Sex differences in androgen-regulated expression of cytochrome P450 aromatase in the rat brain

The basis of functional gender differences in adult responsiveness to testosterone (T) is not yet understood. Conversion of T to estradiol by cytochrome P450 aromatase in the medial preoptic area is required for the full expression of male sexual behavior in rats. High levels of aromatase are found in the medial preoptic nucleus (MPN) and in an interconnected group of sexually dimorphic nuclei which mediate masculine sexual behavior. Within this neural circuit, aromatase is regulated by T, acting through an androgen receptor (AR)-mediated mechanism. This arrangement constitutes a feedforward system because T is both the regulator and the major substrate of aromatase. Preoptic aromatase is thus more active in adult males than in females because of normal sex differences in circulating androgen levels. However, the mechanism of enzyme induction also appears to be sexually dimorphic because equivalent physiological doses of T stimulate aromatase to a greater extent in males than in females. Dose-response studies indicate that the sex difference is apparent over a range of circulating T concentrations and constitute a gender difference in T efficacy, but not potency. Sex differences in aromatase correlate with sex differences in nuclear AR concentrations in most regions of the sexually dimorphic neural circuit, but not in MPN. These results suggest that males may have larger populations of target cells in which aromatase is regulated by androgen, but the lack of a gender difference in AR levels in the MPN suggests that differences in post-receptor mechanisms could also be involved. Measurements of aromatase mRNA in androgen-treated gonadectomized rats demonstrate that sex difference in regulation is exerted pretranslationally. Taken together these results demonstrate a sexually dimorphic mechanism that could potentially limit the action of T in females, and may relate to the enhanced expression of T-stimulated sexual behaviors in males.     

Sex differences and androgen-dependent regulation of aromatase (CYP19) mRNA expression in the developing and adult rat brain

Sex differences, androgen dependence and asymmetries of aromatase activity have been reported during ontogeny of the rat. It remains to be elucidated, however, whether the changes in aromatase activity are reflected by similar changes in specific mRNA levels. In addition, very little is known regarding mechanism(s) underlying such differential regulation of aromatase expression. To address these questions, we have employed the in situ hybridization (ISH) technique to examine specific mRNA levels in the brain of both male and female rats at selected stages of development. In prenatal stages of development, at gestational day (GD) 18 and 20, aromatase mRNA was detected in several hypothalamic and limbic brain regions. Semiquantitative analysis of aromatase mRNA did not reveal statistically significant sex differences in any of these regions (except in one experiment at GD20, when a sex difference was found in the medial preoptic nucleus). In contrast, clear sex differences were determined at postnatal day (PN) 2; male animals contained significantly more aromatase mRNA in the bed nucleus of the stria terminalis (BST) and thesexually dimorphic nucleus of the preoptic area (SDN) compared to female rats. Four days later in development, at PN6, sex differences of aromatase mRNA signals were observed in the BST, but were no longer detectable in the SDN. At PN15 and in adult animals, no sex differences could be determined. The effect of flutamide treatment (50 mg/kg/day) was investigated in GD20 fetuses as well as in adult rats. No statistically significant changes in aromatase mRNA expression were found in either case. In summary, our results suggest that differential regulation of aromatase mRNA expression during the critical period of sexual differentiation might, in part, account for the establishment of some of the many sexually dimorphic parameters of the rat brain. The role of androgens in the regulation of the sex-specific and developmental expression of aromatase mRNA in the rat brain remains to be clarified.     

Sexual dimorphism in the developmental regulation of brain aromatase

Steroid sex hormones have an organizational role in gender-specific brain development. Aromatase (cytochrome P450_AR), converting testosterone (T) to estradiol-17β (E₂) is a key enzyme in brain development and the regulation of aromatase determines the availability of E₂ effective for neural differentiation. Gender differences in brain development and behaviour are likely to be influenced by E₂ acting during sensitive periods. This differentiating action has been demonstrated in rodent and avian species, but also probably occurs in primates including humans. In rodents, E₂ is formed in various hypothalamic areas of the brain during fetal and postnatal development. The question considered here is whether hypothalamic aromatase activity is gender-specific during sensitive phases of behavioural and brain development, and when these sensitive phases occur. In vitro preoptic and limbic aromatase activity has been measured in two strains of wild mice, genetically selected for behavioural aggression based on attack latency, and in the BALB/c mouse. Short attack latency males show a different developmental pattern of aromatase activity in hypothalamus and amygdala to long attack latency males. Using primary brain cell cultures of the BALB/c mouse, sex differences in hypothalamic aromatase activity during both early embryonic and later perinatal development can be demonstrated, with higher E₂ formation in males. The sex dimorphisms are brain region specific, since no differences between male and female are detectable in cultured cortical cells. Immunoreactive staining with a polyclonal aromatase antibody identifies a neuronal rather than an astroglial localization of the enzyme. T increases fetal brain aromatase activity and numbers of aromatase-immunoreactive hypothalamic neuronal cell bodies. T appears to influence the growth of hypothalamic neurons containing aromatase. Differentiation of sexually dimorphic brain mechanisms may involve maturation of a gender-specific network of estrogen-forming neurons which are steroid-sensitive in early development.     

Sex differences in cortisol's regulation of affiliative behavior

A contribution to a special issue on Hormones and Human Competition.A stress perspective is used to illuminate how competitive defeat and victory shape biology and behavior. We report a field study examining how change in cortisol following perceived defeat (vs. victory) in a competition—in this case, a dog agility competition—relates to affiliative behavior. Following competition, we measured cortisol change and the extent to which dog handlers directed affiliative behaviors toward their dogs. We found striking sex differences in affiliation. First, men were more affiliative toward their dogs after victory, whereas women were more affiliative after defeat. Second, the greater a female competitor's increase in cortisol, the more time she spent affiliating with her dog, whereas for men, the pattern was the exact opposite: the greater a male competitor's increase in cortisol, the less time he spent affiliating with his dog. This pattern suggests that, in the wake of competition, men and women's affiliative behavior may serve different functions—shared celebration for men; shared consolation for women. These sex differences show not only that men and women react very differently to victory and defeat, but also that equivalent changes in cortisol across the sexes are associated with strikingly different behavioral consequences for men and women.     

Sex differences in neuropeptide distribution in the rat brain

We have investigated possible sex differences in the regional concentrations of neuropeptides in the rat brain. Immunoreactive neurotensin (NT), neurokinin A (NKA), galanin (GAL), calcitonin gene-related peptide (CGRP), substance P (SP) and neuropeptide Y (NPY) were measured by radioimmunoassay in frontal cortex, occipital cortex, hippocampus, striatum, hypothalamus and pituitary in male and female pre- and postpubertal rats. Sex differences were found for NPY (p < 0.001), NT (p < 0.01) and GAL (p < 0.05), in particular in hippocampus, striatum, hypothalamus and pituitary, but not for CGRP, SP and NKA. Results from analysis of neuropeptides in one sex may not be entirely applicable to the other.     

Analysis of spatiotemporal regulation of aromatase in the brain using transgenic mice

Brain aromatase is widely distributed in the vertebrates, from fish to mammals, and plays important roles in functional reproductive behavior through production of estrogen as a neurosteroid. It is expressed only in the nerve cells of specific brain regions with a transient peak in the neonatal period when sexual behavior becomes organized, and therefore provides a good model system to study regulatory mechanism of cell-specific, brain region-specific, and developmental stage-specific expression.

To elucidate spatiotemporal regulation of brain aromatase, we prepared transgenic mice carrying a reporter gene under the promoter of brain-specific exon 1f of the mouse aromatase gene. The reporter transgene carrying a 6.5 kb upstream region of the brain-specific promoter accurately reproduced the spatiotemporal expression patterns of aromatase in mouse brain, whereas transgenes carrying smaller fragments of the promoter showed ambiguous or inconsistent expression patterns.

The binding sites of Aro-AI, Aro-AII, and Aro-B for nuclear factors were also identified in the proximal region of the exon 1f brain-specific promoter. Introduction of a mutation into the Aro-AII site in the reporter transgene carrying −6.5 kb promoter region of exon 1f caused complete alteration of the spatiotemporal expression pattern of the reporter gene in the transgenic mice.

These results indicate that the −6.5 kb promoter region of exon 1f is the minimal essential element for brain-specific regulation, with both proximal and distal promoter regions required for accurate spatiotemporal expression of aromatase in the mouse brain.     

Sex differences in microglial colonization of the developing rat brain

Microglia are the resident immune cells within the brain and their production of immune molecules such as cytokines and chemokines is critical for the processes of normal brain development including neurogenesis, axonal migration, synapse formation, and programmed cell death. Notably, sex differences exist in many of these processes throughout brain development; however, it is unknown whether a sex difference concurrently exists in the colonization, number, or morphology of microglia within the developing brain. We demonstrate for the first time that the number and morphology of microglia throughout development is dependent upon the sex and age of the individual, as well as the brain region of interest. Males have overall more microglia early in postnatal development [postnatal day (P) 4], whereas females have more microglia with an activated/amoeboid morphology later in development, as juveniles and adults (P30-60). Finally, gene expression of a large number of cytokines, chemokines and their receptors shifts dramatically over development, and is highly dependent upon sex. Taken together, these data warrant further research into the role that sex-dependent mechanisms may play in microglial colonization, number, and function, and their potential contribution to neural development, function, or potential dysfunction.     

Sex differences in hormonal responses of vasopressin pathways in the rat brain

Vasopressin (AVP) immunoreactivity in cells and projections of the bed nucleus of the stria terminalis (BST) and medial amygdaloid nucleus (MA) depends on gonadal steroids. In addition, the AVP projections from the BST show denser fiber staining in males than in females. To study whether these differences depend on different hormone levels in adulthood, male and female rats were gonadectomized and similarly treated with testosterone for 4 weeks prior to sacrifice. Immunocytochemistry showed that males had significantly more AVP-immunoreactive (AVP-IR) cells in the BST and significantly denser AVP-IR projections from this nucleus to the lateral septum, lateral habenular nucleus, and periaqueductal central gray than did females. The number of AVP-IR cells in the MA nucleus was not statistically different, but denser AVP-IR fiber networks were found in the MA and ventral hippocampus, which receives its input from the MA. No differences were found in the anteroventral portion of the periventricular nucleus and the dorsomedial nucleus of the hypothalamus that receive their AVP innervation from the suprachiasmatic nucleus. These results indicate that the sex difference in the steroid-sensitive AVP pathways depends on other factors besides circulating hormone levels in adulthood.     

Expression of estrogen receptor and aromatase mRNAs in embryonic and posthatch zebra finch brain

Male zebra finches sing and females normally do not. This sexually dimorphic behavior is mediated by a sexually dimorphic series of interconnected nuclei that are larger and more developed in males. Estradiol administered to females as early as the day of hatching (P1) causes profound masculinization of this song system. The exact timing of estrogen action is unknown, and there is little information concerning the times and sites of expression of estrogen receptors and aromatase before P5. We measured the expression of mRNAs encoding these proteins in brain during late embryogenesis and on P1 to determine if estrogen synthesis or receptor-mediated actions on the song system, as part of the program of sexual differentiation, might be possible during this period. Using highly sensitive and specific in situ hybridization procedures for mRNAs encoding ERalpha, ERbeta, and aromatase, we detected mRNA for ERs in archistriatal regions as early as embryonic stage 34, and in diencephalic regions as early as embryonic stage 30. ERalpha mRNA was also detected in the dorsal mesencephalon at P1. Aromatase mRNA expression was present as early as embryonic stage 30 in diencephalic and mesencephalic regions. No obvious sex differences in the spatio-temporal pattern of mRNA expression were detected. Our results suggest that estrogen can influence cell growth and differentiation in zebra finch brain well before hatching and into posthatching life. The results fail to provide support for the hypothesis that sexual differentiation of the song system is mediated by sex differences in the expression of these mRNAs at these ages.     

Sex change strategy and the aromatase genes

Sequential hermaphroditism is a common reproductive strategy in many teleosts. Steroid production is known to mediate both the natural and induced sex change, yet beyond this the physiology directing this process has received little attention. Cytochrome P450 aromatase is a key enzyme in the hormonal pathway catalysing the conversion of sex steroids, androgens to oestrogens, and thus is highly relevant to the process of sex change. This study reports the isolation of cDNA sequences for aromatase isoforms CYP19A1 and CYP19A2 from teleost species representing three forms of sexual hermaphroditism: Lates calcarifer (protandry), Cromileptes altivelis (protogyny), and Gobiodon histrio (bi-directional). Deduced amino acid analysis of these isoforms with other reported isoforms from gonochoristic (single sex) teleosts revealed 56–95% identity within the same isoform while only 48–65% identity between isoforms irrespective of species and sexual strategy. Phylogenetic analysis supported this result separating sequences into isoform exclusive clades in spite of species apparent evolutionary distance. Furthermore, this study isolates 5′ flanking regions of all above genes and describes putative cis-acting elements therein. Elements identified include steroidogenic factor 1 binding site (SF-1), oestrogen response element (ERE), progesterone response element (PRE), androgen response element (ARE), glucocorticoid response elements (GRE), peroxisome proliferator-activated receptor /retinoid X receptor heterodimer responsive element (PPAR/RXR), nuclear factor kappaβ (NF-kappaβ), SOX 5, SOX 9, and Wilms tumor suppressor (WTI). A hypothetical in vivo model was constructed for both isoforms highlighting potential roles of these putative cis-acting elements with reference to normal function and sexual hermaphroditism.     

Sex differences in the brain: The relation between structure and function

In the fifty years since the organizational hypothesis was proposed, many sex differences have been found in behavior as well as structure of the brain that depend on the organizational effects of gonadal hormones early in development. Remarkably, in most cases we do not understand how the two are related. This paper makes the case that overstating the magnitude or constancy of sex differences in behavior and too narrowly interpreting the functional consequences of structural differences are significant roadblocks in resolving this issue.     

Sex differences in estrogen and androgen receptors in hamster brain.

The present study was conducted to measure the levels of estrogen and androgen receptors (ER and AR, receptively) simultaneously in the anterior pituitary (AP), and various brain regions from adult male and proestrous female hamsters. Medial preoptic area (MPOA), medial basal hypothalamus (MBH), lateral hypothalamus (LH), medial forebrain bundle (MFB), and amygdala (AMG) were identified and removed from 200-microns frozen brain sections by the Palkovits punch-out technique. ER and AR were determined by the in vitro binding assay using [3H]-estradiol and [3H]-methyltrienolone as the binding ligands. In males, high levels of AR were found in the MPOA, MBH, and AP. In females, the MPOA, MBH, LH, and AP contained high levels of ER. The males exhibited significantly higher levels of AR than females in the MPOA, MBH, and LH, whereas the ER levels in these areas were higher in females. In males, ER and AR contents in the AP were higher, but the contents in the AMG were lower as compared to those of females. The calculated ER/AR ratio in MPOA, MBH, and LH were lowest in males. On the contrary, the ratio in these areas were highest in females. These data suggest that sex differences in response to estrogen and androgen may in part be due to sex differences in ER and AR contents in specific brain regions.     

Sex reversal and aromatase in chicken.

Aromatase inhibitors administered before sexual differentiation of the gonads can induce sex reversal in female chickens. To analyze the process of sex reversal, we have followed for several months the changes induced by Fadrozole, a nonsteroidal aromatase inhibitor, in gonadal aromatase activity and in morphology and structure of the female genital system. Fadrozole was injected into eggs on day four of incubation, and its effects were examined during the embryonic development and for eight months after hatching. In control females, aromatase activity in the right and the left gonad was high in the middle third of embryonic development, and then decreased up to hatching. After hatching, aromatase activity increased in the left ovary, in particular during folliculogenesis, whereas in the right regressing gonad, it continued to decrease to reach testicular levels at one month. In treated females, masculinization of the genital system was characterized by the maintenance of the right gonad and its differentiation into a testis, and by the differentiation of the left gonad into an ovotestis or a testis; however, in all individuals, the left Müllerian duct and the posterior part of the right Müllerian duct were maintained. In testes and ovotestes, aromatase activity was lower than in gonads of control females (except in the right gonad as of one month after hatching) but remained higher than in testes of control and treated males. Moreover, in ovotestes, aromatase activity was higher in parts displaying follicles than in parts devoid of follicles. The main structural changes in the gonads during sex reversal were partial (in ovotestes) or complete (in testes) degeneration of the cortex in the left gonad, and formation of an albuginea and differentiation of testicular cords/tubes in the two gonads. Testicular cords/tubes transdifferentiated from ovarian medullary cords and lacunae whose epithelium thickened and became Sertolian. Transdifferentiation occurred all along embryonic and postnatal development; thus, new testicular cords/tubes were continuously formed while others degenerated. The sex reversed gonads were also characterized by an abundant fibrous interstitial tissue and abnormal medullary condensations of lymphoid-like cells; in the persisting testicular cords/tubes, spermatogenesis was delayed and impaired. Related to aromatase activity, persistence of too high levels of estrogens can explain the presence of oviducts, gonadal abnormalities and infertility in sex reversed females.     

Sex differences and effects of neonatal aromatase inhibition on masculine and feminine copulatory potentials in prairie voles

Copulatory behaviors in most rodents are highly sexually dimorphic, even when circulating hormones are equated between the sexes. Prairie voles (Microtus ochrogaster) are monomorphic in their display of some social behaviors, including partner preferences and parenting, but differences between the sexes in their masculine and feminine copulatory behavior potentials have not been studied in detail. Furthermore, the role of neonatal aromatization of testosterone to estradiol on the development of prairie vole sexual behavior potentials or their brain is unknown. To address these issues, prairie vole pups were injected daily for the first week after birth with 0.5 mg of the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD) or oil. Masculine and feminine copulatory behaviors in response to testosterone or estradiol were later examined in both sexes. Males and females showed high mounting and thrusting in response to testosterone, but only males reliably showed ejaculatory behavior. Conversely, males never showed feminine copulatory behaviors in response to estradiol. Sex differences in these behaviors were not affected by neonatal ATD, but ATD-treated females received fewer mounts and thrusts than controls, possibly indicating reduced attractiveness to males. In other groups of subjects, neonatal ATD demasculinized males' tyrosine hydroxylase expression in the anteroventral periventricular preoptic area, and estrogen receptor alpha expression in the medial preoptic area. Thus, although sexual behavior in both sexes of prairie voles is highly masculinized, aromatase during neonatal life is necessary only for females' femininity. Furthermore, copulatory behavior potentials and at least some aspects of brain development in male prairie voles are dissociable by their requirement for neonatal aromatase.     

Sex differences in regional brain response to aversive pelvic visceral stimuli

To explore sex differences in the response of seven brain regions to an aversive pelvic visceral stimulus, functional magnetic resonance images were acquired from 13 healthy adults (6 women) during 15 s of cued rectal distension at two pressures: 25 mmHg (uncomfortable), and 45 mmHg (mild pain), as well as during an expectation condition (no distension). Random-effects analyses combining subject data voxelwise found 45-mmHg pressure significantly activated the insular and anterior cingulate cortices in both sexes. In men only, the left thalamus and ventral striatum were also activated. Although all activations appeared more extensive in men, no sex difference attained significance. To explore the presence of deactivations, which are generally cancelled by more numerous activations when subjects are combined for each voxel, the number of activated voxels, number of deactivated voxels, and ratio of deactivated voxels to total voxels affected were assessed via random-effects, mixed-model analyses combining subject data at the region level. Greater insula activation in men compared with women was seen during the expectation condition and during the 25-mmHg distension. Greater deactivations in women were seen in the amygdala (25-mmHg distension) and midcingulate (45-mmHg distension). Women had a significantly higher proportion of deactivated voxels than men in all four subcortical structures during 25-mmHg distension. Greater familiarity of females with physiological pelvic visceral discomfort may have enhanced brain systems that dampen arousal networks during lower levels of discomfort.     

Sex differences and the development of the rabbit brain: effects of vinclozolin

The preoptic/anterior hypothalamic area (POA/AH) is one of the most sexually dimorphic areas of the vertebrate brain and plays a pivotal role in regulating male sexual behavior. Vinclozolin is a fungicide thought to be an environmental antiandrogen, which disrupts masculine sexual behavior when administered to rabbits during development. In this study, we examined several characteristics of the rabbit POA/AH for sexual dimorphism and endocrine disruption by vinclozolin. Pregnant rabbits were dosed orally with vinclozolin (10 mg/kg body weight) or carrot paste vehicle once daily for 6 wk beginning at midgestation and continuing through nursing until Postpartum Week 4. At 6 wk, offspring were perfused with 4% paraformaldehyde and brains processed for immunocytochemical localization of tyrosine hydroxylase, calbindin, gonadotropin-releasing hormone (GnRH), or Nissl stain. There were significant sex differences in the distribution of calbindin in the POA/AH and the size of cells in the dorsal POA/AH (values greater in females than in males), but not in the number or distribution of tyrosine hydroxylase or GnRH neurons. In both sexes, exposure to vinclozolin significantly increased calbindin expression in the ventral POA/AH and significantly decreased number of GnRH neurons selectively in the region of the organum vasculosum of the lamina terminalis (OVLT) but not more caudally in the POA/AH. This is the first documentation of a sexually dimorphic region in the rabbit brain, and further supports the use of this species as a model for studying the influence of vinclozolin on reproductive development with potential application to human systems.