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.     

雌激素受体α和β免疫反应在雄性和雌性棕色田鼠脑区分布的比较

单配制和多配制动物社会行为有差异,这些差异可能与雌激素受体类型有关（ERs）。虽然多配制大鼠和小鼠中枢神经雌激素受体α（ERα）和β（ERβ）免疫反应在大脑的分布已有报道,单配制雄性草原田鼠中枢神经ERα的分布也有报道,但单配制田鼠ERα和（或）ERβ在雌性和雄性分布差异未见报道。本研究对雄性和雌性棕色田鼠前脑区域ERα和ERβ免疫反应（IR）细胞数量进行比较。研究结果表明：（1）免疫反应主要分布在细胞核中。 （2）ERα-IR和ERβ-IR细胞广泛分布于整个雌性和雄性前脑区域,在许多脑区表达有重叠。然而,不同受体在雌雄不同脑核中的分布数量是不同的。（3）ERα 和ERβ的分布存在性别差异。例如,雌性ERα在视前核中部（MPN）,终纹床和（BNST）和杏仁内侧核（MeA）比雄性多,相反雄性ERβ在MPN和BNST比雌性多。这些研究结果可能为我们理解如何通过ERα和ERβ调节动物的社会行为,及雌性和雄性社会行为的差异提供一个重要的神经解剖学基础。     

Changes in androgen receptor, estrogen receptor alpha, and sexual behavior with aging and testosterone in male rats

Reproductive aging in males is characterized by a diminution in sexual behavior beginning in middle age. We investigated the relationships among testosterone, androgen receptor (AR) and estrogen receptor alpha (ERα) cell numbers in the hypothalamus, and their relationship to sexual performance in male rats. Young (3 months) and middle-aged (12 months) rats were given sexual behavior tests, then castrated and implanted with vehicle or testosterone capsules. Rats were tested again for sexual behavior. Numbers of AR and ERα immunoreactive cells were counted in the anteroventral periventricular nucleus and the medial preoptic nucleus, and serum hormones were measured. Middle-aged intact rats had significant impairments of all sexual behavior measures compared to young males. After castration and testosterone implantation, sexual behaviors in middle-aged males were largely comparable to those in the young males. In the hypothalamus, AR cell density was significantly (5-fold) higher, and ERα cell density significantly (6-fold) lower, in testosterone- than vehicle-treated males, with no age differences. Thus, restoration of serum testosterone to comparable levels in young and middle-aged rats resulted in similar preoptic AR and ERα cell density concomitant with a reinstatement of most behaviors. These data suggest that age-related differences in sexual behavior cannot be due to absolute levels of testosterone, and further, the middle-aged brain retains the capacity to respond to exogenous testosterone with changes in hypothalamic AR and ERα expression. Our finding that testosterone replacement in aging males has profound effects on hypothalamic receptors and behavior has potential medical implications for the treatment of age-related hypogonadism in men.     

Estrogen receptor-alpha gene expression in the cortex: Sex differences during development and in adulthood

17β-estradiol is a hormone with far-reaching organizational, activational and protective actions in both male and female brains. The organizational effects of early estrogen exposure are essential for long-lasting behavioral and cognitive functions. Estradiol mediates many of its effects through the intracellular receptors, estrogen receptor-alpha (ERα) and estrogen receptor-beta (ERβ). In the rodent cerebral cortex, estrogen receptor expression is high early in postnatal life and declines dramatically as the animal approaches puberty. This decline is accompanied by decreased expression of ERα mRNA. This change in expression is the same in both males and females in the developing isocortex and hippocampus. An understanding of the molecular mechanisms involved in the regulation of estrogen receptor alpha (ERα) gene expression is critical for understanding the developmental, as well as changes in postpubertal expression of the estrogen receptor. One mechanism of suppressing gene expression is by the epigenetic modification of the promoter regions by DNA methylation that results in gene silencing. The decrease in ERα mRNA expression during development is accompanied by an increase in promoter methylation. Another example of regulation of ERα gene expression in the adult cortex is the changes that occur following neuronal injury. Many animal studies have demonstrated that the endogenous estrogen, 17β-estradiol, is neuroprotective. Specifically, low levels of estradiol protect the cortex from neuronal death following middle cerebral artery occlusion (MCAO). In females, this protection is mediated through an ERα-dependent mechanism. ERα expression is rapidly increased following MCAO in females, but not in males. This increase is accompanied by a decrease in methylation of the promoter suggesting a return to the developmental program of gene expression within neurons. Taken together, during development and in adulthood, regulation of ERα gene expression in the cortex can occur by DNA methylation and in a sex-dependent fashion in the adult brain.     

Progesterone receptor in the forebrain of female gray short-tailed opossums: Effects of exposure to male stimuli

Progesterone receptor immunoreactivity (PRir) in brain areas involved in reproductive behavior in eutherian species was examined for the first time in a female marsupial, the gray short-tailed opossum (Monodelphis domestica, hereinafter, opossum). PRir in nuclei of neurons, measured as area covered by stained nuclei, was seen in the arcuate nucleus (Arc); anteroventral periventricular nucleus (AVPv); bed nucleus of the stria terminalis (BST); medial preoptic area (MPOA), and ventromedial hypothalamus (VMH), but not in control areas adjacent to the hypothalamus or cortex. Female opossums are induced into cytological, urogenital sinus (UGS), estrus by male pheromones and into behavioral estrus, i.e., receptivity, by pairing with a male, and both estradiol (E) and progesterone (P) are involved in induction of receptivity in intact and ovariectomized females. PRir in the AVPv, MPOA, and VMH was very low in females that had never been exposed to males or their scent marks, i.e., naïve anestrous (NVA) females, and either previous or current exposure to males or their scent marks was associated with elevated PRir. PRir was significantly higher in the AVPv and MPOA of anestrous females with previous but no current exposure to males and their scent marks, i.e., experienced anestrous (EXPA) females, than in NVA females, but PRir was significantly lower in the MPOA and VMH of EXPA females than in females that were behaviorally receptive and had recently copulated, i.e., behavioral receptive estrous (BRE) females. PRir was higher in the VMH of both UGS estrous (UGSE) and BRE females compared to that in EXPA animals, but PRir did not differ between UGSE and BRE females in any of the 3 brain areas examined, including the MPOA These results provide evidence that pheromonal induction of estrus and sexual receptivity in opossums is associated with elevation of PRir in the VMH and MPOA and that prior exposure to males or their pheromones, even in the absence of current male stimuli, is associated with persistent elevation of PRir in the AVPv and MPOA.     

Progesterone receptors and the sexual differentiation of the medial preoptic nucleus

The central component of the medial preoptic nucleus (MPNc) of the rat has served as an excellent model of sexual differentiation. The MPNc is larger in adult males than in females, and its development is regulated by perinatal gonadal hormones. Although testosterone (T) and its metabolite estradiol (E) sexually differentiate this region, the exact mechanism by which they act during development is not known. There is a dramatic sex difference in the expression of progesterone receptors (PR) in the MPN during development; perinatal males express higher levels of PR than females. Additionally, PR expression during this time is dependent on exposure to T. Thus, PR induction may be one mechanism by which T sexually differentiates the MPN. The present study investigated the potential role of PR in the sexual differentiation of the MPNc. Anatomical examination of PR distribution within the MPN of neonatal males revealed the presence of PR immunoreactive cells within the MPNc, suggesting a direct route of action for PR in the development of the MPNc. Additionally, we measured the effects of neonatal RU486 treatment, a progesterone and glucocorticoid receptor antagonist, on subsequent MPNc volume in neonatally T-treated females and neonatally castrated males, given T. RU486 treatment reduced the MPNc volume of T-treated females while it increased the volume in T-treated, neonatally castrated males. These results, taken together with the expression of PR in the MPNc, suggest that PR may influence the sexual differentiation of the MPNc volume.     

Co-localization of Estrogen Receptor and Aromatase Enzyme Immunoreactivities in Adult Musk Shrew Brain

Estrogens are produced by the aromatization of androgens. These steroids exert their actions after binding to their receptors. Past studies have shown that estrogen receptors (ER) and aromatase enzyme (AROM) reside in many of the same brain regions. Few studies, however, have examined the neural co-localization of these important components involved in estrogen-activated behaviors. In the present study we examined the co-localization of ER and AROM immunoreactive (ir) neurons in musk shrew (Suncus murinus) brains. Data were collected from a representative section from three neural regions, the bed nucleus of the stria terminalis (BNST), medial preoptic area (mPOA), and ventromedial nucleus of the hypothalamus (VMN). Here we report a sex difference in the number of ER-ir neurons from the analyzed section of the mPOA and BNST. Females have more ER-ir neurons in the mPOA and males have more in the BNST. In the sections we examined, males tended to have more aromatase containing neurons than females. Although there were no significant differences in the numbers of double-labeled cells, the VMN contains the greatest percentage of these cells in both males and females; followed by the mPOA and the BNST. In addition, in the mPOA of both sexes, a distinct nucleus of aromatase containing neurons which was devoid of ER immunoreactivity was noted. Area measurements of the AROM-ir nucleus showed that it was significantly larger in males than in females. Taken together, these data suggest that there is not extensive cellular co-localization of estrogen receptors and aromatase enzyme in the musk shrew brain. However, the presence of other genomic forms of ER (membrane and/or ERβ) in AROM containing neurons has not been ruled out by this study. Thus, we hypothesize that estrogens produced in brain affect behavior by binding to ER in neurons other than those that contain aromatase enzyme.     

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.     

Estrogen receptor-alpha antisense decreases brain estrogen receptor levels and affects ventilation in male and female rats.

We hypothesized that administration of an antisense oligodeoxynucleotide (ODN) to estrogen receptor (ER)-alpha mRNA decreases the ER protein in the neonatal rat brain, alters the sex-specific ventilatory responses to aspartic acid in rats, and counteracts the effects of testosterone proportionate (TP) in females. One-day-old rat pups were injected intraventricularly with vehicle, antisense ER ODN, or scrambled ODN control. Additional groups of females received TP or vehicle and one of the three treatments. Brain ER protein levels were decreased by 65% at 6 h and 35% at 24 h after antisense ODN. Aspartic acid decreased ventilation in all groups of weanling males and females except ER ODN-treated females and TP-vehicle-treated females. Aspartic acid decreased ventilation in all groups of adult females except those given TP and in males. Weanling ER ODN-treated rats were shorter and weighed less than controls. Only adult ER ODN-treated males exhibited these traits. Thus neonatal ER affects aspartic acid modulation of breathing and body growth in a sex-specific and developmental manner.     

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.     

Estrogen receptor alpha is required in GABAergic,but not glutamatergic,neurons to masculinize behavior

Masculinization of the altricial rodent brain is driven by estrogen signaling during a perinatal critical period. Genetic deletion of estrogen receptor alpha (Esr1/ERα) results in altered hypothalamic-pituitary-gonadal (HPG) axis signaling and a dramatic reduction of male sexual and territorial behaviors. However, the role of ERα in masculinizing distinct classes of neurons remains unexplored. We deleted ERα in excitatory or inhibitory neurons using either a Vglut2 or Vgat driver and assessed male behaviors. We find that Vglut2-Cre;Esr1^lox/lox mutant males lack ERα in the ventrolateral region of the ventromedial hypothalamus (VMHvl) and posterior ventral portion of the medial amygdala (MePV). These mutants recapitulate the increased serum testosterone levels seen with constitutive ERα deletion, but have none of the behavioral deficits. In contrast, Vgat-Cre;Esr1^lox/lox males with substantial ERα deletion in inhibitory neurons, including those of the principal nucleus of the bed nucleus of the stria terminalis (BNSTpr), posterior dorsal MeA (MePD), and medial preoptic area (MPOA) have normal testosterone levels, but display alterations in mating and territorial behaviors. These mutants also show dysmasculinized expression of androgen receptor (AR) and estrogen receptor beta (Esr2). Our results demonstrate that ERα masculinizes GABAergic neurons that gate the display of male-typical behaviors.     

Tamoxifen and ICI 182,780 activate hypothalamic G protein-coupled estrogen receptor 1 to rapidly facilitate lordosis in female rats

In the female rat, sexual receptivity (lordosis) can be facilitated by sequential activation of estrogen receptor (ER) α and G protein-coupled estrogen receptor 1 (GPER) by estradiol. In the estradiol benzoate (EB) primed ovariectomized (OVX) rat, EB initially binds to ERα in the plasma membrane that complexes with and transactivates metabotropic glutamate receptor 1a to activate β-endorphin neurons in the arcuate nucleus of the hypothalamus (ARH) that project to the medial preoptic nucleus (MPN). This activates MPN μ-opioid receptors (MOP), inhibiting lordosis. Infusion of non-esterified 17β-estradiol into the ARH rapidly reduces MPN MOP activation and facilitates lordosis via GPER. Tamoxifen (TAM) and ICI 182,780 (ICI) are selective estrogen receptor modulators that activate GPER. Therefore, we tested the hypothesis that TAM and ICI rapidly facilitate lordosis via activation of GPER in the ARH. Our first experiment demonstrated that injection of TAM intraperitoneal, or ICI into the lateral ventricle, deactivated MPN MOP and facilitated lordosis in EB-primed rats. We then tested whether TAM and ICI were acting rapidly through a GPER dependent pathway in the ARH. In EB-primed rats, ARH infusion of either TAM or ICI facilitated lordosis and reduced MPN MOP activation within 30 min compared to controls. These effects were blocked by pretreatment with the GPER antagonist, G15. Our findings demonstrate that TAM and ICI deactivate MPN MOP and facilitate lordosis in a GPER dependent manner. Thus, TAM and ICI may activate GPER in the CNS to produce estrogenic actions in neural circuits that modulate physiology and behavior.     

Changes in the content of progesterone receptor isoforms and estrogen receptor alpha in the chick brain during embryonic development

Progesterone and estradiol participate in the regulation of several reproductive functions through interaction with intracellular progesterone receptors (PR) and estrogen receptors (ER), respectively. In this work, we determined PR and ER-alpha isoforms content in the brain of chicks of both sexes on days 8 and 13 of embryonic development as well as on the day of hatching by Western blot analysis. PR isoforms protein content increased during embryonic development in both female and male chick brain. The highest PR isoforms content was observed on the day of hatching in both sexes. Interestingly, PR-A content was higher in the brain of chick males than in that of females on day 8 of embryonic development. PR-A/PR-B ratio was higher in the brain of males than in that of females at all ages. We found two ER-alpha isoforms of 66 and 52 kDa; the content of both isoforms was higher in the brain of females than in that of males on days 8 and 13 of embryonic development. An opposite pattern of ER-alpha isoforms content was observed. In males, ER-alpha content increased during embryonic development whereas in the females it decreased during this process. These results indicate that the content of PR and ER-alpha isoforms is related to the degree of brain development in chicks, and suggest that PR and ER-alpha isoforms should exhibit sexual dimorphism in the brain of chicks during embryonic development.     

Androgen receptor immunoreactivity in the male and female Syrian hamster brain.

To investigate potential mechanisms for sex differences in the physiologic response to androgens, the present study compared the hormonal regulation of intracellular androgen receptor partitioning and the distribution of androgen receptor immunoreactivity in select brain regions from male and female hamsters. Androgen receptors were visualized on coronal brain sections. Two weeks after castration, androgen receptor immunoreactivity filled the neuronal nuclei and cytoplasm in males and females. In gonad-intact males and females, androgen receptor immunoreactivity was limited to the cell nucleus. Whereas exogenous dihydrotestosterone prevented cytoplasmic immunoreactivity, estrogen at physiologic levels did not. These results suggest that nuclear androgen receptor immunoreactivity in gonad-intact females is maintained by endogenous androgens, and that androgens have the potential to influence neuronal activity in either sex. However, sex differences in the number and staining intensity of androgen-responsive neurons were apparent in select brain regions. In the ventral premammillary nucleus, ventromedial nucleus of the hypothalamus, and medial amygdaloid nucleus, androgen receptor staining was similar in gonadectomized males and females. In the lateral septum, posteromedial bed nucleus of the stria terminalis (BNSTpm), and medial preoptic nucleus, the number of androgen receptor-immunoreactive neurons was significantly lower in females (p < .05). Moreover, the integrated optical density/cell in BNSTpm was significantly less in females (1.28+/-0.3 units) than in males (2.21+/-0.2 units; p < .05). These sex differences in the number and staining intensity of androgen-responsive neurons may contribute to sex differences in the behavioral and neuroendocrine responses to androgens.     

Localization of estrogen receptor‐α and ‐βmRNA in brain areas controlling sexual behavior in Japanese quail

Two estrogen receptors (ERs), denoted ERα and ERβ, have been identified in humans and various animal species, including the Japanese quail. Estrogens play a key role in sexual differentiation and in activation of sexual behavior in Japanese quail. The distribution of ERα in the brain of male and female adult quail has previously been studied using immunohistochemistry, whereas in situ hybridization has been employed to study the distribution of ERβ mRNA in males only. In this article, we used in situ hybridization to study the distribution of mRNAs for both ERα and ERβ in brain areas controlling sexual behavior of Japanese quail. Our results show that both ERα mRNA and ERβ mRNA are localized in areas important for sexual behavior, such as the preoptic area and associated limbic areas, in both males and females. Moreover, we found differences in distribution of mRNA for the two receptors in these areas. The results of this article support previously reported data and provide novel data on localization of ER mRNAs in adult quail brain of both sexes. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

Androgen receptor immunoreactivity in the male and female Syrian hamster brain

To investigate potential mechanisms for sex differences in the physiologic response to androgens, the present study compared the hormonal regulation of intracellular androgen receptor partitioning and the distribution of androgen receptor immunoreactivity in select brain regions from male and female hamsters. Androgen receptors were visualized on coronal brain sections. Two weeks after castration, androgen receptor immunoreactivity filled the neuronal nuclei and cytoplasm in males and females. In gonad‐intact males and females, androgen receptor immunoreactivity was limited to the cell nucleus. Whereas exogenous dihydrotestosterone prevented cytoplasmic immunoreactivity, estrogen at physiologic levels did not. These results suggest that nuclear androgen receptor immunoreactivity in gonad‐intact females is maintained by endogenous androgens, and that androgens have the potential to influence neuronal activity in either sex. However, sex differences in the number and staining intensity of androgen‐responsive neurons were apparent in select brain regions. In the ventral premammillary nucleus, ventromedial nucleus of the hypothalamus, and medial amygdaloid nucleus, androgen receptor staining was similar in gonadectomized males and females. In the lateral septum, posteromedial bed nucleus of the stria terminalis (BNSTpm), and medial preoptic nucleus, the number of androgen receptor–immunoreactive neurons was significantly lower in females (p < .05). Moreover, the integrated optical density/cell in BNSTpm was significantly less in females (1.28 ± 0.3 units) than in males (2.21 ± 0.2 units; p < .05). These sex differences in the number and staining intensity of androgen‐responsive neurons may contribute to sex differences in the behavioral and neuroendocrine responses to androgens. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 359–370, 1999     

Neuroanatomical distribution of aromatase mRNA in the rat brain: indications of regional regulation

Aromatase, the enzyme responsible for the conversion of testosterone to estradiol, is found in the rat brain and is present in regions of the preoptic area, hypothalamus, and limbic system. Gonadal steroid hormones regulate aromatase activity levels in many brain regions, but not all. Using in situ hybridization, we examined the distribution of aromatase mRNA in the adult male forebrain, as well as the levels of aromatase mRNA in the brains of males and females, and the regulation by gonadal steroid hormones. In the adult male, many heavily labelled cells were found in the encapsulated bed nucleus of the stria terminalis (BNST), the medial preoptic nucleus (MPN), the ventro-medial nucleus (VMN), the medial amygdala (mAMY) and the cortical amygdala (CoAMY). The regional distribution of aromatase mRNA was similar in males and females, but males tended to have a greater number of aromatase mRNA-expressing cells in each region compared to females. Aromatase mRNA levels in the BNST, MPN, VMN and mAMY tended to be lower in castrated males than in intact males, whereas aromatase mRNA levels were unaltered by castration in the CoAMY. Further analysis of individual cells expressing aromatase mRNA suggests that aromatase mRNA may be regulated by steroid hormones differentially in specific populations of cells in regions where enzyme activity levels are steroid-hormone-dependent.