Estrogen's sex-specific effects on ischemic cell death and estrogen receptor mRNA expression in rat cortical organotypic explants.

Estrogens, such as the biologically active 17-ß estradiol (E2), regulate not only reproductive behaviors in adults, but also influence neurodevelopment and neuroprotection in both females and males. E2, contingent upon the timing and concentration of the therapy, is neuroprotective in female and male rodent models of stroke. In Vivo studies suggest that E2 may partially mediate this neuroprotection, particularly in the cortex, via ERα. In Vitro studies, utilizing a chemically induced ischemic injury in cortical explants from both sexes, suggest that ERα or ERß signaling is needed to mediate the E2 protection. Since we know that the timing and concentration of E2 therapy may be sex-specific, we examined if E2 (1 nM) mediates neuroprotection when female and male cortical explants are separately isolated from postnatal day (PND) 3-4 rat. Changes in basal levels ERα, ERß, and AR mRNA expression are compared across early post-natal development in the intact cortex and the corresponding days in vitro (DIV) for cortical explants. Following ischemic injury at 7 DIV, cell death and ERα, ERß and AR mRNA expression was compared in female and male cortical explants. We provide evidence that E2-mediated protection is maintained in isolated cortical explants from females, but not male rats. In female cortical explants, the E2-mediated protection at 24 h occurs secondarily to a blunted transient increase in ERα mRNA at 12 h. These results suggest that cortical E2-mediated protection is influenced by sex and supports data to differentially treat females and males following ischemic injury.

Characteristic Response to Chemosensory Signals in GABAergic Cells of Medial Amygdala Is Not Driven by Main Olfactory Input.

Chemosensory stimuli from same species (conspecific) and different species (heterospecific) elicit categorically different immediate-early gene (IEG) response patterns in medial amygdala in male hamsters and mice. All heterospecific stimuli activate anterior medial amygdala (MeA) but only especially salient heterospecific stimuli, such as those from predators activate posterior medial amygdala (MeP). We previously reported that characteristic patterns of response in separate populations of cells in MeA and MeP distinguish between different conspecific stimuli. Both gamma aminobutyric acid (GABA)-immunoreactive (ir) cells and GABA-receptor-ir cells make this distinction. Here, using zinc sulfate lesions of the main olfactory epithelium, we show evidence that main olfactory input does not contribute to the characteristic patterns of response in GABA-ir cells of male hamster amygdala, either for conspecific or heterospecific stimuli. Some GABAergic cells are output neurons carrying information from medial amygdala to behavioral executive regions of basal forebrain. Thus, the differential response to different conspecific signals can lead to differential activation of downstream circuits based on nonolfactory input. Finally, we show that an intact vomeronasal organ is necessary and sufficient to produce the characteristic patterns of response to conspecific and heterospecific chemosensory stimuli in hamster medial amygdala. Although main olfactory input may be critical in species with less prominent vomeronasal input for equivalent medial amygdala responses, work presented here suggests that hamster medial amygdala uses primarily vomeronasal input to discriminate between important unlearned conspecific social signals, to distinguish them from the social signals of other species, and may convey that information to brain circuits eliciting appropriate social behavior.

GABAergic mechanisms contributing to categorical amygdala responses to chemosensory signals.

Chemosensory stimuli from conspecific and heterospecific animals, elicit categorically different immediate-early gene response-patterns in medial amygdala in male hamsters and mice. We previously showed that conspecific signals activate posterior (MeP) as well as anterior medial amygdala (MeA), and especially relevant heterospecific signals such as chemosensory stimuli from potential predators also activate MeP in mice. Other heterospecific chemosignals activate MeA, but not MeP. Here we show that male hamster amygdala responds significantly differentially to different conspecific signals, by activating different proportions of cells of different phenotype, possibly leading to differential activation of downstream circuits. Heterospecific signals that fail to activate MeP do activate GABA-immunoreactive cells in the adjacent caudal main intercalated nucleus (mICNc) and elicit selective suppression of MeP cells bearing GABA-Receptors, suggesting GABA inhibition in MeP by GABAergic cells in mICNc. Overall, work presented here suggests that medial amygdala may discriminate between important conspecific social signals, distinguish them from the social signals of other species and convey that information to brain circuits eliciting appropriate social behavior.

Regulation of estrogen receptor alpha gene expression in the mouse prefrontal cortex during early postnatal development.

Estrogens have many functions in the developing rodent brain, and most of these depend on the presence of estrogen receptors. Understanding how expression of these receptors are regulated is crucial for understanding the roles of estradiol in the male and female brain during development In rodents, the prefrontal cortex (PFC) has been shown to be involved in working memory, attention, and behavioral inhibition. Many studies have demonstrated an effect of estradiol on sex difference in these functions attributed to differences in the PFC. We have previously demonstrated that estrogen receptor alpha (ERα) expression decreases in the isocortex across early postnatal development. This decrease corresponds with an increase in methylation of many sites along the ERα promoter. Here we have examined both ERα and ERß mRNA expression in the PFC to determine if methylation also plays a role in this important brain region. We investigated expression of alternate promoters for ERα and methylation of CpG sites along two of these promoters. We found that the pattern of ERα mRNA expression in PFC was similar to the pattern of ERα expression in the isocortex and that there were no sex differences in the level of expression across development. We did, however, find subtle differences in promoter expression and methylation that may indicate a sex-specific difference in PFC during development resulting in a difference in adult response.

Epigenetic regulation of estrogen receptor beta expression in the rat cortex during aging.

During aging, there is an increase in neurodegenerative diseases and a decrease in cognitive performance. Postmenopausal women are more vulnerable as their estrogen levels decline, but most hormone replacement therapies do not prevent cognitive decline. One potential reason is that the timing of hormone replacement is critical and changes in the estrogen receptor expression may over-ride hormonal intervention. In rodents, estrogen receptor ß (ERß) mRNA decreases in the cortex with age. One mechanism by which ERß mRNA could be regulated is by epigenetic modification of ERß promoter. Here, we show an increase in methylation of ERß promoter corresponding to decrease in ERß mRNA in the cortex of an aging female.

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

17ß-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 (ERß). 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, 17ß-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.

Epigenetic regulation of estrogen receptor alpha gene expression in the mouse cortex during early postnatal development.

Estrogens play a critical role in brain development by acting on areas that express estrogen receptors. In the rodent cortex, estrogen receptor alpha (ER alpha) mRNA expression is high early in postnatal development but declines starting at postnatal day (PND) 10 and is virtually absent in the adult cortex. The mechanisms controlling this regulation are largely unknown. Methylation is important for gene silencing during development in many tissues, including the brain. In the present study, we examined the methylation status of ER alpha 5' untranslated exons during early postnatal development in male and female mice using methylation-specific PCR and pyrosequencing. Several regions of ER alpha promoter displayed a significant increase in methylation at PND 18 and 25 compared with PND 4. DNA methyltransferases (DNMT) are important for the initiation and maintenance of methylation. Real-time PCR showed that DNMT3A, the de novo DNMT peaked at PND 10 and was decreased by PND 25. DNMT1, which is important for maintenance of methylation, increased across development and stayed high in adult cortex. The methyl-CpG-binding protein 2 (MeCP2) is also important for stabilization of methylation. A chromatin immunoprecipitation assay showed a correlation between association of MeCP2 with ER alpha promoter and the increase in methylation and decrease in ER alpha expression after PND 10. In mice containing a mutant MeCP2 protein, ER alpha mRNA expression and promoter methylation patterns across development were different compared with wild-type mice. These data suggest that methylation of ER alpha promoters regulates ER alpha mRNA expression in the cortex during postnatal development in a MeCP2-dependent fashion.

Androgen resistance in squirrel monkeys (Saimiri spp.).

The goal of this study was to understand the basis for high androgen levels in squirrel monkeys (Saimiri spp.). Mass spectrometry was used to analyze serum testosterone, androstenedione, and dihydrotestosterone of male squirrel monkeys during the nonbreeding (n = 7) and breeding (n = 10) seasons. All hormone levels were elevated compared with those of humans, even during the nonbreeding season; the highest levels occurred during the breeding season. The ratio of testosterone to dihydrotestosterone in squirrel monkeys is high during the breeding season compared to man. Squirrel monkeys may have high testosterone to compensate for inefficient metabolism to dihydrotestosterone. We also investigated whether squirrel monkeys have high androgens to compensate for low-activity androgen receptors (AR). The response to dihydrotestosterone in squirrel monkey cells transfected with AR and AR-responsive reporter plasmids was 4-fold, compared with 28-fold in human cells. This result was not due to overexpression of cellular FKBP51, which causes glucocorticoid and progestin resistance in squirrel monkeys, because overexpression of FKBP51 had no effect on dihydrotestosterone-stimulated reporter activity in a human fibroblast cell line. To test whether the inherently low levels of FKBP52 in squirrel monkeys contribute to androgen insensitivity, squirrel monkey cells were transfected with an AR expression plasmid, an AR-responsive reporter plasmid, and a plasmid expressing FKBP52. Expression of FKBP52 decreased the EC50 or increased the maximal response to dihydrotestosterone. Therefore, the high androgen levels in squirrel monkeys likely compensate for their relatively low 5 alpha-reductase activity during the breeding season and AR insensitivity resulting from low cellular levels of FKBP52.

Dynamic regulation of estrogen receptor-alpha gene expression in the brain: a role for promoter methylation?

Estrogen has long been known to play an important role in coordinating the neuroendocrine events that control sexual development, sexual behavior and reproduction. Estrogen actions in other, non-reproductive areas of the brain have also been described. It is now known that estrogen can also influence learning, memory, and emotion and has neurotrophic and neuroprotective properties. The actions of estrogen are largely mediated through at least two intracellular estrogen receptors. Both estrogen receptor-alpha and estrogen receptor-beta are expressed in a wide variety of brain regions. Estrogen receptor-alpha (ERalpha), however, undergoes developmental and brain region-specific changes in expression. The precise molecular mechanisms that regulate its expression at the level of gene transcription are not well understood. Adding to the complexity of its regulation, the estrogen receptor gene contains multiple promoters that drive its expression. In the cortex in particular, the ERalpha mRNA expression is dynamically regulated during postnatal development and again following neuronal injury. Epigenetic modification of chromatin is increasingly being understood as a mechanism of neuronal gene regulation. This review examines the potential regulation of the ERalpha gene by such epigenetic mechanisms.

Cortisol metabolism in the Bolivian squirrel monkey (Saimiri boliviensis boliviensis).

New World squirrel monkeys (Saimiri spp.) have high circulating cortisol levels but normal electrolytes and blood pressures. The goal of the present study was to gain insight into adaptive mechanisms used by Bolivian squirrel monkeys to minimize the effects of high cortisol on mineralocorticoid receptor (MR) activity and electrolyte and water balance. Aldosterone levels in serum from 10 squirrel monkeys were 17.7 +/- 3.4 ng/dl (normal range in humans, 4 to 31 ng/dl), suggesting that squirrel monkeys do not exhibit a compensatory increase in aldosterone. The squirrel monkey MR was cloned and expressed in COS-7 cells and found to have similar responsiveness to cortisol and aldosterone as human MR, suggesting that squirrel monkey MR is not inherently less responsive to cortisol. To determine whether altered metabolism of cortisol might contribute to MR protection in squirrel monkeys, serum and urinary cortisol and cortisone were measured, and a comprehensive urinary corticosteroid metabolite profile was performed in samples from anesthetized and awake squirrel monkeys. The levels of cortisone exceeded those of cortisol in serum and urine, suggesting increased peripheral 11beta-hydroxysteroid dehydrogenase 2 activity in squirrel monkeys. In addition, a significant fraction (approximately 20%) of total corticosteroids excreted in the urine of squirrel monkeys appeared as 6beta-hydroxycortisol, compared with that in man (1%). Therefore, changes in cortisol metabolism likely contribute to adaptive mechanisms used by Bolivian squirrel monkeys to minimize effects of high cortisol.

Ovarian stimulation of squirrel monkeys (Saimiri boliviensis boliviensis) using pregnant mare serum gonadotropin.

The application of assisted reproductive technologies (ART) to nonhuman primates has created opportunities for improving reproductive management in breeding colonies, and for creation of new animal models by genetic modification. One impediment to the application of ART in Saimiri spp. has been the lack of an effective gonadotropin preparation for ovarian stimulation. Pregnant mare serum gonadotropin (PMSG) is inexpensive and readily available, but its repeated use in rhesus monkeys has been associated with induction of a refractory state. We have compared PMSG to recombinant human follicle stimulating hormone (rhFSH) for controlled ovarian stimulation in Bolivian squirrel monkeys. Groups of mature squirrel monkeys received rhFSH (75 IU daily) or PMSG (250 IU twice daily) by subcutaneous injection for 4 d during the breeding season (November to January) or nonbreeding season (March to September). Serum estradiol (E2) was measured daily. Follicular growth was monitored by abdominal ultrasound. During the breeding season, PMSG induced a higher E2 response than did rhFSH, with mean E2 levels being significantly higher within 3 d of stimulation. Superior follicular development in PMSG animals was confirmed by abdominal ultrasonography. During the nonbreeding season, PMSG elicited a similar increase in serum E2 levels despite the fact that basal serum E2 is typically low during the nonbreeding season. Repeated use of PMSG (< or = 3 cycles of administration) produced no attenuation of the E2 response. We conclude that PMSG is highly effective for repeated cycles of controlled ovulation stimulation in the squirrel monkey.

Distinctive responses in the medial amygdala to same-species and different-species pheromones.

Chemosignals related to reproductive and social status (pheromones) carry messages between opposite-sex and same-sex individuals in many species. Each individual must distinguish signals relevant to its own social behavior with conspecifics from signals used by other (heterospecific) species relevant to their social behavior. In male hamsters, the medial amygdala responded in a categorically different way to conspecific stimuli (socially relevant) and heterospecific stimuli (not socially relevant but serving similar purposes for other species), and may play an important role in this decision. Immediate-early gene responses to conspecific chemosignals and heterospecific chemosignals were characteristically different. The categorical responses, generated by chemosensory input from the vomeronasal organ and (probably) GABA inhibition within the amygdala, were not apparent at more peripheral sensory levels. This is the first evidence for an important role of the amygdala, a limbic structure known to be involved in social and emotional behavior, in discrimination of species specificity in chemosignals.

Pre-exposure to female chemosignals or intracerebral GnRH restores mating behavior in naive male hamsters with vomeronasal organ lesions.

Chemosensory input is essential for mating in male hamsters and the vomeronasal organ is critical to mating in naive males. In studies to investigate the convergence of vomeronasal chemosensory input and the neurohormone gonadotrophin releasing hormone (GnRH), we have unexpectedly found that pre-exposure to pheromone-containing chemosignals from female hamsters will also eliminate mating deficits normally seen in naive male hamsters with vomeronasal organs removed (VNX). In the present studies, naive-intact and naive-VNX male hamsters were given intracerebroventricular injections of GnRH or saline and exposed to female pheromones found in hamster vaginal fluid (HVF) or to water 40 min prior to a 5 min mating test. VNX males given saline injections and exposed to water had severe mating deficits, but VNX males given saline injections and exposed to HVF mated normally. As shown previously, males given GnRH injections and exposed to water also mated normally. HVF exposure prior to a mating test apparently acted to compensate for the lack of vomeronasal input in these males.