Sex differences in progesterone receptor immunoreactivity in neonatal mouse brain depend on estrogen receptor alpha expression

Around the time of birth, male rats express higher levels of progesterone receptors in the medial preoptic nucleus (MPN) than female rats, suggesting that the MPN may be differentially sensitive to maternal hormones in developing males and females. Preliminary evidence suggests that this sex difference depends on the activation of estrogen receptors around birth. To test whether estrogen receptor alpha (ER alpha) is involved, we compared progesterone receptor immunoreactivity (PRir) in the brains of male and female neonatal mice that lacked a functional ER alpha gene or were wild type for the disrupted gene. We demonstrate that males express much higher levels of PRir in the MPN and the ventromedial nucleus of the neonatal mouse brain than females, and that PRir expression is dependent on the expression of ER alpha in these regions. In contrast, PRir levels in neocortex are not altered by ER alpha gene disruption. The results of this study suggest that the induction of PR via ER alpha may render specific regions of the developing male brain more sensitive to progesterone than the developing female brain, and may thereby underlie sexual differentiation of these regions.     

Sex differences in progesterone receptor immunoreactivity in neonatal mouse brain depend on estrogen receptor α expression

Around the time of birth, male rats express higher levels of progesterone receptors in the medial preoptic nucleus (MPN) than female rats, suggesting that the MPN may be differentially sensitive to maternal hormones in developing males and females. Preliminary evidence suggests that this sex difference depends on the activation of estrogen receptors around birth. To test whether estrogen receptor alpha (ERα) is involved, we compared progesterone receptor immunoreactivity (PRir) in the brains of male and female neonatal mice that lacked a functional ERα gene or were wild type for the disrupted gene. We demonstrate that males express much higher levels of PRir in the MPN and the ventromedial nucleus of the neonatal mouse brain than females, and that PRir expression is dependent on the expression of ERα in these regions. In contrast, PRir levels in neocortex are not altered by ERα gene disruption. The results of this study suggest that the induction of PR via ERα may render specific regions of the developing male brain more sensitive to progesterone than the developing female brain, and may thereby underlie sexual differentiation of these regions. © 2001 John Wiley & Sons, Inc. J Neurobiol 47: 176–182, 2001     

Sex-role reversal is reflected in the brain of African black coucals (Centropus grillii)

In most bird species males compete over access to females and have elevated circulating androgen levels when they establish and defend a breeding territory or guard a mate. Testosterone is involved in the regulation of territorial aggression and sexual display in males. In few bird species the traditional sex-roles are reversed and females are highly aggressive and compete over access to males. Such species represent excellent models to study the hormonal modulation of aggressive behavior in females. Plasma sex steroid concentrations in sex-role reversed species follow the patterns of birds with "traditional" sex-roles. The neural mechanisms modulating endocrine secretion and hormone-behavior interactions in sex-role reversed birds are currently unknown. We investigated the sex differences in the mRNA expression of androgen receptors, estrogen receptor alpha, and aromatase in two brain nuclei involved in reproductive and aggressive behavior in the black coucal, the nucleus taeniae and the bed nucleus of the stria terminalis. In the bed nucleus there were no sex differences in the receptor or aromatase expression. In the nucleus taeniae, however, we show for the first time, that females have a higher mRNA expression of androgen receptors than males. These results suggest that the expression of agonistic and courtship behavior in females does not depend on elevated blood hormone levels, but may be regulated via increased steroid hormone sensitivity in particular target areas in the brain. Hence, aggression in females and males may indeed be modulated by the same hormones, but regulated at different levels of the neuroendocrine cascade.     

Sex differences and similarities in the neuroendocrine regulation of social behavior in an African cichlid fish

An individual's position in a social hierarchy profoundly affects behavior and physiology through interactions with community members, yet little is known about how the brain contributes to status differences between and within the social states or sexes. We aimed to determine sex-specific attributes of social status by comparing circulating sex steroid hormones and neural gene expression of sex steroid receptors in dominant and subordinate male and female Astatotilapia burtoni, a highly social African cichlid fish. We found that testosterone and 17β-estradiol levels are higher in males regardless of status and dominant individuals regardless of sex. Progesterone was found to be higher in dominant individuals regardless of sex. Based on pharmacological manipulations in males and females, progesterone appears to be a common mechanism for promoting courtship in dominant individuals. We also examined expression of androgen receptors, estrogen receptor α, and the progesterone receptor in five brain regions that are important for social behavior. Most of the differences in brain sex steroid receptor expression were due to sex rather than status. Our results suggest that the parvocellular preoptic area is a core region for mediating sex differences through androgen and estrogen receptor expression, whereas the progesterone receptor may mediate sex and status behaviors in the putative homologs of the nucleus accumbens and ventromedial hypothalamus. Overall our results suggest sex differences and similarities in the regulation of social dominance by gonadal hormones and their receptors in the brain.     

Sexually dimorphic androgen and estrogen receptor mRNA expression in the brain of túngara frogs

Sex steroid hormones are potent regulators of behavior and they exert their effects through influences on sensory, motor, and motivational systems. To elucidate where androgens and estrogens can act to regulate sex-typical behaviors in the túngara frog (Physalaemus pustulosus), we quantified expression of the androgen receptor (AR), estrogen receptor alpha (ERα), and estrogen receptor beta (ERβ) genes in the brains of male and females. To do so, we cloned túngara-specific sequences for AR, ERα, and ERβ, determined their distribution in the brain, and then quantified their expression in areas that are important in sexual communication. We found that AR, ERα, and ERβ were expressed in the pallium, limbic forebrain (preoptic area, hypothalamus, nucleus accumbens, amygdala, septum, striatum), parts of the thalamus, and the auditory midbrain (torus semicircularis). Males and females had a similar distribution of AR and ER expression, but expression levels differed in some brain regions. In the auditory midbrain, females had higher ERα and ERβ expression than males, whereas males had higher AR expression than females. In the forebrain, females had higher AR expression than males in the ventral hypothalamus and medial pallium (homolog to hippocampus), whereas males had higher ERα expression in the medial pallium. In the preoptic area, striatum, and septum, males and females had similar levels of AR and ER expression. Our results suggest that sex steroid hormones have sexually dimorphic effects on auditory processing, sexual motivation, and possibly memory and, therefore, have important implications for sexual communication in this system.     

Comparison of sociability,parental care and central estrogen receptor alpha expression between two populations of mandarin voles (<Emphasis Type="Italic">Microtus mandarinus</Emphasis>)

The socially monogamous mandarin vole (Microtus mandarinus) shows significant behavioral plasticity. We examined whether levels of sociability, parental care and central expression of estrogen receptor alpha differed between two populations with different ecologies. Our results show that males from the Chengcun population display significantly more amicable and less aggressive behaviors towards novel same-sex individuals compared to males from the second population of Xinzheng. Chengcun voles directed more licking behavior towards neonatal pups than did Xinzheng voles. Differences were also found in the number of estrogen receptor alpha-immunoreactive neurons. For example, Xinzheng males displayed significantly higher immunoreactivity than Chengcun males in the medial amygdala, medial preoptic area and ventromedial nucleus of the hypothalamus. Xinzheng females expressed higher levels of estrogen receptor alpha-immunoreactivity than Chengcun females in the medial preoptic area. Chengcun females exhibited significantly more estrogen receptor alpha expression than Xinzheng females in the bed nucleus of the stria terminalis. Our results indicate that mandarin voles from the Chengcun site possess monogamous traits, and animals from Xinzheng possess polygamous traits. It also appears that different social behavior and levels of parental care in these two populations may be associated with differences in estrogen receptor alpha-immunoreactive neurons.     

Conserved aquaporin 4 levels associated with reduction of brain edema are mediated by estrogen in the ischemic brain after experimental stroke

Aquaporin 4 (AQP4), the most abundant water channel protein in the brain, is involved in brain edema induced by ischemic insults. To evaluate whether the neuroprotective effects of estrogen are associated with AQP4 expression and edema formation, changes in AQP levels and ischemic edema were examined in the brains of male and female mice subjected to transient middle cerebral artery occlusion. Infarct volume and edema formation were markedly less in females than in males. AQP4 expression in the ischemic cortex of females was relatively well preserved, whereas it was significantly decreased in males. These effects disappeared in ovariectomized females but were reversed by estrogen replacement. Furthermore, AQP4 expression was decreased with increased brain edema in females treated with ICI182,780, an estrogen receptor antagonist. These findings suggest that the estrogen effect on the reduction of ischemic brain edema is associated with the preserved level of AQP4 that is partly mediated by estrogen receptors.     

Evidence for extended action of gonadal hormones on the organization of sexually dimorphic behavior and morphology in gray short-tailed opossums (Monodelphis domestica)

Male and female gray short-tailed opossums were gonadectomized (GDX), or treated with the estrogen receptor antagonist tamoxifen citrate (TX), or corn oil (OIL) (control) during the 5th postnatal week, a time period equivalent to the 3rd postnatal week in rats and associated with high levels of circulating gonadal hormones and neural aromatase activity in this marsupial species. In adulthood following gonadectomy (for animals not previously gonadectomized) and replacement therapy with estradiol or testosterone, GDX males showed less male-typical scent marking and had shorter phalluses than OIL and TX males. Following replacement therapy with estradiol, GDX females were more likely to fight with and less likely to mate with stimulus males than TX females; OIL females were intermediate in these measures. Along with previous findings, these results suggest that gonadal hormones act over an extended postnatal period to organize sexually dimorphic behavior and morphology in male gray opossums and may have some effect on the organization of aggressive behavior in females of this species.     

Depot differences in steroid receptor expression in adipose tissue: possible role of the local steroid milieu

Sex hormones play an important role in adipose tissue metabolism by activating specific receptors that alter several steps of the lipolytic and lipogenic signal cascade in depot- and sex-dependent manners. However, studies focusing on steroid receptor status in adipose tissue are scarce. In the present study, we analyzed steroid content [testosterone (T), 17beta-estradiol (17beta-E2), and progesterone (P4)] and steroid receptor mRNA levels in different rat adipose tissue depots. As expected, T levels were higher in males than in females (P = 0.031), whereas the reverse trend was observed for P4 (P < 0.001). It is noteworthy that 17beta-E2 adipose tissue levels were higher in inguinal than in the rest of adipose tissues for both sexes, where no sex differences in 17beta-E2 tissue levels were noted (P = 0.010 for retroperitoneal, P = 0.005 for gonadal, P = 0.018 for mesenteric). Regarding steroid receptor levels, androgen (AR) and estrogen receptor (ER)alpha and ERbeta densities were more clearly dependent on adipose depot location than on sex, with visceral depots showing overall higher mRNA densities than their subcutaneous counterparts. Besides, expression of ERalpha predominated over ERbeta expression, and progesterone receptor (PR-B form and PR-A+B form) mRNAs were identically expressed regardless of anatomic depot and sex. In vitro studies in 3T3-L1 cells showed that 17beta-E2 increased ERalpha (P = 0.001) and AR expression (P = 0.001), indicating that estrogen can alter estrogenic and androgenic signaling in adipose tissue. The results highlighted in this study demonstrate important depot-dependent differences in the sensitivity of adipose tissues to sex hormones between visceral and subcutaneous depots that could be related to metabolic situations observed in response to sex hormones.     

Methylation at global LINE-1 repeats in human blood are affected by gender but not by age or natural hormone cycles

Previously, we reported on inter-individual and gender specific variations of LINE-1 methylation in healthy individuals. In this study, we investigated whether this variability could be influenced by age or sex hormones in humans. To this end, we studied LINE-1 methylation in vivo in blood-derived DNA from individuals aged 18 to 64 years and from young healthy females at various hormone levels during the menstrual cycle. Our results show that no significant association with age was observed. However, the previously reported increase of LINE-1 methylation in males was reconfirmed. In females, although no correlation between LINE-1 or Alu methylation and hormone levels was observed, a significant stable individual specific level of methylation was noted. In vitro results largely confirmed these findings, as neither estrogen nor dihydrotestosterone affected LINE-1 or Alu methylation in Hek293T, HUVEC, or MDA-kb2 cell lines. In contrast, a decrease in methylation was observed in estrogen-treated T47-Kbluc cell lines strongly expressing estrogen receptor. The very low expression of estrogen receptor in blood cells could explain the observed insensitivity of methylation at LINE-1 to natural hormonal variations in females. In conclusion, neither natural cycle of hormones nor age has a detectable effect on the LINE-1 methylation in peripheral blood cells, while gender remains an important factor.     

Role of aromatase and androgen receptor expression in gonadal sex differentiation of ZW/ZZ-type frogs,Rana rugosa

To elucidate the mechanisms of amphibian gonadal sex differentiation, we examined the expression of aromatase and androgen receptor (AR) mRNAs for days 17-31 after fertilization. The effects of inhibitors and sex steroid hormones were also examined. In ZZ males, expression of AR decreased after day 19, while aromatase expression was low throughout the sampling period. Males treated with 17beta-estradiol (E2) showed increasing aromatase expression after day 21, and formed ovaries. AR antagonist treatment also induced high-level aromatase expression and ovarian differentiation. In males co-treated with an aromatase inhibitor and E2, the undifferentiated gonads developed into testes despite high-level aromatase expression. Males treated with androgen and E2 before and during an estrogen sensitive period, respectively, also formed testes. In ZW females, AR expression persisted at a low-level, while aromatase expression increased after day 18. Short-term treatment with an aromatase inhibitor was ineffective in preventing ovarian differentiation, whereas long-term treatment resulted in testes developing from ovarian structure. Compared with the ZZ males and ZW females, WW females did not exhibit detectable expression of AR, suggesting that the active AR gene(s) itself, or a putative gene regulating AR gene expression, is located on Z chromosomes. From the time lag of aromatase expression between ZW females and ZZ males treated with E2 and the effect of AR antagonist, it was found that in males elevated AR expression suppresses aromatase expression directly or indirectly. Consequently, endogenous androgens, accumulated by blocking estrogen biosynthesis, induced testicular differentiation. The gonadogenesis of males is dependent on sex hormone, whereas that of females has evolved to hormone-independence.     

Multihormonal regulation of hepatic sinusoidal Ntcp gene expression

Bile acids are efficiently removed from sinusoidal blood by a number of transporters including the Na+-taurocholate-cotransporting polypeptide (Ntcp). Na+-dependent bile salt uptake, as well as Ntcp, are expressed twofold higher in male compared with female rat livers. Also, estrogen administration to male rats decreases Ntcp expression. The aims of this study were to determine the hormonal mechanism(s) responsible for this sexually dimorphic expression of Ntcp. We examined castrated and hypophysectomized rats of both sexes. Sex steroid hormones, growth hormone, thyroid, and glucocorticoids were administered, and livers were examined for changes in Ntcp messenger RNA (mRNA). Ntcp mRNA and protein content were selectively increased in males. Estradiol selectively decreased Ntcp expression in males, whereas ovariectomy increased Ntcp in females, confirming the importance of estrogens in regulating Ntcp. Hypophysectomy decreased Ntcp mRNA levels in males and prevented estrogen administration from decreasing Ntcp, indicating the importance of pituitary hormones. Although constant infusion of growth hormone to intact males reduced Ntcp, its replacement alone after hypophysectomy did not restore the sex differences. In contrast, thyroid hormone and corticosterone increased Ntcp mRNA in hypophysectomized rats. Sex differences in Ntcp mRNA levels were produced only when the female pattern of growth hormone was administered to animals also receiving thyroid and corticosterone. Thyroid and dexamethasone also increased Ntcp mRNA in isolated rat hepatocytes, whereas growth hormone decreased Ntcp. These findings demonstrate the essential role that pituitary hormones play in the sexually dimorphic control of Ntcp expression in adult rat liver and in the mediation of estrogen effects.     

Social dominance regulates androgen and estrogen receptor gene expression

In Astatotilapia burtoni, dominant males have higher levels of sex steroid hormones than subordinate males. Because of the complex regulatory interactions between steroid hormones and receptors, we asked whether dominance is also associated with variation in sex steroid receptor gene expression. Using quantitative PCR, we compared the expression of specific subtypes of androgen (AR) and estrogen (ER) receptor genes between dominant and subordinated males in 3 divisions of the brain, the pituitary, and the testes. We measured mRNA levels of AR-alpha, AR-beta, ER-alpha, ER-betaa, and ER-betab, gonadotropin-releasing hormone 1 (GnRH1), and GnRH receptor 1 (GnRH-R1) relative to 18S rRNA. In the anterior part of the brain, we found that dominant males had higher mRNA expression of AR-alpha, AR-beta, ER-betaa, and ER-betab, but not ER-alpha, compared to subordinate males. This effect of dominance was reflected in a positive correlation between testes size and AR-alpha, AR-beta, ER-betaa, and ER-betab in the anterior brain. In addition, mRNA levels of all ARs and ERs in the anterior brain were positively correlated with mRNA level of GnRH1. In the middle and posterior portions of the brain, as well as the testes, steroid receptor mRNA levels were similar among dominants and subordinates. In the pituitary, ER-alpha mRNA level was positively correlated with testes size and AR-alpha mRNA was positively correlated with GnRH-R1 mRNA level. These data suggest that dominant male brains could be more sensitive to sex steroids, which may contribute to the increased complexity of the behavioral repertoires of dominant males.     

Estrogen receptor in the thymus of the castrated mice.

Sucrose density gradient ultracentrifugation and dextran-coated charcoal adsorption permitted us to characterize the estrogen-binding proteins in cytosols obtained from the thymus, spleen and mesenteric lymph node of the castrated male and female mice of C57BL strain. The thymic cytosol from both sexes incubated with 3H-estradiol-17 beta in the presence of excess unlabeled steroids showed a specific estrogenbinding 4 S protein with its binding capacity of 10(-14) moles/mg protein for males and 4 x 10(-15) moles/mg protein for females, respectively. The dissociation constant was of 4 x 10(-10) M for males and 3 x 10(-10) M for females, respectively. No specific binding was, however, found in the cytosols of the spleen and mesenteric lymph node. Steroid analysis by thin-layer chromatography of the thymic cytosols after incubation of them with 3H-estradiol-17 beta showed that a fair amount (around 60%) of radioactivity was from the undegradated radioactive steroid still bound to 4 S binder in both sexes. Enzyme study and heat experiment revealed that the estrogen specific 4 S binding component in the thymic cytosols bears at least protein in nature and is of heat-labile nature. These results strongly suggest that the thymus of the castrated mice contain a specific estrogen receptor, the nature of which is in part protein and heat-labile.     

Regulation of reproduction- and biomarker-related gene expression by sex steroids in the livers and ovaries of adult female western mosquitofish (Gambusia affinis)

To assess the adverse toxicological effects of steroid hormones on western mosquitofish (Gambusia affinis), 180 adult females were exposed to individual or binary combinations of progesterone (1μg/L), testosterone (1μg/L) and 17β-estradiol (1μg/L) for eight days. The expression patterns of vitellogenin, estrogen receptor, androgen receptor, metallothionein, and cytochrome P450 1A genes in mosquitofish varied according to tissue as well as the specificity of steroids. Treatment by progesterone or testosterone alone inhibited target gene expression in the livers. The expression levels of both vitellogenin A and vitellogenin B mRNAs were up-regulated by17β-estradiol, and a parallel induction of estrogen receptor α mRNA expression was also observed in the livers. In addition, 17β-estradiol treatment alone suppressed androgen receptor α, metallothionein and cytochrome P450 1A mRNA expression in the livers. In general, multiple hormone treatments had different effects on target gene expression compared with corresponding hormone alone. The results demonstrate that steroid hormones cause multiple biological responses including the expression of vitellogenin, estrogen receptor and androgen receptor mRNA in the hormone signaling pathways and the expression of metallothionein and cytochrome P450 1A mRNA in the xenobiotic signaling pathway.     

Thymic sensitivity to sex hormones develops post-natally; an in vivo and an in vitro study

In vitro thymic organ cultures were used to examine the effects of the sex hormones estradiol and dihydrotestosterone on thymocytes. In contrast with the marked loss of cortical thymocytes seen in vivo with these hormones, no effect was apparent in vitro even at concentrations up to 10(-6) M. The glucocorticoid dexamethasone caused severe depletion in vivo and in vitro. Thymic androgen and estrogen receptors were determined; in the newborn animals up to 2 wk of age, receptor levels were barely detectable. The possibility of indirect modulation of thymic function by steroids in vivo was investigated by culturing thymic lobes in media containing serum from animals treated with these hormones. Only sera from dexamethasone-injected animals caused changes in cell size, number, viability, or phenotype in the culture system. The mechanism for the previously reported effects of sex steroids on the neonatal thymus therefore remains to be elucidated.     

Estrogen-dependent modifications to hippocampal plasticity in paternal California mice (Peromyscus californicus)

In many biparental species, mothers and fathers experience similar modifications to circulating hormones. With these modifications come alterations in neural structure and function suggesting that neuroendocrine mechanisms may underlie postpartum plasticity in both males and females. In the biparental California mouse (Peromyscus californicus), adult neurogenesis is maintained and anxiety-like behavior is attenuated in fathers during the mid-postpartum period. Given a causal relationship between estrogen and regulation of both adult neurogenesis and anxiety, we aimed to elucidate the role of estrogen-dependent mechanisms in paternal experience-related modifications to hippocampal neuroplasticity in California mice. In Experiment 1, hippocampal estrogen receptor beta (ERβ) mRNA expression, along with circulating estradiol concentrations, were determined throughout the postpartum period. An upregulation in ERβ expression was observed in postnatal day 16 males compared to virgins. Additionally, a rise in circulating estradiol concentrations was detected on postnatal day 2 compared to virgins; levels began to decline toward virgin levels on postnatal day 16 and postnatal day 30. In Experiment 2, we determined the role of estrogen-dependent mechanisms in adult neurogenesis and anxiety-like behavior by treating virgin and paternal males with saline or the selective estrogen receptor modulator, tamoxifen (TMX), during the time of axon extension (i.e., one week after bromodeoxyuridine injection). While TMX failed to alter elevated plus maze performance, TMX treatment inhibited survival of adult born neurons but only in paternal mice. These findings highlight the potential for estrogen-dependent pathways to mediate hippocampal adult neurogenesis in paternal mice.     

Estrogen-Inducible Progesterone Receptors in the Rat Lumbar Spinal Cord: Regulation by Ovarian Steroids and Fluctuation across the Estrous Cycle

Ovarian hormones influence the physiology of the spinal cord through incompletely understood cellular mechanisms. To date, there has been little compelling evidence for progesterone receptors in spinal cord neurons. Using two antibodies specific for progesterone receptors in an immunohistochemical investigation, we now report the presence of estrogen-inducible progesterone receptors in the spinal cord. Estrogen-inducible progesterone receptors were observed in the neurons of lamina X and the interomedialateral cell column, which are also known to express estrogen receptors. Estrogen-inducible progesterone receptors similar to those observed in females were also apparent in lamina X and interomediolateral cell column neurons in the spinal cords of males treated with estradiol. Furthermore, the density of progesterone receptors in lamina X was observed to fluctuate across the estrous cycle in female rats, with the highest progesterone receptor expression levels occurring late in proestrus, following the estradiol surge and coincident with high circulating progesterone levels. The lowest progesterone receptor expression levels were observed late in estrus following the progesterone surge. Together, these results demonstrate that estrogen-sensitive progestin targets exist in the spinal cord, and their possible role in the nervous control of reproduction and ovarian steroid modulation of nociception is discussed.     

Hormonal differentiation of sexual behavior in Japanese quail

The effect of hormones on the development of Japanese quail during the postembryonic period was examined. First, subcutaneous implants of estradiol monobenzoate (EB) and testosterone propionate (TP) were implanted 6–12 hr after hatching. EB and TP had no effect on the differentiation of sexual behavior in genetic males or females. However, EB had marked feminizing effects on plumage in genetic males. Second, the role of gonadal hormones during development was examined by gonadectomizing males and females 6–12 hr after hatching and treating them intramuscularly with EB or TP as adults. EB-treated adult females displayed sexual behavior typical of the genetic female and developed female plumage. A significant proportion of TP-treated females (57%) displayed male sexual behavior patterns. Cloacal gland development and male-type vocalizations were induced. EB-treated males displayed either male or female sexual patterns depending on the stimulus conditions. Third, to test whether bisexuality in gonadectomized males and females is maintained despite steroid treatment and expression of sexual behavior in adulthood, gonadectomized quail which were originally treated with EB received TP and vice versa. The results indicate that in the absence of gonadal hormones after hatching female quail remain bisexual until exposed to estrogen, whereas gonadectomized male quail retain behavioral bisexuality irrespective of prior estrogen or androgen exposure.