. Fried G, Andersson E, Csoregh L, Enmark E, Gustafsson JA, Aanesen A, Osterlund C. Estrogen receptor beta is expressed in human embryonic brain cells and is regulated by 17beta-estradiol. The European journal of neuroscience. 2004;20(9):2345–2354. [PubMed]
64. Gorski RA, Gordon JH, Shryne JE, Southam AM. Evidence for a morphological sex difference within the medial preoptic area of the rat brain. Brain research. 1978;148(2):333–346. [PubMed]
65. Grafstrom RH, Yuan R, Hamilton DL. The characteristics of DNA methylation in an in vitro DNA synthesizing system from mouse fibroblasts. Nucleic Acids Res. 1985;13(8):2827–2842. [PMC free article] [PubMed]
66. Gu GB, Simerly RB. Projections of the sexually dimorphic anteroventral periventricular nucleus in the female rat. The Journal of comparative neurology. 1997;384(1):142–164. [PubMed]
67. Guillamon A, Segovia S, del Abril A. Early effects of gonadal steroids on the neuron number in the medial posterior region and the lateral division of the bed nucleus of the stria terminalis in the rat. Brain research Developmental brain research. 1988;44(2):281–290. [PubMed]
68. Handa RJ, Ogawa S, Wang JM, Herbison AE. Roles for oestrogen receptor beta in adult brain function. Journal of neuroendocrinology. 2012;24(1):160–173. [PMC free article] [PubMed]
69. Herman JP, Cullinan WE, Watson SJ. Involvement of the bed nucleus of the stria terminalis in tonic regulation of paraventricular hypothalamic CRH and AVP mRNA expression. Journal of neuroendocrinology. 1994;6(4):433–442. [PubMed]
70. Hestiantoro A, Swaab DF. Changes in estrogen receptor-alpha and -beta in the infundibular nucleus of the human hypothalamus are related to the occurrence of Alzheimer's disease neuropathology. The Journal of clinical endocrinology and metabolism. 2004;89(4):1912–1925. [PubMed]
71. Hines M, Allen LS, Gorski RA. Sex differences in subregions of the medial nucleus of the amygdala and the bed nucleus of the stria terminalis of the rat. Brain research. 1992;579(2):321–326. [PubMed]
72. Hines M, Davis FC, Coquelin A, Goy RW, Gorski RA. Sexually dimorphic regions in the medial preoptic area and the bed nucleus of the stria terminalis of the guinea pig brain: a description and an investigation of their relationship to gonadal steroids in adulthood. The Journal of neuroscience : the official journal of the Society for Neuroscience. 1985;5(1):40–47. [PubMed]
73. Hisasue S, Seney ML, Immerman E, Forger NG. Control of cell number in the bed nucleus of the stria terminalis of mice: role of testosterone metabolites and estrogen receptor subtypes. J Sex Med. 2010;7(4 Pt 1):1401–1409. [PubMed]
74. Hofman MA, Swaab DF. The sexually dimorphic nucleus of the preoptic area in the human brain: a comparative morphometric study. Journal of anatomy. 1989;164:55–72. [PubMed]
75. Honda K, Sawada H, Kihara T, Urushitani M, Nakamizo T, Akaike A, Shimohama S. Phosphatidylinositol 3-kinase mediates neuroprotection by estrogen in cultured cortical neurons. Journal of neuroscience research. 2000;60(3):321–327. [PubMed]
76. Honda K, Shimohama S, Sawada H, Kihara T, Nakamizo T, Shibasaki H, Akaike A. Nongenomic antiapoptotic signal transduction by estrogen in cultured cortical neurons. Journal of neuroscience research. 2001;64(5):466–475. [PubMed]
77. Hutton LA, Gu G, Simerly RB. Development of a sexually dimorphic projection from the bed nuclei of the stria terminalis to the anteroventral periventricular nucleus in the rat. The Journal of neuroscience : the official journal of the Society for Neuroscience. 1998;18(8):3003–3013. [PubMed]
78. Ishunina TA, Kruijver FP, Balesar R, Swaab DF. Differential expression of estrogen receptor alpha and beta immunoreactivity in the human supraoptic nucleus in relation to sex and aging. The Journal of clinical endocrinology and metabolism. 2000;85(9):3283–3291. [PubMed]
79. Ishunina TA, Swaab DF. Decreased alternative splicing of estrogen receptor-alpha mRNA in the Alzheimer's disease brain. Neurobiology of aging. 2012;33(2):286–296. e283. [PubMed]
80. Ishunina TA, Unmehopa UA, van Heerikhuize JJ, Pool CW, Swaab DF. Metabolic activity of the human ventromedial nucleus neurons in relation to sex and ageing. Brain research. 2001;893(1–2):70–76. [PubMed]
81. Jacobson CD, Arnold AP, Gorski RA. Steroid autoradiography of the sexually dimorphic nucleus of the preoptic area. Brain research. 1987;414(2):349–356. [PubMed]
82. Jacobson CD, Davis FC, Gorski RA. Formation of the sexually dimorphic nucleus of the preoptic area: neuronal growth, migration and changes in cell number. Brain research. 1985;353(1):7–18. [PubMed]
83. Jacobson CD, Gorski RA. Neurogenesis of the sexually dimorphic nucleus of the preoptic area in the rat. The Journal of comparative neurology. 1981;196(3):519–529. [PubMed]
84. Jacobson CD, Shryne JE, Shapiro F, Gorski RA. Ontogeny of the sexually dimorphic nucleus of the preoptic area. The Journal of comparative neurology. 1980;193(2):541–548. [PubMed]
85. Jager RJ, Anvret M, Hall K, Scherer G. A human XY female with a frame shift mutation in the candidate testis-determining gene SRY. Nature. 1990;348(6300):452–454. [PubMed]
86. Jakab RL, Harada N, Naftolin F. Aromatase- (estrogen synthetase) immunoreactive neurons in the rat septal area. A light and electron microscopic study. Brain research. 1994;664(1–2):85–93. [PubMed]
87. Janowsky JS, Finlay BL. Cell degeneration in early development of the forebrain and cerebellum. Anatomy and embryology. 1983;167(3):439–447. [PubMed]
88. Ju G, Swanson LW. Studies on the cellular architecture of the bed nuclei of the stria terminalis in the rat: ICytoarchitecture. The Journal of comparative neurology. 1989;280(4):587–602. [PubMed]
89. Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wideranging implications in tissue kinetics. British journal of cancer. 1972;26(4):239–257. [PMC free article] [PubMed]
90. Klose RJ, Bird AP. Genomic DNA methylation: the mark and its mediators. Trends BiochemSci. 2006;31(2):89–97. [PubMed]
91. Kolbinger W, Trepel M, Beyer C, Pilgrim C, Reisert I. The influence of genetic sex on sexual differentiation of diencephalic dopaminergic neurons in vitro and in vivo. Brain research. 1991;544(2):349–352. [PubMed]
92. Koopman P. The genetics and biology of vertebrate sex determination. Cell. 2001;105(7):843–847. [PubMed]
93. Kruijver FP, Balesar R, Espila AM, Unmehopa UA, Swaab DF. Estrogen-receptorbeta distribution in the human hypothalamus: similarities and differences with ER alpha distribution. The Journal of comparative neurology. 2003;466(2):251–277. [PubMed]
94. Kruijver FP, Zhou JN, Pool CW, Hofman MA, Gooren LJ, Swaab DF. Male-to-female transsexuals have female neuron numbers in a limbic nucleus. The Journal of clinical endocrinology and metabolism. 2000;85(5):2034–2041. [PubMed]
95. Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA. Cloning of a novel receptor expressed in rat prostate and ovary. Proceedings of the National Academy of Sciences of the United States of America. 1996;93(12):5925–5930. [PubMed]
96. Kurian JR, Bychowski ME, Forbes-Lorman RM, Auger CJ, Auger AP. Mecp2 organizes juvenile social behavior in a sex-specific manner. J Neurosci. 2008;28(28):7137–7142. [PMC free article] [PubMed]
97. Kurian JR, Olesen KM, Auger AP. Sex differences in epigenetic regulation of the estrogen receptor-alpha promoter within the developing preoptic area. Endocrinology. 2010;151(5):2297–2305. [PubMed]
98. Lauber AH, Mobbs CV, Muramatsu M, Pfaff DW. Estrogen receptor messenger RNA expression in rat hypothalamus as a function of genetic sex and estrogen dose. Endocrinology. 1991;129(6):3180–3186. [PubMed]
99. LeVay S. A difference in hypothalamic structure between heterosexual and homosexual men. Science (New York, NY) 1991;253(5023):1034–1037. [PubMed]
100. Lisciotto CA, Morrell JI. Circulating gonadal steroid hormones regulate estrogen receptor mRNA in the male rat forebrain. Brain research Molecular brain research. 1993;20(1–2):79–90. [PubMed]
101. Liu YC, Salamone JD, Sachs BD. Lesions in medial preoptic area and bed nucleus of stria terminalis: differential effects on copulatory behavior and noncontact erection in male rats. The Journal of neuroscience : the official journal of the Society for Neuroscience. 1997;17(13):5245–5253. [PubMed]
102. MacLusky NJ, Philip A, Hurlburt C, Naftolin F. Estrogen formation in the developing rat brain: sex differences in aromatase activity during early post-natal life. Psychoneuroendocrinology. 1985;10(3):355–361. [PubMed]
103. Maerkel K, Durrer S, Henseler M, Schlumpf M, Lichtensteiger W. Sexually dimorphic gene regulation in brain as a target for endocrine disrupters: developmental exposure of rats to 4-methylbenzylidene camphor. ToxicolApplPharmacol. 2007;218(2):152–165. [PubMed]
104. Manin M, Baron S, Goossens K, Beaudoin C, Jean C, Veyssiere G, Verhoeven G, Morel L. Androgen receptor expression is regulated by the phosphoinositide 3-kinase/Akt pathway in normal and tumoral epithelial cells. The Biochemical journal. 2002;366(Pt 3):729–736. [PubMed]
105. Matsumoto A, Arai Y. Sex difference in volume of the ventromedial nucleus of the hypothalamus in the rat. Endocrinologia japonica. 1983;30(3):277–280. [PubMed]
106. Matsumoto A, Arai Y. Male-female difference in synaptic organization of the ventromedial nucleus of the hypothalamus in the rat. Neuroendocrinology. 1986;42(3):232–236. [PubMed]
107. Mayer A, Lahr G, Swaab DF, Pilgrim C, Reisert I. The Y-chromosomal genes SRY and ZFY are transcribed in adult human brain. Neurogenetics. 1998;1(4):281–288. [PubMed]
108. Mayer A, Mosler G, Just W, Pilgrim C, Reisert I. Developmental profile of Sry transcripts in mouse brain. Neurogenetics. 2000;3(1):25–30. [PubMed]
109. McDonald PG, Doughty C. Effect of neonatal administration of different androgens in the female rat: correlation between aromatization and the induction of sterilization. The Journal of endocrinology. 1974;61(1):95–103. [PubMed]
110. McEwen BS, Lieberburg I, Chaptal C, Krey LC. Aromatization: important for sexual differentiation of the neonatal rat brain. Hormones and behavior. 1977;9(3):249–263. [PubMed]
111. Mensah PL. An electron microscopical study of neuronal cell clustering in postnatal mouse striatum, with special emphasis on neuronal cell death. Anatomy and embryology. 1982;164(3):387–401. [PubMed]
112. Metivier R, Gallais R, Tiffoche C, Le PC, Jurkowska RZ, Carmouche RP, Ibberson D, Barath P, Demay F, Reid G, Benes V, Jeltsch A, Gannon F, Salbert G. Cyclical DNA methylation of a transcriptionally active promoter. Nature. 2008;452(7183):45–50. [PubMed]
113. Micevych PE, Park SS, Akesson TR, Elde R. Distribution of cholecystokininimmunoreactive cell bodies in the male and female rat: IHypothalamus. The Journal of comparative neurology. 1987;255(1):124–136. [PubMed]
114. Morris JA, Jordan CL, Breedlove SM. Sexual differentiation of the vertebrate nervous system. Nature neuroscience. 2004;7(10):1034–1039. [PubMed]
115. Mosselman S, Polman J, Dijkema R. ER beta: identification and characterization of a novel human estrogen receptor. FEBS letters. 1996;392(1):49–53. [PubMed]
116. Mott NN, Pak TR. Characterisation of human oestrogen receptor beta (ERbeta) splice variants in neuronal cells. Journal of neuroendocrinology. 2012;24(10):1311–1321. [PMC free article] [PubMed]
117. Murray EK, Hien A, De Vries GJ, Forger NG. Epigenetic control of sexual differentiation of the bed nucleus of the stria terminalis. Endocrinology. 2009;150(9):4241–4247. [PubMed]
118. Naftolin F, Horvath TL, Jakab RL, Leranth C, Harada N, Balthazart J. Aromatase immunoreactivity in axon terminals of the vertebrate brain. An immunocytochemical study on quail, rat, monkey and human tissues. Neuroendocrinology. 1996;63(2):149–155. [PubMed]
119. Nanney DL. Epigenetic control systems. ProcNatlAcadSciUSA. 1958;44(7):712–717. [PubMed]
120. Nishizuka M, Arai Y. Regional difference in sexually dimorphic synaptic organization of the medial amygdala. Experimental brain research Experimentelle Hirnforschung Experimentation cerebrale. 1983;49(3):462–465. [PubMed]
121. Nishizuka M, Sumida H, Kano Y, Arai Y. Formation of neurons in the sexually dimorphic anteroventral periventricular nucleus of the preoptic area of the rat: effects of prenatal treatment with testosterone propionate. Journal of neuroendocrinology. 1993;5(5):569–573. [PubMed]
122. Nottebohm F, Arnold AP. Sexual dimorphism in vocal control areas of the songbird brain. Science (New York, NY) 1976;194(4261):211–213. [PubMed]
123. Olster DH, Blaustein JD. Development of progesterone-facilitated lordosis in female guinea pigs: relationship to neural estrogen and progestin receptors. Brain research. 1989;484(1–2):168–176. [PubMed]
124. Oppenheim RW. Cell death during development of the nervous system. Annual review of neuroscience. 1991;14:453–501. [PubMed]
125. Orikasa C, Sakuma Y. Estrogen configures sexual dimorphism in the preoptic area of C57BL/6J and ddN strains of mice. The Journal of comparative neurology. 2010;518(17):3618–3629. [PubMed]
126. Osterlund M, Kuiper GG, Gustafsson JA, Hurd YL. Differential distribution and regulation of estrogen receptor-alpha and -beta mRNA within the female rat brain. Brain research Molecular brain research. 1998;54(1):175–180. [PubMed]
127. Osterlund MK, Gustafsson JA, Keller E, Hurd YL. Estrogen receptor beta (ERbeta) messenger ribonucleic acid (mRNA) expression within the human forebrain: distinct distribution pattern to ERalpha mRNA. The Journal of clinical endocrinology and metabolism. 2000;85(10):3840–3846. [PubMed]
128. Pak TR, Chung WC, Hinds LR, Handa RJ. Arginine vasopressin regulation in preand postpubertal male rats by the androgen metabolite 3beta-diol. American journal of physiology Endocrinology and metabolism. 2009;296(6):E1409–E1413. [PubMed]
129. Pakkenberg B, Gundersen HJ. Neocortical neuron number in humans: effect of sex and age. The Journal of comparative neurology. 1997;384(2):312–320. [PubMed]
130. Patisaul HB, Whitten PL, Young LJ. Regulation of estrogen receptor beta mRNA in the brain: opposite effects of 17beta-estradiol and the phytoestrogen, coumestrol. Brain research Molecular brain research. 1999;67(1):165–171. [PubMed]
131. Pfaff DW, Sakuma Y. Deficit in the lordosis reflex of female rats caused by lesions in the ventromedial nucleus of the hypothalamus. The Journal of physiology. 1979;288:203–210. [PubMed]
132. Pfaff DW, Sakuma Y. Facilitation of the lordosis reflex of female rats from the ventromedial nucleus of the hypothalamus. The Journal of physiology. 1979;288:189–202. [PubMed]
133. Phoenix CH, Goy RW, Gerall AA, Young WC. Organizing action of prenatally administered testosterone propionate on the tissues mediating mating behavior in the female guinea pig. Endocrinology. 1959;65:369–382. [PubMed]
134. Polanco JC, Koopman P. Sry and the hesitant beginnings of male development. Developmental biology. 2007;302(1):13–24. [PubMed]
135. Pozzo Miller LD, Aoki A. Stereological analysis of the hypothalamic ventromedial nucleus. II. Hormone-induced changes in the synaptogenic pattern. Brain research Developmental brain research. 1991;61(2):189–196. [PubMed]
136. Raisman G, Field PM. Sexual dimorphism in the preoptic area of the rat. Science (New York, NY) 1971;173(3998):731–733. [PubMed]
137. Raisman G, Field PM. Sexual dimorphism in the neuropil of the preoptic area of the rat and its dependence on neonatal androgen. Brain research. 1973;54:1–29. [PubMed]
138. Rakic S, Zecevic N. Programmed cell death in the developing human telencephalon. The European journal of neuroscience. 2000;12(8):2721–2734. [PubMed]
139. Ramsahoye BH, Biniszkiewicz D, Lyko F, Clark V, Bird AP, Jaenisch R. Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a. ProcNatlAcadSciUSA. 2000;97(10):5237–5242. [PubMed]
140. Razin A, Riggs AD. DNA methylation and gene function. Science (New York, NY) 1980;210(4470):604–610. [PubMed]
141. Robyr D, Wolffe AP, Wahli W. Nuclear hormone receptor coregulators in action: diversity for shared tasks. Molecular endocrinology (Baltimore, Md) 2000;14(3):329–347. [PubMed]
142. Sasano H, Takashashi K, Satoh F, Nagura H, Harada N. Aromatase in the human central nervous system. Clinical endocrinology. 1998;48(3):325–329. [PubMed]
143. Savic I, Berglund H, Gulyas B, Roland P. Smelling of odorous sex hormone-like compounds causes sex-differentiated hypothalamic activations in humans. Neuron. 2001;31(4):661–668. [PubMed]
144. Shammah-Lagnado SJ, Beltramino CA, McDonald AJ, Miselis RR, Yang M, de Olmos J, Heimer L, Alheid GF. Supracapsular bed nucleus of the stria terminalis contains central and medial extended amygdala elements: evidence from anterograde and retrograde tracing experiments in the rat. The Journal of comparative neurology. 2000;422(4):533–555. [PubMed]
145. Sharma M, Chuang WW, Sun Z. Phosphatidylinositol 3-kinase/Akt stimulates androgen pathway through GSK3beta inhibition and nuclear beta-catenin accumulation. The Journal of biological chemistry. 2002;277(34):30935–30941. [PubMed]
146. Shughrue P, Scrimo P, Lane M, Askew R, Merchenthaler I. The distribution of estrogen receptor-beta mRNA in forebrain regions of the estrogen receptor-alpha knockout mouse. Endocrinology. 1997;138(12):5649–5652. [PubMed]
147. Shupnik MA, Pitt LK, Soh AY, Anderson A, Lopes MB, Laws ER., Jr Selective expression of estrogen receptor alpha and beta isoforms in human pituitary tumors. The Journal of clinical endocrinology and metabolism. 1998;83(11):3965–3972. [PubMed]
148. Sibug R, Kuppers E, Beyer C, Maxson SC, Pilgrim C, Reisert I. Genotype-dependent sex differentiation of dopaminergic neurons in primary cultures of embryonic mouse brain. Brain research Developmental brain research. 1996;93(1–2):136–142. [PubMed]
149. Simerly RB, Chang C, Muramatsu M, Swanson LW. Distribution of androgen and estrogen receptor mRNA-containing cells in the rat brain: an in situ hybridization study. The Journal of comparative neurology. 1990;294(1):76–95. [PubMed]
150. Simerly RB, Swanson LW, Gorski RA. Reversal of the sexually dimorphic distribution of serotonin-immunoreactive fibers in the medial preoptic nucleus by treatment with perinatal androgen. Brain research. 1985;340(1):91–98. [PubMed]
151. Simerly RB, Young BJ. Regulation of estrogen receptor messenger ribonucleic acid in rat hypothalamus by sex steroid hormones. Molecular endocrinology (Baltimore, Md) 1991;5(3):424–432. [PubMed]
152. Simonati A, Tosati C, Rosso T, Piazzola E, Rizzuto N. Cell proliferation and death: morphological evidence during corticogenesis in the developing human brain. Microscopy research and technique. 1999;45(6):341–352. [PubMed]
153. Sinclair AH, Berta P, Palmer MS, Hawkins JR, Griffiths BL, Smith MJ, Foster JW, Frischauf AM, Lovell-Badge R, Goodfellow PN. A gene from the human sexdetermining region encodes a protein with homology to a conserved DNA-binding motif. Nature. 1990;346(6281):240–244. [PubMed]
154. Suzuki S, Handa RJ. Estrogen receptor-beta, but not estrogen receptor-alpha, is expressed in prolactin neurons of the female rat paraventricular and supraoptic nuclei: comparison with other neuropeptides. The Journal of comparative neurology. 2005;484(1):28–42. [PubMed]
155. Swaab DF, Chung WC, Kruijver FP, Hofman MA, Ishunina TA. Structural and functional sex differences in the human hypothalamus. Hormones and behavior. 2001;40(2):93–98. [PubMed]
156. Swaab DF, Fliers E. A sexually dimorphic nucleus in the human brain. Science (New York, NY) 1985;228(4703):1112–1115. [PubMed]
157. Swaab DF, Hofman MA. Sexual differentiation of the human brain. A historical perspective. Progress in brain research. 1984;61:361–374. [PubMed]
158. Swaab DF, Hofman MA. Sexual differentiation of the human hypothalamus: ontogeny of the sexually dimorphic nucleus of the preoptic area. Brain research Developmental brain research. 1988;44(2):314–318. [PubMed]
159. Swaab DF, Hofman MA. Sexual differentiation of the human hypothalamus in relation to gender and sexual orientation. Trends in neurosciences. 1995;18(6):264–270. [PubMed]
160. Tetel MJ, Auger AP, Charlier TD. Who's in charge? Nuclear receptor coactivator and corepressor function in brain and behavior. Front Neuroendocrinol. 2009;30(3):328–342. [PMC free article] [PubMed]
161. Tobet SA, Zahniser DJ, Baum MJ. Sexual dimorphism in the preoptic/anterior hypothalamic area of ferrets: effects of adult exposure to sex steroids. Brain research. 1986;364(2):249–257. [PubMed]
162. Toft D, Gorski J. A receptor molecule for estrogens: isolation from the rat uterus and preliminary characterization. Proceedings of the National Academy of Sciences of the United States of America. 1966;55(6):1574–1581. [PubMed]
163. Treit D, Aujla H, Menard J. Does the bed nucleus of the stria terminalis mediate fear behaviors? Behavioral neuroscience. 1998;112(2):379–386. [PubMed]
164. Tremblay GB, Tremblay A, Copeland NG, Gilbert DJ, Jenkins NA, Labrie F, Giguere V. Cloning, chromosomal localization, and functional analysis of the murine estrogen receptor beta. Molecular endocrinology (Baltimore, Md) 1997;11(3):353–365. [PubMed]
165. Tsai HW, Grant PA, Rissman EF. Sex differences in histone modifications in the neonatal mouse brain. Epigenetics. 2009;4(1):47–53. [PMC free article] [PubMed]
166. van Kesteren PJ, Gooren LJ, Megens JA. An epidemiological and demographic study of transsexuals in The Netherlands. Archives of sexual behavior. 1996;25(6):589–600. [PubMed]
167. van VJ, Oates NA, Whitelaw E. Epigenetic mechanisms in the context of complex diseases. Cell MolLife Sci. 2007;64(12):1531–1538. [PubMed]
168. Vreeburg JT, van der Vaart PD, van der Schoot P. Prevention of central defeminization but not masculinization in male rats by inhibition neonatally of oestrogen biosynthesis. The Journal of endocrinology. 1977;74(3):375–382. [PubMed]
169. Wang Z, Hulihan TJ, Insel TR. Sexual and social experience is associated with different patterns of behavior and neural activation in male prairie voles. Brain research. 1997;767(2):321–332. [PubMed]
170. Weaver IC, Cervoni N, Champagne FA, D'Alessio AC, Sharma S, Seckl JR, Dymov S, Szyf M, Meaney MJ. Epigenetic programming by maternal behavior. NatNeurosci. 2004;7(8):847–854. [PubMed]
171. Weisz J, Ward IL. Plasma testosterone and progesterone titers of pregnant rats, their male and female fetuses, and neonatal offspring. Endocrinology. 1980;106(1):306–316. [PubMed]
172. Westberry JM, Trout AL, Wilson ME. Epigenetic Regulation of Estrogen Receptor {alpha} Gene Expression in the Mouse Cortex during Early Postnatal Development. Endocrinology. 2009 [PubMed]
173. Westberry JM, Trout AL, Wilson ME. Epigenetic regulation of estrogen receptor beta expression in the rat cortex during aging. Neuroreport. 2011;22(9):428–432. [PMC free article] [PubMed]
174. Wiegand SJ, Terasawa E. Discrete lesions reveal functional heterogeneity of suprachiasmatic structures in regulation of gonadotropin secretion in the female rat. Neuroendocrinology. 1982;34(6):395–404. [PubMed]
175. Xu J, Disteche CM. Sex differences in brain expression of X- and Y-linked genes. Brain research. 2006;1126(1):50–55. [PubMed]
176. Yamamoto Y, Carter CS, Cushing BS. Neonatal manipulation of oxytocin affects expression of estrogen receptor alpha. Neuroscience. 2006;137(1):157–164. [PubMed]
177. Yao HH, Whoriskey W, Capel B. Desert Hedgehog/Patched 1 signaling specifies fetal Leydig cell fate in testis organogenesis. Genes & development. 2002;16(11):1433–1440. [PubMed]
178. Yokosuka M, Okamura H, Hayashi S. Postnatal development and sex difference in neurons containing estrogen receptor-alpha immunoreactivity in the preoptic brain, the diencephalon, and the amygdala in the rat. JComp Neurol. 1997;389(1):81–93. [PubMed]
179. Yoon HG, Chan DW, Reynolds AB, Qin J, Wong J. N-CoR mediates DNA methylation-dependent repression through a methyl CpG binding protein Kaiso. MolCell. 2003;12(3):723–734. [PubMed]
180. Zhou JN, Hofman MA, Gooren LJ, Swaab DF. A sex difference in the human brain and its relation to transsexuality. Nature. 1995;378(6552):68–70. [PubMed]