Although considerable progress has been made in our understanding of brain function, many questions remain unanswered. The ultimate goal of studying the brain is to understand the connection between brain structure and function and behavioural outcomes. Since sex differences in brain morphology were first observed, subsequent studies suggest different functional organization of the male and female brains in humans. Sex and gender have been identified as being a significant factor in understanding human physiology, health and disease, and the biological differences between the sexes is not limited to the gonads and secondary sexual characteristics, but also affects the structure and, more crucially, the function of the brain and other organs. Significant variability in brain structures between individuals, in addition to between the sexes, is factor that complicates the study of sex differences in the brain. In this review, we explore the current understanding of sex differences in the brain, mostly focusing on preclinical animal studies.

INTRODUCTION

The brain is undoubtedly the most complicated organ and although considerable progress has been made in the understanding of brain function, many unanswered questions remain. Different approaches are used to study brain function with the ultimate aim of understanding how brain structure and function are connected with the behavioural outcomes of the individual. Pioneering studies on sex differences in the brain started in the 1960s when sex differences in brain morphology and some structural differences were observed in model animals. Later, such studies were also introduced into clinical medicine, and many studies in humans suggest that the functional organization of the brain is not entirely alike in men and women [1]. In 2001, the Institute of Medicine at National academy of Sciences in the US issued a statement that sex has a significant importance for understanding health and disease, and for understanding human physiology generally [2]. This was an important step forward in recognizing that males and females are biologically different not only with regards to gonads and secondary sexual characteristics but also in the structure and, more importantly, the function of many other organs including the brain [1]. In addition to methodological difficulties in studying the human brain, significant individual variability in brain structures complicates studies of sex differences in the brain, as some differences might differ more between individuals than between sexes, as was recently demonstrated in a report by Joel et al. [3]. Because of these large differences between individuals within one sex/gender, the term sexual allomorphism is usually used instead of sexual dimorphism in regard to the brain, taking into account this vast individual variability in brain structure. In this article, we will review the current understanding of sex differences in the brain, mostly focusing on preclinical animal studies. Firstly, the conventional hypothesis of brain sexual differentiation will be described followed by newer contributions to this field, focusing mainly on hormone-independent sex differences in the brain. The article is focused mostly on the limbic system, although sex differences have been described in other parts of the brain including the cortex and cerebellum [48], but these are beyond the scope of this review.

CONVENTIONAL VIEW OF BRAIN SEXUAL DIFFERENTIATION

Mammalian sex is determined at the time of conception with the combination of two sex chromosomes, XX for females and XY for males. Although sex differences were described in very early embryos with male embryos growing faster than female embryos [9], the onset of sexual differentiation is usually considered the initiation of expression of the Sry gene, which is located on the Y chromosome [10]. The Sry gene initiates the development of the testis from undifferentiated gonad, and this is considered as primary sexual differentiation. Shortly after primary sexual differentiation, Leydig cells in the testes start to produce testosterone, and this circulating testosterone is responsible for secondary sexual differentiation, which includes all male secondary sexual characteristics including the male pattern of the brain function. The conventional view of brain sexual differentiation follows this paradigm, suggesting that the masculine or feminine organization of the brain depends on the presence or absence of early gonadal steroid exposure (Figure 1). According to this hypothesis, testosterone is responsible for masculinization and de-feminization of the brain, although feminization in female fetuses takes place in the absence of gonadal steroids since the ovaries are inactive during fetal development [11]. The foundation of this theory lies in a series of experiments beginning almost 80 years ago. The pioneering studies started with Alfred Jost [12], whose work in the 1940s and 1950s demonstrated that male characteristics are imposed on the fetus by testicular hormones testosterone and the anti-Mullerian hormone (AMH). In the absence or inactivity of these two hormones, the fetus becomes phenotypically female [12]. This hypothesis was extended to the brain by the work of Pfeiffer [13] and especially with behavioural studies on guinea pigs by Phoenix et al. [14]. Pfeiffer [13] showed that the pattern of pituitary secretion in adulthood depends on testicular secretions during early development. Female rats ovariectomized shortly after birth can be masculinized by testes transplanted into the neck whereas castration of males at birth resulted in feminine patterns of gonadotropin release from the pituitary [13]. A similar concept of masculinization was proposed by Phoenix et al. [14]. In their experiments, pregnant guinea pigs were primed with testosterone propionate (TP) to explore the effect of masculinization on the offspring. This simple approach demonstrated that female offspring prenatally exposed to TP phenotypically resemble males. They had enlarged labia vulvae and clitorises and increased anogenital distance. In adulthood, the treatment of these female guinea pigs with oestrogens and progesterone, which normally induce sexual receptivity in females, failed to induce lordosis, a typical posture of sexually receptive female rodents. In contrast, no effects of prenatal testosterone treatment were observed in male offspring [14]. From this experiment, the hypothesis of masculinization and de-feminization by androgens was extended to the brain, and the theory of organizational and activational effects of gonadal steroids in the brain was developed. In mammals, organizational effects thus represent the early developmental changes of the CNS, which are permanent and irreversible, even with different hormonal treatments in adulthood. In contrast, the activational effects are temporary effects of steroid hormones in the brain during adult life and could be induced, reversed or removed in adulthood. Thus, it is postulated that organizational effects prepare the brain for appropriate function in adulthood, which is often dependent on the further activational effects of sex steroid hormones [11].

Schematic representation of the conventional theory of brain sexual differentiation

Figure 1
Schematic representation of the conventional theory of brain sexual differentiation
Figure 1
Schematic representation of the conventional theory of brain sexual differentiation

HORMONALLY DRIVEN SEXUAL DIFFERENTIATION OF THE BRAIN

Numerous data prove that the main hormone involved in the process of sexual differentiation of the CNS in mammals is testicular testosterone. Exposure to testosterone early during fetal development can permanently change the structure and function of the brain [1]. For instance, different sexual characteristics of the brain like differences in the volume of the medial preoptic area (SDN MPOA) [15] or vasopressin expressing fibres [16] could be reversed in females by testosterone exposure prenatally and/or immediately after birth. At the behavioural level, the same effects of testosterone in females can be observed in aggressive behaviour during social interactions [17,18], which is not normally expressed in females, although the neonatal defeminizing effects of testosterone caused an impairment of lordosis behaviour [14,18]. Interestingly, some earlier reports have shown that exposure to oestrogens early in development can affect feminine brain development in the same way as testosterone does. Female rat fetuses exposed to oestrogens during gestation or shortly after birth show an anovulatory sterility in adulthood that closely resembles sterility observed after perinatal testosterone administration [1921]. In accordance with the aromatization hypothesis that was first proposed by MacLusky and Naftolin [11], it is now well established that, at least in the rodent brain, local production of oestrogens mediated by cytochrome P450 aromatase plays a crucial role in sexual differentiation of the brain [22,23]. To date, this hypothesis has been tested and confirmed in numerous studies with rodent models with functional disruption of different steroidogenic receptors or enzymes such as rats with testicular feminization (TFM) and mice lacking oestrogenic receptors (ERKO) or the aromatase enzyme (ArKO) [2432].

The confirmation of oestradiol as the main masculinizing hormone raised another question. If oestrogens’ formation within the brain plays a vital role in sexual differentiation of the male brain, female fetuses must be protected against circulating oestrogens coming from the maternal bloodstream and placenta [33]. In rats and mice, this is achieved by alpha-fetoprotein, an oestrogen-binding protein synthesized in the developing yolk sac and fetal liver. Alpha-fetoprotein is present at high concentrations in fetal blood during the latter part of gestation and then gradually disappears over the first week of postnatal life [34,35]. Alpha-fetoprotein binds oestrogens with high affinity and prevents them from entering the female brain. As alpha-fetoprotein only binds oestrogens and does not bind testosterone, testosterone can enter the brain where it is then converted to oestradiol and interacts with oestrogen receptors [33]. The inactivation of alpha-fetoprotein using specific antibodies can masculinize female rat fetuses [36], and alpha-fetoprotein knockout female mice are masculinized [37,38], confirming the role of alpha-fetoprotein in the prevention of masculinization of the female brain.

In humans, it is less clear whether oestrogens are the main sex steroid hormones responsible for masculinization of the male brain. These processes are difficult to study in humans, and their main findings come from different clinical conditions affecting fetal steroid production or steroid receptor functions, as well as from studying correlations between masculine traits and levels of testosterone in amniotic fluid [3941]. Some insights, in particular from TFM/androgen receptor insensitivity syndrome (TFM), suggest that oestrogens have less importance in humans, and it is likely that testosterone, acting through androgen receptors, is more important for the masculinization of the human brain. Genetically male (XY) individuals with dysfunctional androgen receptor gene develop testes that secrete testosterone, but a body without functional androgen receptors develops a feminine phenotype. The complete TFM syndrome is usually undetected at birth, and those individuals are raised as girls, and as adults they identify themselves as women [42,43]. If oestrogens are equally necessary for the masculinization of the human brain as they are in rodents, patients with TFM syndrome should have masculinized brain since they have fully functional oestrogen receptors. However, these genetically male patients do not appear to have problems with their sexual identity (being raised as women) and do not show typical male behaviours [43], suggesting that testosterone has a more important role in the masculinization of the human brain.

CRITICAL PERIODS FOR ORGANIZATIONAL EFFECTS OF SEX STEROID HORMONES

The organizational effects of sex steroid hormones only occur during periods of hormone sensitivity when the genitalia and brain will respond to the gonadal hormones. These developmental periods of hormone sensitivity are unique for each species and are commonly referred to as critical periods of sexual differentiation. In most cases, these critical periods exist prenatally or soon after birth [11]. In mice [44,45] and similarly in rats [46], two peaks of gonadal hormone secretion during development are essential for sexual differentiation, the first peak at around day 17 of gestation and the second peak immediately after birth. If these peaks of testosterone secretion are disturbed, masculinization of the brain could be affected and cause behavioural deficits in adult life. Intermale aggression that normally occurs in male mice can only be induced in females by testosterone treatment soon after birth, and intermale aggression is diminished in male mice castrated immediately after birth [17,47].

In humans, fetal testes start to produce testosterone between the 8th and 9th weeks of gestation with the masculinization process starting around the 10th, and ending between the 12th and 14th weeks of gestation. However, shortly after birth, the second surge of testosterone produced by the testes is observed, and it is not yet clear what the consequences of this postnatal testosterone surge are [48,49]. Normal inter-individual variation in prenatal testosterone levels, estimated from amniotic fluid measurements, correlate with later sex-typed behaviour [50,51]. For example, fetal testosterone levels correlate with behavioural traits such as eye contact, vocabulary size, empathy and systemizing, which are expressed in a more male manner in individuals with higher amniotic testosterone [39,52]. Similarly, girls with congenital adrenal hyperplasia (CAH), a disorder in which adrenals start to produce an excessive amount of androgens early in development, more frequently engage in male play behaviour [53].

In addition to the neonatal time, puberty is another period that is now recognized as a critical period, not only to activate some sex differences by sex steroids but also for further organization of the CNS. Studies in animals indicate that interruptions of gonadal hormone secretion during puberty have long lasting effect on some behaviours in adulthood even in the presence of the activational effects of gonadal hormones [54]. Castration before puberty thus greatly impairs male sexual behaviour in Syrian hamsters even in the presence of appropriate activational testosterone [55]. The effect of puberty was also observed in our study on maternal behaviour as female mice gonadectomized prior to puberty show decreases in maternal behaviour [56]. Two other examples of behaviours that are organized during adolescence are territorial scent marking in tree shrews [57] and social interaction in novel environments in rats [58,59]. In both cases, the absence of gonadal hormones or the pharmacological blockade of their action during puberty suppressed the typical expression of the behaviour in adult animals, even if the hormones were replaced in adulthood. Moreover, a recent study by Koss et al. [60] suggests that gonadectomy before puberty could have different effects between the sexes on brain morphology. Prepubertal ovariectomy in rats resulted in higher numbers of neurons in the medial prefrontal cortex and a larger volume of white matter in comparison with sham control females whereas a similar effect of gonadectomy was not observed in males.

As previously described, the conventional theory suggests that the female brain develops intrinsically in the absence of gonadal hormones, since the ovaries are prenatally inactive. However, some recent studies indicate that (at least in rats and mice) postnatal, prepubertal exposure to low levels of oestrogens secreted by the ovaries is needed for the active feminization of the female brain [61,62]. The most compelling evidence comes from studies with aromatase knockout mice (ArKO). When tested for female sexual behaviour, ArKO females showed impairment of lordosis; thus, they are not entirely feminized [37]. In these mice, female sexual behaviour could be at least partially restored by treating mice with oestrogens between days 15 and 25 postnatally [63]. Furthermore, our unpublished studies with SF-1 KO mice suggest that similar postnatal feminization might be required for proper expression of maternal behaviour as agonadal SF-1 KO male and female mice show lower levels of parental behaviour in adult life in comparison with prepubertally (around day 23) gonadectomized WT females (N. Grgurevic, unpublished work).

Different studies suggest that both neonatal and pubertal periods are essential for the organizational effects of sex steroid hormones on the CNS. However, the outcome of the organization of the entire neuroendocrine system is most likely beyond the simple hormone effects and is a result of the interplay of environment, hormones, and genes, which will be discussed later.

ORGANIZATIONAL HORMONAL EFFECTS ON STRUCTURAL DIFFERENCES IN THE BRAIN AND EXPRESSION OF DIFFERENT GENES/PROTEINS

After the landmark paper of Phoenix et al. [14], the most obvious potential answer to the question why males and females differ in their behaviours was that sex steroid hormones must impose structural differences within the brain. Indeed, sexual dimorphisms in the CNS have been described at different levels: the molecular, ultrastructural, cellular, anatomical and neural systems levels. Differences are present as differences in the brain nuclei volume, cell numbers, number of synapses and differences in the expression of different genes/proteins [64]. These structural differences, leading to sex differences in behaviour, are spread throughout the nervous system rather than concentrated in a single structure or a system. Sex differences were initially found in the hypothalamus, one of the most sexually dimorphic areas of the brain [6467]. One of the first observed sexual dimorphic areas was the preoptic area of the hypothalamus, more precisely the medial preoptic area (MPOA), part of the brain that regulates reproductive behaviours [15,68]. Within this area, there is a sexual dimorphic nucleus (SDN), which is bigger in males than in females [15] in different species including humans [64,66]. Surprisingly, unlike in rats, many mice strains do not exhibit sexual dimorphism in SDN MPOA as shown with Nissl staining of the brain slices [69,70]. Nevertheless, studies in mice showed the difference in SDN MPOA in the number of neurons expressing the cellular marker calbindin D-28k, a calcium-binding protein, with males having more calbindin-expressing cells in the MPOA than females [71,72]. Sex difference in calbindin expression is strongly influenced by the organizational effects of sex steroids early in development [72,73]. SDN is believed to be involved in the regulation of male sexual behaviour [74] although its precise role has not yet been determined. Interestingly, some studies also suggested SDN's role in a partner preference in rams [75] and even in humans [76], although differences in SDN volume between homosexual and heterosexual men were not confirmed by follow-up studies, and it is now accepted that SDN does not have a role in sexual orientation in humans (reviewed in [77]).

Another part of the hypothalamus that is sexually dimorphic is anteroventral periventricular nucleus (AVPV), a part of the neural circuit controlling ovulation. In AVPV, sex steroids (testosterone/oestradiol) promote cell death in males [78]. In contrast with the SDN MPOA, AVPV is larger in females than in males [79,80]. In AVPV, the sex difference was described in the number of cells expressing alpha and β oestrogen receptors, with females having more immunopositive cells for both receptors [8184], and tyrosine hydroxylase, a marker for dopaminergic cells, which are also present in higher numbers in female AVPV [85,86]. Sex differences were also observed in the expression of kisspeptin, a regulator of GnRH secretion essential for the onset of puberty. Female mice have up to 10 times more kisspeptin immunopositive cell bodies than male mice in AVPV [87]. Similarly, GABA/glutamate neurons appear to be sexually differentiated in the AVPV, with females having twice as many dual-phenotype (GABA and glutamate expressing) neurons than males do [88]. These sex differences in the AVPV are believed to be connected with the regulation of the GnRH secretion and LH surge, mediated by oestrogens and kisspeptin neurons [89]. Therefore, it is not surprising that this area is sexually dimorphic as patterns of gonadotropin secretion are well known to differ between sexes [90].

The ventromedial nucleus of the hypothalamus (VMH) was first described as sexually dimorphic by Dorner and Staudt [91], who found that the cellular nuclei of the VMH neurons were larger in female than in male rats. However, the overall volume of the nucleus was larger in males than in females [91]. Several other characteristics of the VMH are different between the two sexes. Sex differences have been described in the expression of both oestrogen receptors and progesterone receptors (PR). Female rats have more cells expressing oestrogen receptors α [92] and β [81] in the VMH than male rats do. Interestingly, in neonatal mice, there are many more PR immunopositive cells in the male VMH in comparison with females [93], whereas there is no sex difference in the PR expression in the neonatal VMH in rats [94]. However, reversed sex difference is observed postnatally and in adulthood, with females having more PR immunopositive cells in the VMH than males do, in both mice and rats [9496]. In rodents, VMH has an important role in the regulation of lordosis reflex, which can be induced by oestradiol and progesterone. Therefore, it is believed that these sex differences are connected with the regulation of female sexual behaviour [97]. In humans, one study also showed a direct involvement of the VMH in sexually differentiated behavioural response. After exposure to an odour that includes androgen or oestrogen-like compounds, different parts of the hypothalamus were activated in men and women. In women, smelling the androgen-like compound activates VMH whereas in men the oestrogen-like compound activates the dorsomedial nucleus and not the VMH [98].

In extra-hypothalamic brain areas the most consistent sex differences are found in the lateral septum (LS), bed nucleus of the stria terminalis (BNST) and amygdala [64]. The projections of vasopresin immunoreactive fibres (VP-ir fibres) to the LS are one of the more robust sex differences in vertebrates in adult animals, firstly recognized by De Vries and colleagues [99,100]. The presence of vasopressin in males but not in females is dependent on both the organizational and activational effects of testosterone since sex difference is not present in gonadectomized animals [100]. Vasopressin is produced in cells of the BNST, which project to the LS where protein is released [99]. Early studies in rats found sex differences in the medial posterior portion of the BNST with males having a larger volume of this nucleus [101] and a higher number of neurons [102] than females do. The other region of the BNST, lateral-anterior BNST, is sexually dimorphic in the opposite direction with females having a higher number of neurons than males do [102]. Consistent with the sex difference in the LS, male BNST has more cells expressing a vasopressin precursor, propressophysin, in comparison with female BNST [103,104]. The presence of sex differences in BNST in VP-ir fibres is less clearly defined in primates and humans, which could also be due to the limited number of studies. No sex differences were found in macaques [105] whereas in marmosets [106] males had a higher number of VP-ir fibres than females. In the same brain area, no sex differences were found in humans [107], whereas, within the hypothalamus, sex differences were found in the paraventricular and supraoptic nucleus with males having a higher number of VP-ir neurons than females [108]. The functional significance of sexual dimorphisms in vasopressin is still largely not clear, although it is believed that vasopressin is involved in the regulation of different social behaviours in many species [109111].

Within the amygdala, sex differences were reported mostly in the medial nucleus, with male rats having larger nuclear volume, higher synaptic input and higher dendritic spine density [112114]. Similarly, the number of vasopressin [115] and substance P-producing neurons [116] is also higher in male rat amygdala when compared with the female amygdala. A study by Morris et al. [117] suggests that amygdalar sex differences in mice mirror those in rats in both regional volume and soma size, which are all greater in males than in females. Oestrogens acting through oestrogen receptors play an important role in the organization of sex difference in the amygdala. This process was shown as temporal and region specific as oestrogen receptors alpha are expressed in the postnatal amygdala in a sexually dimorphic manner only in the amygdalo-hippocampal area [118]. In the adult human brain, the amygdala is larger in males than in females when total brain size is considered [119]. Although exact behavioural correlations with the size of the amygdala and other structural sex differences are not known, it is plausible to assume that structural sex differences are connected with sex differences in emotion processing. Different studies suggest that emotional memory tends to be stronger (memories can be recalled more quickly, and recalls are more intense) in women than in men [120]. Moreover, several studies reported gender-specific differences in the activation between the left and right amygdala. The activity of the left amygdala is correlated with the level of emotional memory in women whereas in the same context, the right amygdala is preferentially activated in man [121,122]. Furthermore, in males amygdala showed greater activation than in females when subjects were viewing sexually stimulating pictures [123].

In addition to the previously described structures, which are all part of the limbic system mostly related to body homoeostasis, sex differences were also consistently described in other areas of the brain such as the hippocampus, cerebellum, cortex and thickness of the grey matter, suggesting that sex differences are not only present in the parts of the brain regulating autonomic processes, but also in the higher brain areas; these are described elsewhere [48].

SEX CHROMOSOMES AND SEXUAL DIFFERENTIATION OF THE MAMMALIAN BRAIN

Although sex steroid hormones account for most aspects of brain sexual differentiation, a growing amount of literature has also raised an important question about the direct role of genes on sex chromosomes separate from the actions of sex steroid hormones [65,124126]. It is evident that sex chromosomes differ by sex and start the process of sexual differentiation, but the extent to which they might cause sex differences in the brain is still debated. The direct roles of sex chromosome genes in the development of sex differences are not well studied. This is mainly due to two reasons; the first is that the conventional theory remains dominant, and the second is that only a few good animal models exist that allow testing the influence of sex chromosomes separately from the effect of sex hormones [65,124]. The important question is, therefore, which genetic mechanisms powered by different genetic factors in males compared with females could influence sexual differentiation of the brain. The first and the most obvious is the presence of the Y chromosome in males, which is absent from females. In contrast with males, females have a double dose of genes from X chromosomes, although one X chromosome in females is inactivated during early development [127]. Nevertheless, some genes on the inactive X chromosome escape inactivation and are present in double dosage in female cells in comparison with male cells [128]. The process of X chromosome inactivation in individual cells is random during embryonic development, and this results in allelic mosaicism since females express the maternal X alleles in about half of their cells but paternal alleles in the other half. In contrast, males always inherit X chromosome from their mothers. Therefore, active genes from X chromosomes in male cells are always of maternal origin whereas in female cells, they are either from the maternal or paternal X chromosome. Interestingly, the mosaicism of the X chromosomes in humans has been suggested to lead to sex differences in mortality and susceptibility to disease [129].

Studies in vitro were the first studies describing sex differences in the brain that develop independently of gonadal hormones and are most probably influenced by sexually dimorphic genetic factors. In 1991, Kolbinger et al. [130] described 30% larger soma size in tyrosine hydroxylase immunoreactive (TH-ir) neurons from female diencephalonic tissue culture in comparison with male cells. Treatment with sex steroids did not affect the sex difference in soma size, suggesting that sexual differentiation of dopaminergic neurons may be under primary genetic control. Subsequent in vitro studies consistently reported hormone-independent sex differences also in mesencephalic cells, where similar sex differences were noted in the number of TH-positive neurons and different measurements of dopamine production [131,132]. Since Sry transcripts were detected in the hypothalamus and in mesencephalon (midbrain) it was suggested that Sry gene might directly influence the dimorphic sexual development of dopaminergic neurons [133].

MOUSE MODELS FOR STUDYING HORMONE-INDEPENDENT BRAIN SEXUAL DIFFERENTIATION

In vivo studies separating genetic from hormonal influences are more difficult. Some models are available for such studies; these include ERKO, ARKO, TFM and ArKO mice that have impaired steroid hormone production or the function of receptors for sex steroid hormones. Although these are important models, in these animals, sex chromosome effect cannot be studied independently of sex steroid hormones although different sex steroids are still present and this is complicating the interpretation of the results. To overcome these limitations, various models for evaluating sex chromosome effects have been developed; to date, most answers have been provided by two special models: agonadal steroidogenic factor-1 (SF-1) knockout mice and mice with direct manipulation of X and Y sex chromosomes, called four core genotype (FCG) mice.

FOUR CORE GENOTYPE MODEL

FCG mice carry two critical genetic modifications: deletion of the testis-determining factor Sry from the Y chromosome and insertion of the Sry transgene on to the autosome in the same mouse. The FCG model yields four genotypes with differences in gonads and sex chromosomes: two genetically different females (F), XXF and XYF (with a deleted Sry gene from the Y chromosome), and two genetically different males (M), XXM and XYM, both carrying the Sry gene on the autosomes (reviewed by Arnold [124,134]; Figure 2). The presence of the Sry gene on the autosome is sufficient to cause testis development and consequently most male-specific sex characteristics including male external genitalia in XX mice. Nevertheless, the gonads of XX and XY males differ in morphology and function. Although XXM testes produce testosterone, spermatogenesis in adult males is incomplete due to the absence of regulatory genes on the Y chromosome, and such male mice are infertile [135]. Several studies have shown that there are no differences in the levels of testosterone between XX and XY adult males or in oestradiol between XX and XY females [136138]. A recent study by Itoh et al. [139] also suggests that perinatal exposure to androgens does not differ between XX and XY males since anogenital distance is similarly masculinized in both genotypes and significantly larger when compared with XX and XY females. Comparisons of phenotypes of mice with the same type of gonad XXF compared with XYF and XXM compared with XYM but different sex chromosomes provide information about differential effects of XX and XY genomes. It is important to note that gonadal hormones are not eliminated in this model, and FCG mice still experience the organizational and activational effects of sex steroid hormones, although the activational effects could be removed with gonadectomy [124].

Schematic representation of WT and FCG mice

Figure 2
Schematic representation of WT and FCG mice

FCG mice lack the sry gene on the Y chromosome, but have the sry gene translocated to the one of the autosomes.

Figure 2
Schematic representation of WT and FCG mice

FCG mice lack the sry gene on the Y chromosome, but have the sry gene translocated to the one of the autosomes.

To date, FCG mice have been analysed for numerous neural and non-neural phenotypes. Sexually different traits are listed in Tables 1 and 2 and reviewed by Arnold [124,134]. The earliest studies focused on brain and behavioural phenotypes representing some classic sexually dimorphic traits to define whether the chromosome complement played any role in sexual differentiation of the brain and behaviour. For this purpose, mice were gonadectomized in adulthood and treated with testosterone when experiments were performed [140]. Many of the sexually dimorphic traits previously described in this article were found in FCG mice as a result of the organizational effects of gonadal hormones and were not influenced by the presence of different genetic factors from the sex chromosomes [140,141]. However, a study by De Vries et al. [140] and a subsequent study by Getewood et al. [136] showed that well-established sexual dimorphism in the density of vasopressin fibres in the LS is partially influenced by sex chromosomes. Within groups of the same gonadal sex, the difference in the vasopressin expression was present with mice carrying Y chromosome (XYM and XYF) having more vasopressin immunopositive fibres than mice carrying two X chromosomes (XXM and XXF). Nevertheless, the organizational effect of gonadal steroids was also present as males (XXM and XYM), regardless of sex chromosomes, had more immunopositive vasopressin fibres than females of both genotypes (XXF and XYF).

Table 1
Sex differences in brain structures and expression of cellular markers in FCG mice model

Abbreviations: GDX, gonadectomy; T, testosterone; F, gonadal females of both genotypes XX and XY; M, gonadal males of both genotypes XX and XY.

Sex differences inthe brainGonadal/hormonal statusResultGonadal hormones effectSex chromosome effectsReferences
Number of PR immunopositive neurons in AVPV, mPOA, VMH Gonads present M > F Yes No [141
Number of motor neurons in spinal nucleus of the bulbocavernosus GDX +T M > F Yes No [140
Cerebral cortex thickness Gonads present M > F Yes No [4
Number of TH positive cells in AVPV GDX +T F > M Yes No [140
Vasopressin fibre density in the lateral septum GDX +T XYF > XXF XYM > XXM M > F Yes Yes [136,140
Oestrogen positive feedback in post-pubertal hypothalamic astrocytes culture (in vitroGonads present XXM and XYM lack feedback Yes No [156
Number of TH-positive cells in embryonic mesencephalon culture (in vitroNo hormones XYM, XYF > XXM, XXF No Yes [157
Sex differences inthe brainGonadal/hormonal statusResultGonadal hormones effectSex chromosome effectsReferences
Number of PR immunopositive neurons in AVPV, mPOA, VMH Gonads present M > F Yes No [141
Number of motor neurons in spinal nucleus of the bulbocavernosus GDX +T M > F Yes No [140
Cerebral cortex thickness Gonads present M > F Yes No [4
Number of TH positive cells in AVPV GDX +T F > M Yes No [140
Vasopressin fibre density in the lateral septum GDX +T XYF > XXF XYM > XXM M > F Yes Yes [136,140
Oestrogen positive feedback in post-pubertal hypothalamic astrocytes culture (in vitroGonads present XXM and XYM lack feedback Yes No [156
Number of TH-positive cells in embryonic mesencephalon culture (in vitroNo hormones XYM, XYF > XXM, XXF No Yes [157
Table 2
Sex differences in different behavioural traits in FCG mice model

Abbreviations: GDX, gonadectomy; T, testosterone; F, gonadal females of both genotypes XX and XY; M, gonadal males of both genotypes XX and XY.

Sex differences inbehavioural traitGonadal/hormonal statusResultGonadal hormones effectSex chromosome effectsReferences
Male copulatory behaviour GDX +T M > F Yes No [140
Social exploration behaviour (time sniffing female) GDX +T M > F Yes No [140
Olfactory preference toward the opposite sex GDX +T XXM and XYM preferred female odour Yes No [136
Aggressive GDX +T XXM, XYM, XYF > XXF Yes Yes [136
Parental GDX XXF > XXM, XYM, XYF Yes Yes [136
Pain sensitivity (latency to escape from a foot shock) GDX F > M Yes No [144
Social interaction GDX     
 (a) Sniffing, grooming  (a) XX mice < XY mice Yes Yes [144
 (b) Digging  (b) XXF > XXM    
 (c) Latency to follow  (c) XXF > XYF    
Adolescent social interaction Gonads inactive     
 (a) Nonsocial with nonsiblings  (a) XXF > XXM, XYF Yes Yes [158
 (b) Social with siblings  (b) XXF > XXM, XYF    
 (c) Solicited play withnonsiblings  (c) XYM > XXM, XYF    
 (d) Solicited play withsiblings  (d) XXF < XXM, XYF    
Habit formation with food reinforcement GDX XX mice > XY mice No Yes [145
Habit formation with alcohol reinforcement Gonads present or GDX XY mice > XX mice No Yes [146
Alcohol consumption ad libitum Gonads present F > M Yes No [146
Sweetened milk consumption ad libitum Gonads present XY mice > XX mice Yes Yes [147
Motivation to obtain reward (sweetened milk) Gonads present XY mice > XX mice Yes Yes [147
Pain sensitivity after thermal or chemical stimulus GDX XX mice > XY mice No Yes [148
Sex differences inbehavioural traitGonadal/hormonal statusResultGonadal hormones effectSex chromosome effectsReferences
Male copulatory behaviour GDX +T M > F Yes No [140
Social exploration behaviour (time sniffing female) GDX +T M > F Yes No [140
Olfactory preference toward the opposite sex GDX +T XXM and XYM preferred female odour Yes No [136
Aggressive GDX +T XXM, XYM, XYF > XXF Yes Yes [136
Parental GDX XXF > XXM, XYM, XYF Yes Yes [136
Pain sensitivity (latency to escape from a foot shock) GDX F > M Yes No [144
Social interaction GDX     
 (a) Sniffing, grooming  (a) XX mice < XY mice Yes Yes [144
 (b) Digging  (b) XXF > XXM    
 (c) Latency to follow  (c) XXF > XYF    
Adolescent social interaction Gonads inactive     
 (a) Nonsocial with nonsiblings  (a) XXF > XXM, XYF Yes Yes [158
 (b) Social with siblings  (b) XXF > XXM, XYF    
 (c) Solicited play withnonsiblings  (c) XYM > XXM, XYF    
 (d) Solicited play withsiblings  (d) XXF < XXM, XYF    
Habit formation with food reinforcement GDX XX mice > XY mice No Yes [145
Habit formation with alcohol reinforcement Gonads present or GDX XY mice > XX mice No Yes [146
Alcohol consumption ad libitum Gonads present F > M Yes No [146
Sweetened milk consumption ad libitum Gonads present XY mice > XX mice Yes Yes [147
Motivation to obtain reward (sweetened milk) Gonads present XY mice > XX mice Yes Yes [147
Pain sensitivity after thermal or chemical stimulus GDX XX mice > XY mice No Yes [148

BEHAVIOURAL STUDIES WITH FCG MICE

From a behavioural perspective, the sex chromosome effect was initially described in aggressive and parental behaviours [136] that are both usually sexually dimorphic [142,143]. In these tests, XXM, XYM and XYF mice showed similar levels of aggressive behaviour, whereas XXF mice were less aggressive. In contrast, in parental behaviour, XXF mice showed highest levels of maternal behaviour in comparison with the other three groups (XYF, XYM and XXM) [136].

In the extensive behavioural study by McPhie-Lalmansingh et al. [144], gonadectomized FCG mice were subjected to several behavioural tests measuring exploratory activity, anxiety-like behaviours, and motor and olfactory functions. In most of the tests, neither a gonadal nor sex chromosome effect was found, except in the active avoidance test where females, regardless of their sex chromosomal complement, escaped faster than males after receiving mild electric foot shocks. In the same study, social interaction was influenced by the presence of different sex chromosome complements. Mice with the XX genotype, irrespective of gonadal sex, spent less time and engaged in fewer bouts of sniffing and grooming the male intruder mice than XY mice did, and XY females were also faster to follow the intruder than XX females were [144].

ADDICTION AND NOCICEPTION

One of the first studies suggesting the importance of the sex chromosome complement in sex differences in addiction was one in which habit formation was observed in FCG mice through food reinforcement [145]. With the training, gonadectomized FCG mice from all groups learned to obtain food pellets from an apparatus. However, when the reward (food) was devaluated by associating the taste of the food with an aversive stimulus, XX mice, regardless of gonadal sex, showed stronger persistence in response to food in comparison with XY mice. The difference between XX and XY mice disappeared with extended training, which suggests that all mice develop habit formation, but XX mice, regardless of gonadal sex, are more sensitive than XY mice [145]. A similar chromosomal effect was found with the alcohol reinforcement [146], although this time XY mice, regardless of their gonadal sex, developed habitual response faster than XX mice. Conversely, when alcohol consumption was measured in an ad libitum context, gonadal status predicted the outcome as females with ovaries (XXF, XYF) consumed more alcohol than gonadal males with testes (XYM, XXM). More recently, another study supported the influence of the sex chromosome complement on the reward system using different dilutions of sweetened condensed milk (SCM) as a reward [147]. When 10% SCM was available to mice ad libitum, an effect of gonadal sex and an effect of sex chromosome complement were found. The SCM was consumed in a greater amount by XY mice than XX mice, but the difference was larger in gonadal females (XXF compared with XYF) than in gonadal males (XXM compared with XYM). This difference disappeared if the concentration of the SCM was increased (to 32%), making the solution even more palatable, or if mice were placed on a mild dietary restriction before the experiment (maintaining 90% of body weight prior to restriction). Under the progressive ratio schedule of the reinforcement, when animals had to put much more effort to obtain the reward, XY mice pressed the lever more often to obtain SCM than XX mice regardless of their gonadal sex, suggesting that XY mice are more sensitive to the rewarding impact of the SCM solution than XX mice are [147].

All these studies [145147] revealed that sex chromosomes significantly influence sex difference in the sensitivity of the reward system, but sex difference also depends on the type of the reinforcer and the context of the reinforcement.

Similar robust sex differences between XX and XY mice were observed in nociception. In the study by Gioiosa et al. [148], two different traits were tested in FCG mice: pain sensitivity after thermal (hot plate) or chemical (injection of the formalin into the footpad) stimuli and the development of tolerance to morphine. All these traits are known to be sexually dimorphic in both rodents and humans [149,150]. To both painful stimuli, thermal or chemical, XX mice reacted with signs of stronger pain than XY mice did. XX mice of both gonadal sexes had shorter latency to escape painful stimuli and were licking the formalin-irritated hind paw more than XY mice of both gonadal sexes were. The difference between XX and XY mice persisted even after seven days of morphine injections. Since all these differences in nociception are noted in the absence of gonadal hormones (gonadectomized animals), and because the effects of chromosome complement were found in both gonadal males and gonadal females, it seems that nociception is strongly influenced by differences in sex chromosome complements [148].

OTHER SEXUALLY DIMORPHIC TRAITS

FCG mice were also intensively studied as a model for different medical conditions and diseases in humans not directly connected with the central nervous system, such as autoimmune diseases [151153], adiposity [154,155] and atherosclerosis [62]. The effects of sex chromosome complement were found in gonadectomized FCG mice models of multiple sclerosis with XX animals showing shorter latencies to develop the disease, and more severe symptoms than XY mice [137,151,153]. Recent data on FCG mice also revealed a significant contribution of the sex chromosome complement to sex differences in weight, adiposity and some metabolic markers. Gonadectomized XX mice of both gonadal sexes had up to 2-fold increased adiposity and greater food intake during daylight in comparison with XY mice. On a high-fat diet, XX mice also had accelerated weight gain, developed more severe fatty liver disease, and had higher elevations in lipid and insulin levels [154].

STEROIDOGENIC FACTOR-1 KNOCKOUT MICE

SF-1, officially designated NR5a1, was discovered to be a regulator of the cytochrome P450 steroidogenic enzymes [159] in the adrenal cortex. SF-1 KO mice are born without adrenal glands and gonads, with dysfunctional pituitary gonadotropes and disorganized VMH [160,161]. Although genital ridges in SF-1 KO mice develop normally until E10.5, rudimentary gonads start to regress and disappear by E12.5 [160]. Due to complete gonadal agenesis, SF-1 KO mice develop in the absence of masculinizing hormones (anti-Mullerian hormone and testosterone). Consequently, they are born with male-to-female sex reversal, with internal and external female reproductive organs and without ovaries [162]. Although phenotypically females, SF-1 KO male and female mice differ in their sex chromosome complement (XX female and XY male). In contrast with FCG mice, in which gonads are present early in development, SF-1 KO males and females are not exposed to endogenous gonadal hormones throughout their entire life. Therefore, the organizational effect of gonadal steroids can be completely separated from activational ones. However, exposure from maternal steroid hormones in utero and from neighbouring male fetuses cannot be excluded [163165], but this type of exposure is not expected to differ systematically between KO males and females. Adrenal insufficiency in newborn SF-1 KO mice results in significantly diminished corticosterone levels and elevated adrenocorticotropic hormone (ACTH) causing death within several hours of birth [162]. Corticosteroid injections followed by adrenal transplantation could rescue these mice so that they survive into adulthood. Another important feature of SF-1 KO mice is disorganized VMH, so compact nucleuses cannot be recognized with Nissl staining.

Studies of adult SF-1 KO mice are important from two perspectives: (i) with highly selective alterations in the VMH organization they present a useful model to study different behaviours and physiological processes that depend on the VMH function, such as feeding and energy balance regulation, as well as aggressive, sexual and affective-like behaviours; (ii) with the lack of gonadal exposure yet the presence of different sex chromosomes SF-1 KO mice present another model to study sex chromosome-dependent sexual differentiation in the brain. By comparing WT and SF-1 KO mice, four groups of mice with different combinations of sex chromosomes and gonads can be observed; WT females (XX, ovaries), WT males (XY, testes, normal prenatal testosterone exposure), SF-1 KO females (XX, no ovaries) and SF-1 KO males (XY, no testes, no prenatal exposure to testosterone, Figure 3). However, like FCG mice, one also has to be careful with the interpretation of the data obtained from these mice. In particular, altered structures of the VMH in SF-1 KO mice could by itself lead to some behavioural discrepancies in comparison with WT mice that are not necessarily due to the absence of gonadal hormones.

Schematic representation of SF-1 KO mice

Figure 3
Schematic representation of SF-1 KO mice

As seen in the diagram, SF-1 KO mice of both sexes are without gonads and are thus phenotypically females, but they still differ in their sex chromosome complements (XX or XY).

Figure 3
Schematic representation of SF-1 KO mice

As seen in the diagram, SF-1 KO mice of both sexes are without gonads and are thus phenotypically females, but they still differ in their sex chromosome complements (XX or XY).

SEXUAL DIFFERENTIATION OF BRAIN AND BEHAVIOUR IN SF-1 KO MICE

In our initial study, we found two neuronal markers that were expressed in a sexually dimorphic manner also in agonadal SF-1 KO mice, neural nitric oxide synthase (nNOS) and calbindin D-28k (Calb) [72]. Both of these proteins were examined in the brain areas known to be sexually dimorphic: the preoptic area (POA) and AVPV, the bed nucleus of stria terminalis (BNST), VMH and the amygdala. In this study, WT control mice were gonadectomized before the onset of puberty and all mice were treated with TP in adulthood before sacrifice. The calbindin immunopositive cell cluster in the MPOA was absent from WT females and in SF-1 KO mice of both sexes, whereas it was distinctly visible in WT males. These results confirm the findings in previous studies that this sex difference is dependent upon prenatal or early postnatal exposure to gonadal hormones. However, the distribution of nNOS immunopositive cells in both POA and AVPV was sexually dimorphic in both genotypes, with WT and SF-1 KO males having a higher number of nNOS positive cells than WT and SF-1 KO females. Since agonadal SF-1 KO males still show a male pattern of nNOS expression, this difference must be regulated by sex chromosomes independently of gonadal secretion. Interestingly, in the AVPV, but not in the POA, a greater difference was observed between WT males and females in comparison with SF-1 KO males and females, which might suggest that the sex difference is initially produced by the action of genes on sex chromosome, but full expression of this difference is only achieved by exposure to sex steroids. In the VMH of SF-1 KO mice, the distribution of both nNOS and calbindin, immunopositive cells were altered as expected due to disorganization of the VMH in SF-1 KO mice. Interestingly, in the VMH calbindin, immunopositive cells showed a significant sex difference in WT mice and in SF-1 KO mice. Males of both genotypes had fewer cells expressing calbindin than females of both genotypes, which again suggests a sex chromosome effect [72].

Recently, the expression of the Kisspeptin1 in AVPV and arcuate nucleus (Arc) were examined in WT and SF-1 KO mice. Kisspeptin1 is a key regulator of gonadotropin-releasing hormone expression and has been implicated in the sexual maturation [166]. In the AVPV, mature female mice have approximately ten times more Kisspeptin1 immunoreactive neurons than males do [87]. Similarly, sex difference was reported before for the Arc, where again Kisspeptin1 mRNA levels are usually higher in females than in males [167]. In SF-1 KO mice, no sex difference between males and females was observed, suggesting that sex difference in Kisspeptin1 expression is solely dependent on the activational effects of sex steroid hormones. However, interestingly, postnatal exposure to oestradiol, which has previously been shown to be necessary for the female pattern of kisspeptin expression [87] was not sufficient to fully restore kisspeptin levels in either SF-1 KO males or females, suggesting that most likely an additional earlier organizational effect is required to enable full expression of kisspeptin in both AVPV and Arc in adult females [168].

To date, two behavioural studies on SF-1 KO mice, one exploring aggressive/social behaviour and another investigating female sexual behaviour were published [96,169]. In the study of aggressive and social behaviour, SF-1 KO mice were compared with WT mice gonadectomized prior to puberty. In this study, only WT males showed aggression (bites and attacks) towards male intruders. Since SF-1 KO mice behaved similarly to WT females despite appropriate hormonal activation, this study suggests that intermale aggression is organized by prenatal and neonatal exposure to gonadal hormones and that sex chromosomes were not sufficient to activate aggression toward gonad-intact males [169]. In the follow-up of these studies, we have observed that postnatal treatment alone, without any prenatal exposure to testosterone, is sufficient to induce aggressive behaviour in SF-1 KO males and females to the levels observed in WT males (Spanic, unpublished observation).

In the second behavioural study, female sexual behaviour was observed in WT mice gonadectomized prior to puberty and in agonadal SF-1 KO mice. All test mice were hormone-primed with oestradiol benzoate and progesterone prior to testing with sexually experienced WT males. Different parameters of female sexual behaviour such as lordosis quotient of the test mice, and penile intromission and thrusting of the stud male during intromission were observed. After testing, PR were visualized by immunostaining. A consistent sex chromosome effect was observed in all parameters of female sexual behaviour. Although WT females showed the highest level of female sexual behaviour, SF-1 KO females had higher lordosis quotients and received a higher number of intromissions and thrusting in comparison with SF-1 KO males. Moreover, the same sex difference was observed in the expression of the PR in the VMH. Although the number of PR immunopositive cells was reduced in the VMH of SF-1 KO mice, a significant sex difference was still observed with SF-1 KO males having fewer cells than SF-1 KO females in the VMH. These results, therefore, suggest that the organization of female sexual behaviour (at least in rodents) is partially regulated by a sex chromosome complement.

CONCLUSION

The conventional view of brain sexual differentiation suggests that neonatal testosterone exposure, at least in rodents converted locally to oestradiol, is responsible for the masculinization of the brain and behaviour. However, studies in the previous two decades have shown that these processes are not so straightforward and are likely much more complex. Several studies suggest that in addition to the neonatal period, later adolescent periods (prepubertal, pubertal) play a major role in the organization of the brain in a sex-specific manner by gonadal hormones. Furthermore, increasing numbers of studies are showing roles of sex chromosome complement in the development and expression of sex differences in the brain, and some recent studies also suggest the involvement of epigenetic effects in the sexual differentiation of the brain. Although sex chromosomal effects and epigenetic influences on the brain are extremely difficult to study in humans, studies in rodents do suggest that these effects should also be considered when discussing sex differences in the human brain.

Therefore, future studies on sex differences in the brain in both health and disease will have to take into account all these contributing factors. Novel descriptions of sexual differentiation of the brain will have to be developed and experimentally confirmed in order to better understand the development of such differences in the brain, sex differences in behaviour and, especially, sex differences in the prevalence and symptoms of many diseases of central nervous system.

FUNDING

This work was supported by Slovenian Research Agency (ARRS) [grant numbers P4-0053, J3-6801 and J7-7226 (to N.G. and G.M.)].

Abbreviations

     
  • AVPV

    anteroventral periventricular nucleus

  •  
  • BNST

    bed nucleus of the stria terminalis

  •  
  • FCG

    four core genotype

  •  
  • LS

    lateral septum

  •  
  • nNOS

    neural nitric oxide synthase

  •  
  • PR

    progesterone receptors

  •  
  • SCM

    sweetened condensed milk

  •  
  • SF-1

    steroidogenic factor-1

  •  
  • TFM

    testicular feminization

  •  
  • TP

    testosterone propionate

  •  
  • VMH

    ventromedial nucleus of the hypothalamus

  •  
  • VP-ir fibres

    vasopresin immunoreactive fibres

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