The medial preoptic area and acute cocaine's stimulant effects in rats: Potential influences of estradiol and biological sex.

The medial preoptic area (mPOA) in the hypothalamus is an important integrator of neuroendocrine signaling and a key regulator of both natural and drug-induced reward. Although the mPOA modulates sex differences in other behaviors, whether it also modulates sex differences in cocaine response remains unclear. To help us better understand the mPOA's role in sex differences associated with cocaine response, we examined cocaine-induced changes in locomotion and neural activity in the mPOA of male and female rats. In addition, neural activity in the striatum, a brain area known to be involved in cocaine response, was examined for comparison purposes. Fos, the protein product of the immediate early gene c-fos, was used as the marker of neural activity. Locomotion chambers were used to measure behavior, radioimmunoassays and vaginal lavages were used to determine hormonal status, and immunohistochemical assays were used to quantify Fos. To account for the effects of gonadal hormones, rats were left gonadally intact and categorized as either 'low-estradiol' or 'high-estradiol' based on their hormonal status on test day. Results indicate that high-estradiol females experienced greater cocaine-induced mPOA Fos-immunoreactivity (Fos-ir) and displayed greater cocaine-induced locomotion than low estradiol females. Conversely, high-estradiol males experienced less cocaine-induced mPOA Fos-ir and displayed less cocaine-induced locomotion than low-estradiol males. Cocaine-induced Fos-ir in the mPOA also correlated with cocaine-induced Fos-ir in areas of the striatum already associated with cocaine response. These findings further support the mPOA's role in the endocrine-mediated response to cocaine. It also identifies the mPOA as a contributor to sex differences in cocaine response and potential differences in vulnerability to developing cocaine use disorders.

Effects of endocrine-disrupting chemicals on hypothalamic oxytocin and vasopressin systems.

Exposures to endocrine disrupting chemicals (EDCs) perturb hormonal systems. EDCs are particularly problematic when exposure happens in the fetus and infant due to the high sensitivity of developing organisms to hormone actions. Previous work has shown that prenatal polychlorinated biphenyl (PCB) exposure disrupts hypothalamic development, reproductive physiology, mate preference behavior, and social behaviors in a sexually dimorphic manner. Based on evidence that EDCs perturb social behaviors in rodents, we examined effects of PCBs on the neuropeptides oxytocin (OXT) and vasopressin (AVP) that are involved in regulating these behaviors. Rats were exposed prenatally (gestational days 16 and 18) to the weakly estrogenic PCB mixture Aroclor 1221 (0.5 or 1 mg/kg), to estradiol benzoate (EB, a positive control), or to the vehicle (3% dimethyl sulfoxide). In adult (~P90) brains, we counted immunolabeled oxytocin and vasopressin cell numbers in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus. EDCs did not change absolute numbers of oxytocin or vasopressin cells in either region, although there were some modest shifts in the rostral-caudal distribution. Second, expression of genes for these nonapeptides (Oxt, Avp), their receptors (Oxtr, Avpr1a), and the estrogen receptor beta (Esr2), was determined by qPCR. In the PVN, there were dose-dependent effects of PCBs in males (Oxt, Oxtr), and effects of EB in females (Avp, Esr2). In the SON, Oxt, and Esr2 were affected by treatments in males. These changes to protein and gene expression caused by prenatal treatments suggest that transcriptional and posttranscriptional mechanisms play roles in mediating how EDCs reprogram hypothalamic development.

Transgenerational effects of polychlorinated biphenyls: 2. Hypothalamic gene expression in rats†.

Polychlorinated biphenyls (PCBs) are endocrine-disrupting chemicals (EDCs) with well-established effects on reproduction and behavior in developmentally-exposed (F1) individuals. Because of evidence for transgenerational effects of EDCs on the neuroendocrine control of reproductive physiology, we tested the hypothesis that prenatal PCB exposure leads to unique hypothalamic gene-expression profiles in three generations. Pregnant Sprague-Dawley rats were treated on gestational days 16 and 18 with the PCB mixture Aroclor 1221 (A1221), vehicle (3% DMSO in sesame oil), or estradiol benzoate (EB, 50 µg/kg), the latter a positive control for estrogenic effects of A1221. Maternal- and paternal-lineage F2 and F3 generations were bred using untreated partners. The anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC), involved in the hypothalamic control of reproduction, were dissected from F1 to F3 females and males, RNA extracted, and gene expression measured in a qPCR array. We detected unique gene-expression profiles in each generation, which were sex- and lineage-specific. In the AVPV, treatment significantly changed 10, 25, and 11 transcripts in F1, F2, and F3 generations, whereas 10, 1, and 12 transcripts were changed in these generations in the ARC. In the F1 AVPV and ARC, most affected transcripts were decreased by A1221. In the F2 AVPV, most effects of A1221 were observed in females of the maternal lineage, whereas only Pomc expression changed in the F2 ARC (by EB). The F3 AVPV and ARC were mainly affected by EB. It is notable that results in one generation do not predict results in another, and that lineage was a major determinant in results. Thus, transient prenatal exposure of F1 rats to A1221 or EB can alter hypothalamic gene expression across three generations in a sex- and lineage-dependent manner, leading to the conclusion that the legacy of PCBs continues for generations.

Exposure to prenatal PCBs shifts the timing of neurogenesis in the hypothalamus of developing rats.

The developing brain is highly sensitive to the hormonal milieu, with gonadal steroid hormones involved in neurogenesis, neural survival, and brain organization. Limited available evidence suggests that endocrine-disrupting chemicals (EDCs) may perturb these developmental processes. In this study, we tested the hypothesis that prenatal exposure to a mixture of polychlorinated biphenyls (PCBs), Aroclor 1221, would disrupt the normal timing of neurogenesis in two hypothalamic regions: the ventromedial nucleus (VMN) and the preoptic area (POA). These regions were selected because of their important roles in the control of sociosexual behaviors that are perturbed in adulthood by prenatal EDC exposure. Pregnant Sprague-Dawley rats were exposed to PCBs from Embryonic Day 8 (E8) to E18, encompassing the period of neurogenesis of all hypothalamic neurons. To determine the birth dates of neurons, bromo-2-deoxy-5-uridine (BrdU) was administered to dams on E12, E14, or E16. On the day after birth, male and female pups were perfused, brains immunolabeled for BrdU, and numbers of cells counted. In the VMN, exposure to PCBs significantly advanced the timing of neurogenesis compared to vehicle-treated pups, without changing the total number of BrdU+ cells. In the POA, PCBs did not change the timing of neurogenesis nor the total number of cells born. This is the first study to show that PCBs can shift the timing of neurogenesis in the hypothalamus, specifically in the VMN but not the POA. This result has implications for functions controlled by the VMN, especially sociosexual behaviors, as well as for sexual selection more generally.

Endocrine-disrupting chemicals alter the neuromolecular phenotype in F2 generation adult male rats.

Endocrine-disrupting chemical (EDC) exposures to the fetus have long-lasting effects on health and disease in adulthood. Such EDC exposure to the F1 fetuses also reaches the germ cells that become the F2 generation. Previously, we demonstrated that adult social and communicative behaviors such as ultrasonic vocalizations and mating behaviors were altered by EDCs in F2 rats, especially males. In the current study, we used the brains of these F2 males to ascertain the underlying molecular changes in the hypothalamus related to these behavioral outcomes. Their progenitors were Sprague-Dawley rat dams, treated on pregnancy days 8 to 18 with one of three treatments: a polychlorinated biphenyl (PCB) mixture, Aroclor 1221, selected because it is weakly estrogenic; the anti-androgenic fungicide vinclozolin (VIN); or the vehicle, 6% dimethylsulfoxide in sesame oil (VEH). In adulthood, F1 male and female offspring were bred with untreated partners to generate paternal or maternal lineages of the F2 offspring, the subjects of molecular work. Quantitative real-time PCR was conducted in the medial preoptic area (POA) and the ventromedial nucleus (VMN) of the hypothalamus, selected for their roles in social and sexual behaviors. Of the genes assessed, steroid hormone receptors (estrogen receptor α, androgen receptor, progesterone receptor) but not dopamine receptors 1 and 2 or DNA methyltransferase 3a expression were altered, particularly in the VIN males. Several significant correlations between behavior and gene expression were also detected. These results suggest that preconceptional exposure of male rats to EDCs at the germ cell stage alters the neuromolecular phenotype in adulthood in a lineage-dependent manner.

Endocrine disruptors and the future of toxicology testing - lessons from CLARITY-BPA.

Five years ago, an ambitious collaboration, the Consortium Linking Academic and Regulatory Insights on Toxicity of BPA (CLARITY-BPA; henceforth CLARITY), was launched by three US agencies. The goal was to provide a definitive evaluation of bisphenol A (BPA) and explain disparities between traditional regulatory studies and findings from independent investigators. BPA or vehicle-treated rats from an FDA facility were used in a guideline study and animals and/or tissues were provided to academic researchers for analysis. An interim summary released in February 2018 by the FDA concluded that currently authorized uses of BPA continue to be safe. We disagree. In this Perspectives, we summarize the goals, design and problems of CLARITY. We conclude that, despite its flaws, CLARITY provides important insight and, taken together, the data provide compelling evidence that low-dose BPA exposure induces marked adverse effects. Indeed, the greatest number of effects were observed at doses 20,000 times lower than the current 'safe' dose of BPA for humans.

Sex differences in effects of gestational polychlorinated biphenyl exposure on hypothalamic neuroimmune and neuromodulator systems in neonatal rats.

Polychlorinated biphenyls (PCBs) are ubiquitous in the environment and exposure to them is associated with immune, endocrine and neural dysfunction. Effects of PCBs on inflammation and immunity are best described in spleen and blood, with fewer studies on neural tissues. This is an important gap in knowledge, as molecules typically associated with neuroinflammation also serve neuromodulatory roles and interact with hormones in normal brain development. The current study used Sprague-Dawley rats to assess whether gestational PCB exposure altered hypothalamic gene expression and serum cytokine concentration in neonatal animals given an immune challenge. Dams were fed wafers containing a mixture of PCBs at an environmentally relevant dose and composition (20 µg/kg, 1:1:1 Aroclor 1242:1248:1254) or oil vehicle control throughout their pregnancy. One day old male and female offspring were treated with an inflammatory challenge (lipopolysaccharide, LPS, 50 µg/kg, sc) or saline vehicle control approximately 3.5 h prior to tissue collection. Across both basal and activated inflammatory states, PCB exposure caused greater expression of a subset of inflammatory genes in the hypothalamus and lower expression of genes involved in dopamine, serotonin, and opioid systems compared to oil controls. PCB exposure also altered reactions to inflammatory challenge: it reversed the normal decrease in Esr2 hypothalamic expression and induced an abnormal increase in IL-1b and IL-6 serum concentration in response to LPS. Many of these effects were sex specific. Given the potential long-term consequences of neuroimmune disruption, our findings demonstrate the need for further research.

Specific effects of prenatal DEHP exposure on neuroendocrine gene expression in the developing hypothalamus of male rats.

Endocrine disrupting chemicals may disrupt developing neuroendocrine systems, especially when the exposure occurs during a critical period. This study aimed to investigate whether prenatal exposure to di-(2-ethylhexyl) phthalate (DEHP), a major component of plasticizers used worldwide, disrupted the development of a network of genes important for neuroendocrine function in male rats. Pregnant rats were treated with corn oil (vehicle control), 2, 10 or 50 mg/kg DEHP by gavage from gestational day 14 to 19. The neuroendocrine gene expressions were quantified using a 48-gene Taqman qPCR array in the whole hypothalamus of neonatal rats (postnatal day 1) and in the anteroventral periventricular nucleus (AVPV), medial preoptic nucleus (MPN) and arcuate nucleus (ARC) of adult rats (postnatal day 70). Immunofluorescent signals of ERα and CYP19 were detected using the confocal microscopy in adult AVPV, MPN and ARC. The results showed that prenatal DEHP exposure perturbed somatic and reproductive development of offspring. Eleven genes were down-regulated in neonatal hypothalamus and showed non-monotonic dose-response relationships, that the 10 mg/kg DEHP dosage was associated with the greatest number of gene expression changes. Different from this, 14 genes were altered in adult AVPV, MPN and ARC and most of alterations were found in the 50 mg/kg DEHP group. Also, 50 mg/kg DEHP reduced ERα expression in the ARC, but no alterations were observed in CYP19 expression. These results indicated that prenatal DEHP exposure may perturb hypothalamic gene programming and the influences are permanent. The effects showed dependence on developmental stages and nuclei region.

Testing the critical window of estradiol replacement on gene expression of vasopressin, oxytocin, and their receptors, in the hypothalamus of aging female rats.

The current study tested the "critical window" hypothesis of menopause that postulates that the timing and duration of hormone treatment determine their potential outcomes. Our focus was genes in the rat hypothalamus involved in social and affiliative behaviors that change with aging and/or estradiol (E2): Avp, Avpr1a, Oxt, Oxtr, and Esr2 in the paraventricular nucleus (PVN) and supraoptic nucleus (SON). Rats were reproductively mature or aging adults, ovariectomized, given E2 or vehicle treatment of different durations, with or without a post-ovariectomy delay. Our hypothesis was that age-related changes in gene expression are mitigated by E2 treatments. Contrary to this, PVN Oxtr increased with E2, and Avpr1a increased with age. In the SON, Avpr1a increased with age, Oxtr with age and timing, and Avp was altered by duration. Thus, chronological age and E2 have independent actions on gene expression, with the "critical window" hypothesis supported by the observed timing and duration effects.

Testing the Critical Window Hypothesis of Timing and Duration of Estradiol Treatment on Hypothalamic Gene Networks in Reproductively Mature and Aging Female Rats.

At menopause, the dramatic loss of ovarian estradiol (E2) necessitates the adaptation of estrogen-sensitive neurons in the hypothalamus to an estrogen-depleted environment. We developed a rat model to test the "critical window" hypothesis of the effects of timing and duration of E2 treatment after deprivation on the hypothalamic neuronal gene network in the arcuate nucleus and the medial preoptic area. Rats at 2 ages (reproductively mature or aging) were ovariectomized and given E2 or vehicle replacement regimes of differing timing and duration. Using a 48-gene quantitative low-density PCR array and weighted gene coexpression network analysis, we identified gene modules differentially regulated by age, timing, and duration of E2 treatment. Of particular interest, E2 status differentially affected suites of genes in the hypothalamus involved in energy balance, circadian rhythms, and reproduction. In fact, E2 status was the dominant factor in determining gene modules and hormone levels; age, timing, and duration had more subtle effects. Our results highlight the plasticity of hypothalamic neuroendocrine systems during reproductive aging and its surprising ability to adapt to diverse E2 replacement regimes.

GnRH neurons of young and aged female rhesus monkeys co-express GPER but are unaffected by long-term hormone replacement.

Menopause is caused by changes in the function of the hypothalamic-pituitary-gonadal axis that controls reproduction. Hypophysiotropic gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus orchestrate the activity of this axis and are regulated by hormonal feedback loops. The mechanisms by which GnRH responds to the primary regulatory sex steroid hormone, estradiol (E2), are still poorly understood in the context of menopause. Our goal was to determine whether the G protein-coupled estrogen receptor (GPER) is co-expressed in adult primate GnRH neurons and whether this changes with aging and/or E2 treatment. We used immunofluorescence double-labeling to characterize the co-expression of GPER in GnRH perikarya and terminals in the hypothalamus. Young and aged rhesus macaques were ovariectomized and given long-term (~2-year) hormone treatments (E2, E2 + progesterone, or vehicle) selected to mimic currently prescribed hormone replacement therapies used for the alleviation of menopausal symptoms in women. We found that about half of GnRH perikarya co-expressed GPER, while only about 12% of GnRH processes and terminals in the median eminence (ME) were double-labeled. Additionally, many GPER-labeled processes were in direct contact with GnRH neurons, often wrapped around the perikarya and processes and in close proximity in the ME. These results extend prior work by showing robust co-localization of GPER in GnRH in a clinically relevant model, and they support the possibility that GPER-mediated E2 regulation of GnRH occurs both in the soma and terminals in nonhuman primates.

Hypothalamic molecular changes underlying natural reproductive senescence in the female rat.

The role of the hypothalamus in female reproductive senescence is unclear. Here we identified novel molecular neuroendocrine changes during the natural progression from regular reproductive cycles to acyclicity in middle-aged female rats, comparable with the perimenopausal progression in women. Expression of 48 neuroendocrine genes was quantified within three hypothalamic regions: the anteroventral periventricular nucleus, the site of steroid positive feedback onto GnRH neurons; the arcuate nucleus (ARC), the site of negative feedback and pulsatile GnRH release; and the median eminence (ME), the site of GnRH secretion. Surprisingly, the majority of changes occurred in the ARC and ME, with few effects in anteroventral periventricular nucleus. The overall pattern was increased mRNA levels with chronological age and decreases with reproductive cycle status in middle-aged rats. Affected genes included transcription factors (Stat5b, Arnt, Ahr), sex steroid hormone receptors (Esr1, Esr2, Pgr, Ar), steroidogenic enzymes (Sts, Hsd17b8), growth factors (Igf1, Tgfa), and neuropeptides (Kiss1, Tac2, Gnrh1). Bionetwork analysis revealed region-specific correlations between genes and hormones. Immunohistochemical analyses of kisspeptin and estrogen receptor-α in the ARC demonstrated age-related decreases in kisspeptin cell numbers as well as kisspeptin-estrogen receptor-α dual-labeled cells. Taken together, these results identify unexpectedly strong roles for the ME and ARC during reproductive decline and highlight fundamental differences between middle-aged rats with regular cycles and all other groups. Our data provide evidence of decreased excitatory stimulation and altered hormone feedback with aging and suggest novel neuroendocrine pathways that warrant future study. Furthermore, these changes may impact other neuroendocrine systems that undergo functional declines with age.

Disruption of reproductive aging in female and male rats by gestational exposure to estrogenic endocrine disruptors.

Polychlorinated biphenyls (PCBs) are industrial contaminants and known endocrine-disrupting chemicals. Previous work has shown that gestational exposure to PCBs cause changes in reproductive neuroendocrine processes. Here we extended work farther down the life spectrum and tested the hypothesis that early life exposure to Aroclor 1221 (A1221), a mixture of primarily estrogenic PCBs, results in sexually dimorphic aging-associated alterations to reproductive parameters in rats, and gene expression changes in hypothalamic nuclei that regulate reproductive function. Pregnant Sprague Dawley rats were injected on gestational days 16 and 18 with vehicle (dimethylsulfoxide), A1221 (1 mg/kg), or estradiol benzoate (50 µg/kg). Developmental parameters, estrous cyclicity (females), and timing of reproductive senescence were monitored in the offspring through 9 months of age. Expression of 48 genes was measured in 3 hypothalamic nuclei: the anteroventral periventricular nucleus (AVPV), arcuate nucleus (ARC), and median eminence (females only) by real-time RT-PCR. Serum LH, testosterone, and estradiol were assayed in the same animals. In males, A1221 had no effects; however, prenatal estradiol benzoate increased serum estradiol, gene expression in the AVPV (1 gene), and ARC (2 genes) compared with controls. In females, estrous cycles were longer in the A1221-exposed females throughout the life cycle. Gene expression was not affected in the AVPV, but significant changes were caused by A1221 in the ARC and median eminence as a function of cycling status. Bionetwork analysis demonstrated fundamental differences in physiology and gene expression between cycling and acyclic females independent of treatment. Thus, gestational exposure to biologically relevant levels of estrogenic endocrine-disrupting chemicals has sexually dimorphic effects, with an altered transition to reproductive aging in female rats but relatively little effect in males.

Molecular profiling of postnatal development of the hypothalamus in female and male rats.

Reproductive function is highly dynamic during postnatal developmental. Here, we performed molecular profiling of gene expression patterns in the hypothalamus of developing male and female rats to identify which genes are sexually dimorphic, to gain insight into a more complex network of hypothalamic genes, and to ascertain dynamic changes in their relationships with one another and with sex steroid hormones during development. Using a low-density PCR platform, we quantified mRNA levels in the preoptic area (POA) and medial basal hypothalamus (MBH), and assayed circulating estradiol, testosterone, and progesterone at six ages from birth through adulthood. Numerous genes underwent developmental change, particularly postnatal increases, decreases, or peaks/plateaus at puberty. Surprisingly, there were few sex differences; only Esr1, Kiss1, and Tac2 were dimorphic (higher in females). Cluster analysis of gene expression revealed sexually dimorphic correlations in the POA but not the MBH from P30 (Postnatal Day 30) to P60. Hormone measurements showed few sex differences in developmental profiles of estradiol; higher levels of progesterone in females only after P30; and a developmental pattern of testosterone with a nadir at P30 followed by a dramatic increase through P60 (males). Furthermore, bionetwork analysis revealed that hypothalamic gene expression profiles and their relationships to hormones undergo dynamic developmental changes that differ considerably from adults. These data underscore the importance of developmental stage in considering the effects of hormones on the regulation of neuroendocrine genes in the hypothalamus. Moreover, the finding that few neuroendocrine genes are sexually dimorphic highlights the need to consider postnatal development from a network approach that allows assessment of interactions and patterns of expression.

Neuroendocrine control of the transition to reproductive senescence: lessons learned from the female rodent model.

The natural transition to reproductive senescence is an important physiological process that occurs with aging, resulting in menopause in women and diminished or lost fertility in most mammalian species. This review focuses on how rodent models have informed our knowledge of age-related changes in gonadotropin-releasing hormone (GnRH) neurosecretory function and the subsequent loss of reproductive capacity. Studies in rats and mice have shown molecular, morphological and functional changes in GnRH cells. Furthermore, during reproductive aging altered sex steroid feedback to the hypothalamus contributes to a decrease of stimulatory signaling and increase in inhibitory tone onto GnRH neurons. At the site of the GnRH terminals where the peptide is released into the portal vasculature, the cytoarchitecture of the median eminence becomes disorganized with aging, and mechanisms of glial-GnRH neuronal communication may be disrupted. These changes can result in the dysregulation of GnRH secretion with reproductive decline. Interestingly, reproductive aging effects on the GnRH circuitry are observed in middle age even prior to any obvious physiological changes in cyclicity. We speculate that the hypothalamus may play a critical role in this mid-life transition. Because there are substantial species differences in these aging processes, we also compare and contrast rodent aging to that in primates. Work discussed herein shows that in order to understand neuroendocrine mechanisms of reproductive senescence, further research needs to be conducted in ovarian-intact models.

Prenatal PCBs disrupt early neuroendocrine development of the rat hypothalamus.

Neonatal exposure to endocrine disrupting chemicals (EDCs) such as polychlorinated biphenyls (PCBs) can interfere with hormone-sensitive developmental processes, including brain sexual differentiation. We hypothesized that disruption of these processes by gestational PCB exposure would be detectable as early as the day after birth (postnatal day (P) 1) through alterations in hypothalamic gene and protein expression. Pregnant Sprague-Dawley rats were injected twice, once each on gestational days 16 and 18, with one of the following: DMSO vehicle; the industrial PCB mixture Aroclor 1221 (A1221); a reconstituted mixture of the three most prevalent congeners found in humans, PCB138, PCB153, and PCB180; or estradiol benzoate (EB). On P1, litter composition, anogenital distance (AGD), and body weight were assessed. Pups were euthanized for immunohistochemistry of estrogen receptor α (ERα) or TUNEL labeling of apoptotic cells or quantitative PCR of 48 selected genes in the preoptic area (POA). We found that treatment with EB or A1221 had a sex-specific effect on developmental apoptosis in the neonatal anteroventral periventricular nucleus (AVPV), a sexually dimorphic hypothalamic region involved in the regulation of reproductive neuroendocrine function. In this region, exposed females had increased numbers of apoptotic nuclei, whereas there was no effect of treatment in males. For ERα, EB treatment increased immunoreactive cell numbers and density in the medial preoptic nucleus (MPN) of both males and females, while A1221 and the PCB mixture had no effect. PCR analysis of gene expression in the POA identified nine genes that were significantly altered by prenatal EDC exposure, in a manner that varied by sex and treatment. These genes included brain-derived neurotrophic factor, GABA(B) receptors-1 and -2, IGF-1, kisspeptin receptor, NMDA receptor subunits NR2b and NR2c, prodynorphin, and TGFα. Collectively, these results suggest that the disrupted sexual differentiation of the POA by prenatal EDC exposures is already evident as early as the day after birth, effects that may change the trajectory of postnatal development and compromise adult reproductive function.

The hypothalamic median eminence and its role in reproductive aging.

The median eminence at the base of the hypothalamus serves as an interface between the neural and peripheral endocrine systems. It releases hypothalamic-releasing hormones into the portal capillary bed for transport to the anterior pituitary, which provides further signals to target endocrine systems. Of specific relevance to reproduction, a group of about 1000 neurons in mammals release the gonadotropin-releasing hormone (GnRH) peptide from neuroterminals in the median eminence. During the life cycle, there are dramatic changes in reproductive demands, and we focus this review on how GnRH terminals in the median eminence change during reproductive senescence. We discuss morphological and functional properties of the median eminence, and how relationships among GnRH terminals and their microenvironment of nerve terminals, glial cells, and the portal capillary vasculature determine the ability of GnRH peptide to be secreted and to reach its target in the anterior pituitary gland.

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 (ERalpha) cell numbers in the hypothalamus, and their relationship to sexual performance in male rats. Young (3months) and middle-aged (12months) 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 ERalpha 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 ERalpha 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 ERalpha 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 ERalpha 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.

Sexual experience changes sex hormones but not hypothalamic steroid hormone receptor expression in young and middle-aged male rats.

Testosterone is well known to regulate sexual behavior in males, but this is dependent upon prior sexual experience. Aging is associated with decreased libido and changes in testosterone, but the role of experience in these age-related processes has not been systematically studied. We examined effects of age and sexual experience on serum hormones (total testosterone, free testosterone, estradiol, LH) and on numbers of androgen receptor (AR) and estrogen receptor alpha (ERalpha) immunoreactive cells in the hypothalamus. Extensive sexual experience was given to male rats at 4 months of age. Rats were euthanized at either 4 months (young) or 12 months (middle-aged (MA)). Comparable sexually naïve male rats were handled and placed into the testing arena but did not receive any sexual experience. Thus, we had four groups: young-naïve, young-experienced, MA-naïve and MA-experienced. Serum hormone levels were assayed, and numbers of AR and ERalpha cells were quantified stereologically in the medial preoptic nucleus (MPN) and the anteroventral periventricular nucleus (AVPV). Sexually experienced males had significantly elevated serum testosterone and free testosterone in both age groups. Both total and free testosterone were higher, and estradiol lower, in middle-aged than young rats. Experience did not alter either AR or ERalpha expression in the preoptic brain regions studied. Aging was associated with increased expression of AR, but no change in ERalpha. These results show that sexual experience can induce short-term and long-term alterations in serum hormones but these effects are not manifested upon their receptors in the hypothalamus.

Developmental programming and endocrine disruptor effects on reproductive neuroendocrine systems.

The ability of a species to reproduce successfully requires the careful orchestration of developmental processes during critical time points, particularly the late embryonic and early postnatal periods. This article begins with a brief presentation of the evidence for how gonadal steroid hormones exert these imprinting effects upon the morphology of sexually differentiated hypothalamic brain regions, the mechanisms underlying these effects, and their implications in adulthood. Then, I review the evidence that aberrant exposure to hormonally-active substances such as exogenous endocrine-disrupting chemicals (EDCs), may result in improper hypothalamic programming, thereby decreasing reproductive success in adulthood. The field of endocrine disruption has shed new light on the discipline of basic reproductive neuroendocrinology through studies on how early life exposures to EDCs may alter gene expression via non-genomic, epigenetic mechanisms, including DNA methylation and histone acetylation. Importantly, these effects may be transmitted to future generations if the germline is affected via transgenerational, epigenetic actions. By understanding the mechanisms by which natural hormones and xenobiotics affect reproductive neuroendocrine systems, we will gain a better understanding of normal developmental processes, as well as develop the potential ability to intervene when development is disrupted.