Influence of estradiol on NADPH diaphorase/neuronal nitric oxide synthase activity and colocalization with progesterone or type II glucocorticoid receptors in ovine hypothalamus

Nitric oxide (NO) has been shown to play an important role in both the neuroendocrine reproductive and stress axes, which are closely linked. Because progesterone (P4) receptors (PRs) and glucocorticoid receptors (GRs) are not found in GnRH neurons and the NOergic system has been implicated in the control of GnRH secretion, this study aimed to ascertain whether steroids altered the NOergic system. Our first objective was to map the distribution of NO synthase (NOS) cells in the ovine preoptic area (POA) and hypothalamus and to determine whether NOS activity is enhanced by estradiol (E2) treatment. Using NADPH diaphorase (NADPHd) histochemistry, we found that NADPHd-positive neurons were spread throughout the ovine POA and hypothalamus, and that all NADPHd cells were immunoreactive for NOS. In response to estradiol, a significant increase in the number of NADPHd cells was noted only in the ventrolateral region of the ventromedial nucleus (VMNvl), with no significant difference in the POA or arcuate nucleus. Progesterone and glucocorticoid receptors were colocalized with NADPHd reactive neurons in the POA, arcuate nucleus, and VMNvl of ewes in both treatment groups. In ewes receiving estradiol, the number of NADPHd-positive cells containing steroid receptors in the POA (PR, 81%; GR, 79%) and arcuate nucleus (PR, 89%; GR, 84%) was similar, but in the VMNvl, fewer NADPHd-positive cells contained GR (PR, 88%, GR, 31%). These data show that estradiol up-regulates NOS activity in a site-specific manner and that the influence and possible interaction of progesterone and corticosteroids on NO producing cells may differ according to the neural location.     

Estrogen receptor and progesterone receptor-immunoreactive cells are not co-localized with gonadotropin-releasing hormone in the brain of the female mink (Mustela vison)

The distribution of gonadal steroid (estrogen, progesterone) receptors in the brain of the adult female mink was mapped by immunocytochemistry. Using a monoclonal rat antibody raised against human estrogen receptor (ER), the most dense collections of ER-immunoreactive (IR) cells were found in the preoptic/anterior hypothalamic area, the mediobasal hypothalamus (arcuate and ventromedial nuclei), and the limbic nuclei (amygdala, bed nucleus of the stria terminalis, lateral septum). Immunoreactivity was mainly observed in the cell nucleus and a marked heterogeneity of staining appeared from one region to another. A monoclonal mouse antibody raised against rabbit uterine progesterone receptor (PR) was used to identify the PR-IR cells in the preoptic/anterior hypothalamic area and the mediobasal hypothalamus (arcuate and ventromedial nuclei). This study also focused on the relationship between cells containing sex-steroid receptors and gonadotropin-releasing hormone (GnRH) neurons on the same sections of the mink brain using a sequential double-staining immunocytochemistry procedure. Although preoptic and hypothalamic GnRH neurons were frequently in close proximity to perikarya containing ER or PR, they did not themselves possess receptor immunoreactivity. The present study provides neuroanatomical evidence that GnRH cells are not the major direct targets for gonadal steroids and confirms for the first time in mustelids the results previously obtained in other mammalian species.     

Studies on the cellular localization and distribution of glucocorticoid receptor and estrogen receptor immunoreactivity in the central nervous system of the rat and their relationship to the monoaminergic and peptidergic neurons of the brain

By means of monoclonal antibodies against the rat liver glucocorticoid receptor (GR) and the human estrogen receptor (ER), in combination with an immunocytochemical analysis, it has been possible to map out GR and ER immunoreactive (IR) neurons in the rat central nervous system and GR IR glial cells in the white matter. The GR IR is located in the cytoplasm and especially in the nucleus while the ER IR is only demonstrated in the nuclei of the neurons. Upon adrenalectomy the GR IR appears to be present exclusively in the cytoplasm, while after castration the ER IR is still exclusively present in the nuclei. Upon corticosterone treatment of the adrenalectomized rat the GR IR is again predominantly found in the nuclei of the neurons. These results indicate that the occupied GR and the unoccupied and occupied ER are located in the nuclei and the unoccupied GR in the cytoplasm. Evidence has been presented that large numbers of monoamine and peptide nerve cell bodies contain GR IR. Furthermore, neuronal GR IR is found in neuronal populations all over the central nervous system, especially in the cerebral cortex, the thalamus and the hypothalamus, indicating a major role of GR in regulating the metabolic and synaptic functions of the brain. The ER IR is instead limited to certain neuronal populations, mainly those of the preoptic area, the bed nucleus of the striae terminalis and the arcuate nucleus, suggesting a specific role in control of LHRH secretion and reproductive behaviour.     

Somatostatin-14 neurons in the ovine hypothalamus: colocalization with estrogen receptor alpha and somatostatin-28(1-12) immunoreactivity,and activation in response to estradiol

Pituitary gland growth hormone (GH) secretion is influenced by two hypothalamic neuropeptides: growth hormone-releasing hormone (GHRH) and somatostatin. Recent data also suggest that estrogen modulates GH release, particularly at the time of the preovulatory luteinizing hormone surge, when a coincident surge of GH is observed in sheep. The GHRH neurons do not possess estrogen receptor alpha (ERalpha), suggesting that estrogen does not act directly on GHRH neurons. Similarly, few somatotropes express ERalpha, suggesting a weak pituitary effect of estradiol on GH. It was hypothesized, therefore, that estradiol may affect somatostatin neurons to modulate GH release from the pituitary. Using immunocytochemical approaches, the present study revealed that although somatostatin neurons were located in several hypothalamic sites, only those in the arcuate nucleus (13% +/- 2%) and ventromedial nucleus (VMN; 29% +/- 1%) expressed ERalpha. In addition, we found that all neurons immunoreactive for somatostatin-14 were also immunoreactive for somatostatin-28(1-12). To determine whether increased GH secretion in response to estradiol is through modulation of GHRH and/or somatostatin neuronal activity, a final study investigated whether c-fos expression increased in somatostatin- and GHRH-immunoreactive cells at the time of the estradiol-induced LH surge in intact anestrous ewes. Estradiol significantly (P < 0.05) increased the percentage of GHRH (estradiol, 75% +/- 3%; no estradiol, 19% +/- 2%) neurons expressing c-fos in the hypothalamus. The percentage of somatostatin-immunoreactive neurons coexpressing c-fos in the estradiol-treated animals was significantly (P < 0.05) higher (periventricular, 44% +/- 3%; arcuate, 72% +/- 5%; VMN, 81% +/- 5%) than in the control animals (periventricular, 22% +/- 1%; arcuate, 29% +/- 3%; VMN, 31% +/- 3%). The present study suggests that estradiol modulates the activity of GHRH and somatostatin neurons but that this effect is most likely mediated through an indirect interneuronal pathway.     

Immunocytochemical colocalization of progestin receptors and β-endorphin or enkephalin in the hypothalamus of female guinea pigs

Double-label immunocytochemistry was used to determine whether estradiol-induced progestin receptors and either β-endorphin or leucine-enkephalin are colocalized in female guinea pig brain. Ovariectomized, adult guinea pigs were implanted with capsules containing estradiol-17β to induce high levels of progestin receptors, and injected intracerebroventricularly with co chicine to improve visualization of the opiate peptides. Sections through the hypothalamus and preoptic area were processed for progestin receptor, followed by β-endorphin or leucine-enkephalin immunocytochemistry. As reported previously, high concentrations of progestin receptor-immunoreactive (PR-IR) cells were found in the preoptic area (medial and periventricular portions, medial preoptic nucleus) and hypothalamus (anterior hypothalamic and arcuate nuclei, ventrolateral area). Many β-endorphin-IR cells contained PR-IR in the arcuate nucleus and its surroundings (33%) and in the dorsomedial area of the hypothalamus (64%). Scattered enkephalin-IR cells were found in the septal nucleus, medial and lateral preoptic area, bed nucleus of the stria terminalis, and the arcuate nucleus. The ventromedial nucleus of the hypothalamus and dorsolateral magnocellular nucleus, respectively, contained moderate and heavy concentrations of enkephalin-IR cells. Although some of these areas also contained PR-IR, enkephalin-IR was colocalized consistently with PR-IR only in a small number of cells in the arcuate nucleus and ventromedial/ventrolateral area of the hypothalamus. These data, taken together with earlier observations that virtually all cells containing estradiol-induced PR-IR also contain estrogen receptor-IR, provide neuroanatomical evidence that hypothalamic actions of progesterone and estradiol may be mediated by β-endorphin and/or enkephalin.     

Immunocytochemical colocalization of progestin receptors and beta-endorphin or enkephalin in the hypothalamus of female guinea pigs

Double-label immunocytochemistry was used to determine whether estradiol-induced progestin receptors and either beta-endorphin or leucine-enkephalin are colocalized in female guinea pig brain. Ovariectomized, adult guinea pigs were implanted with capsules containing estradiol-17 beta to induce high levels of progestin receptors, and injected intracerebroventricularly with colchicine to improve visualization of the opiate peptides. Sections through the hypothalamus and preoptic area were processed for progestin receptor, followed by beta-endorphin or leucine-enkephalin immunocytochemistry. As reported previously, high concentrations of progestin receptor-immunoreactive (PR-IR) cells were found in the preoptic area (medial and periventricular portions, medial preoptic nucleus) and hypothalamus (anterior hypothalamic and arcuate nuclei, ventrolateral area). Many beta-endorphin-IR cells contained PR-IR in the arcuate nucleus and its surroundings (33%) and in the dorsomedial area of the hypothalamus (64%). Scattered enkephalin-IR cells were found in the septal nucleus, medial and lateral preoptic area, bed nucleus of the stria terminalis, and the arcuate nucleus. The ventromedial nucleus of the hypothalamus and dorsolateral magnocellular nucleus, respectively, contained moderate and heavy concentrations of enkephalin-IR cells. Although some of these areas also contained PR-IR, enkephalin-IR was colocalized consistently with PR-IR only in a small number of cells in the arcuate nucleus and ventromedial/ventrolateral area of the hypothalamus. These data, taken together with earlier observations that virtually all cells containing estradiol-induced PR-IR also contain estrogen receptor-IR, provide neuroanatomical evidence that hypothalamic actions of progesterone and estradiol may be mediated by beta-endorphin and/or enkephalin.     

Coumestrol antagonizes neuroendocrine actions of estrogen via the estrogen receptor alpha

The phytoestrogen coumestrol has estrogenic actions on peripheral reproductive tissues. Yet in the brain this compound has both estrogenic and anti-estrogenic effects. We used estrogen receptor alpha knockout mice (ERalphaKO) to determine whether coumestrol has estrogenic actions in mice and also if these effects are mediated by the classic ERalpha. Female wild-type (WT) and ERalphaKO mice were ovariectomized and treated with estradiol (E2), dietary coumestrol, both, or neither compound. Ten days later the animals were sacrificed, blood was collected, and brain tissues were perfused. Fixed brains were sectioned and immunocytochemistry was employed to quantify progesterone receptors (PR) in the medial preoptic (POA) and ventromedial nucleus of the hypothalamus (VMN). Plasma was assayed for luteinizing hormone (LH). Estrogen treatment induced PR immunoreactivity in both regions in brains of WT females. In ERalphaKO mice, lower levels of PR were induced. The stimulatory effects of E2 on PR were attenuated in the POA by cotreatment with coumestrol, and the same trend was noted in the VMN. WT ovariectomized females treated with E2 had low levels of LH, while LH was high in untreated females and even higher in ovariectomized females treated with coumestrol. ERalphaKO females in all treatment groups had high levels of LH. Taken together, the results show that coumestrol has anti-estrogenic actions in the brain and pituitary and that ERalpha mediates these effects.     

Increased estrogen receptor alpha immunoreactivity in the forebrain of sexually satiated rats

Estrogen receptor alpha (ERalpha) participates in the neuroendocrine regulation of male sexual behavior, primarily in brain areas located in the limbic system. Males of many species present a long-term inhibition of sexual behavior after several ejaculations, known as sexual satiety. It has been shown that androgen receptor density is reduced 24 h after a single ejaculation or mating to satiety, in the medial preoptic area, nucleus accumbens and ventromedial hypothalamus. The aim of this study was to analyze if the density of ERalpha was also modified 24 h after a single ejaculation or mating to satiety. Sexual satiety was associated with an increased ERalpha density in the anteromedial bed nucleus of the stria terminalis (BSTMA), ventrolateral septum (LSV), posterodorsal medial amygdala (MePD), medial preoptic area (MPA) and nucleus accumbens core (NAc). A single ejaculation was related to an increase in ERalpha density in the BSTMA and MePD. ERalpha density in the arcuate (Arc) and ventromedial hypothalamic nuclei (VMN), and serum estradiol levels remained unchanged 24 h after one ejaculation or mating to satiety. These data suggest a relationship between sexual activity and an increase in the expression of ERalpha in specific brain areas, independently of estradiol levels in systemic circulation.     

Ultrastructural analysis of estrogen receptor immunoreactive neurons in the medial preoptic area of the female rat brain

Neurons of the medial preoptic area were studied in the brain of the female rat by means of ultrastructural immunocytochemistry using a monoclonal antibody generated against purified estrogen receptor (ER), in order to delineate the morphological correlates of estrogen feedback mechanisms. In addition to the preoptic area, the bed nucleus of the stria terminalis, the arcuate and ventromedial nuclei of the hypothalamus exhibited an intense labelling for estrogen receptor. At the light microscopic level, the cell nuclei were immunoreactive. No major alterations were detected in the ER expression of medial preoptic neurons sampled during the estrous cycle, but proestrous rats did exhibit a slightly increased intensity of staining. At the ultrastructural level, the ER immunoreactivity was primarily confined to the nuclei and associated with the chromatin. Long term steroid deprivation elicited by either ovariectomy or ovariectomy plus adrenalectomy resulted in a marked intensity of nuclear labelling. This pattern was not influenced by acute estradiol replacement. These morphological data indicate that neurons of the medial preoptic area have the capacity to detect estrogens via receptor mechanisms and that changes in the level of the circulating ligand are manifested in an alteration in the staining for the estrogen receptor. The study also supports the revised concept of estrogen receptor action by demonstrating the presence of receptors in the nuclei of the cells, whether or not they are occupied by their ligand.     

Progesterone receptor mRNA expression and distribution in the female rabbit brain

Progesterone regulates diverse functions in the rabbit brain through the interaction with its nuclear receptor (PR). Although PR protein has been detected in some regions of the rabbit forebrain, PR mRNA expression and distribution in the rabbit brain are unknown. Hence, we investigated these issues by in situ hybridization. New Zealand adult female rabbits were ovariectomized and treated with vehicle or estradiol (5 μg/(kg day)) for 3 days. The results show an extended distribution of PR mRNA expression in the rabbit brain. The highest expression was detected in preoptic area and hypothalamic anterior nuclei such as paraventricular, periventricular and arcuate nuclei. A high expression was also detected in thalamic and telencephalic areas, including hippocampus and cerebral cortex. Estradiol treatment induced an increase in PR mRNA expression in many brain areas, particularly in the hippocampus and the hypothalamic and preoptic area regions. The wide distribution of PR mRNA in the rabbit brain suggests that progesterone through PR activation is involved in several functions apart from reproductive behavior in rabbits, and that PR expression is up-regulated by estradiol in the rabbit brain.     

Ultrastructural analysis of estrogen receptor immunoreactive neurons in the medial preoptic area of the female rat brain

Summary Neurons of the medial preoptic area were studied in the brain of the female rat by means of ultrastructural immunocytochemistry using a monoclonal antibody generated against purified estrogen receptor (ER), in order to delineate the morphological correlates of estrogen feedback mechanisms. In addition to the preoptic area, the bed nucleus of the stria terminalis, the arcuate and ventromedial nuclei of the hypothalamus exhibited an intense labelling for estrogen receptor. At the light microscopic level, the cell nuclei were immunoreactive. No major alterations were detected in the ER expression of medial preoptic neurons sampled during the estrous cycle, but proestrous rats did exhibit a slightly increased intensity of staining. At the ultrastructural level, the ER immunoreactivity was primarily confined to the nuclei and associated with the chromatin. Long term steroid deprivation elicited by either ovariectomy or ovariectomy plus adrenalectomy resulted in a marked intensity of nuclear labelling. This pattern was not influenced by acute estradiol replacement.These morphological data indicate that neurons of the medial preoptic area have the capacity to detect estrogens via receptor mechanisms and that changes in the level of the circulating ligand are manifested in an alteration in the staining for the estrogen receptor. The study also supports the revised concept of estrogen receptor action by demonstrating the presence of receptors in the nuclei of the cells, whether or not they are occupied by their ligand.Supported by grants from the IBRO/MacArthur Foundation Network Grant, the National Science Foundation (NSF INT 8703030), the Hungarian Academy of Sciences (OTKA 104), the National Institutes of Health (NS 19266), the National Foundation of Technical Development (OKKFT Tt 286/1986) and the Well-come Trust (14685/1.5)     

Uterine estrogen receptor alpha and progesterone receptor during the follicular and luteal phase in llamas

Estrogen receptor-alpha (ERalpha) and progesterone receptor (PR) were characterized in different endometrial cell types as luminal and glandular epithelium and stroma during the follicular (FP) and the luteal phase (LP) in llamas. Animals were examined daily by transrectal ultrasonography for the determination of the presence of an ovulatory follicle and ovulation was immediately induced by a GnRH injection (Day 0). Endometrial samples were obtained by transcervical biopsies from the left uterine horn on Day 0 (FP) and 9 days after the GnRH injection (Day 9, LP). Blood samples were collected on these days for estradiol 17beta and progesterone determination by RIA. An immunohistochemical technique was used to visualize ERalpha and PR immunostaining which was then analyzed by two independent observers. Total positive area and average staining for ERalpha were affected by the phase of the ovarian activity: in the three cell types there was more positive area and intense staining during the FP than during the LP. Similar findings were observed for PR, more positive stained areas were found during the FP than during the LP in the epithelia. In addition, the three cell types had more intense staining during the FP than during the LP. An effect of the cell type for ERalpha and PR was observed; epithelia (luminal and glandular) had more positive stained areas and greater intensity than stromal cells. In conclusion, the results of the present study suggest that in llamas, like in other ruminants, estradiol has a stimulatory effect while progesterone downregulates the ERalpha and PR and that the receptor is cell type specific.     

Steroid control of gonadotropin-releasing hormone secretion: associated changes in pro-opiomelanocortin and preproenkephalin messenger RNA expression in the ovine hypothalamus

The endogenous opioid peptides have been implicated in mediating the actions of estrogen and progesterone on GnRH release. We used in situ hybridization histochemistry to determine whether steroid-induced changes in GnRH/LH release in the female sheep are associated with changes in the cellular mRNA content of the precursors for beta-endorphin (pro-opiomelanocortin; POMC) and met-enkephalin (pre-proenkephalin; PENK). Two specific hypotheses were tested. First, that the inhibitory actions of progesterone are associated with an increase in opioid gene expression in specific hypothalamic nuclei. Our data support this hypothesis. Thus, an increase in progesterone was associated with increased POMC gene expression in the arcuate nucleus and PENK in the paraventricular nucleus. Further, the increase in POMC was restricted to regions of the arcuate nucleus that contain steroid sensitive beta-endorphin neurons. Our second hypothesis, that gene expression for the two opioid precursors would decrease prior to the start of the estradiol-stimulated GnRH surge, was not supported. Rather, POMC (but not PENK) gene expression in the arcuate nucleus was significantly higher in estradiol-treated animals than controls at the peak of the GnRH surge. These data suggest that beta-endorphin neurons in subdivisions of the arcuate nucleus and enkephalin neurons in the paraventricular nucleus are part of the neural network by which progesterone inhibits LH release. While enkephalin neurons may not play a role in estrogen positive feedback, increases in POMC mRNA in the arcuate nucleus at the time of the GnRH peak may be important for replenishing beta-endorphin stores and terminating estrous behavior.     

Orphanin FQ in the mediobasal hypothalamus facilitates sexual receptivity through the deactivation of medial preoptic nucleus mu-opioid receptors

Sexual receptivity, lordosis, can be induced by sequential estradiol and progesterone or extended exposure to high levels of estradiol in the female rat. In both cases estradiol initially inhibits lordosis through activation of β-endorphin (β-END) neurons of the arcuate nucleus of the hypothalamus (ARH) that activate μ-opioid receptors (MOP) in the medial preoptic nucleus (MPN). Subsequent progesterone or extended estradiol exposure deactivates MPN MOP to facilitate lordosis. Opioid receptor-like receptor-1 (ORL-1) is expressed in ARH and ventromedial hypothalamus (VMH). Infusions of its endogenous ligand, orphanin FQ (OFQ/N, aka nociceptin), into VMH–ARH region facilitate lordosis. Whether OFQ/N acts in ARH and/or VMH and whether OFQ/N is necessary for steroid facilitation of lordosis are unclear. In Exp I, OFQ/N infusions in VMH and ARH that facilitated lordosis also deactivated MPN MOP indicating that OFQ/N facilitation of lordosis requires deactivation of ascending ARH-MPN projections by directly inhibiting ARH β-END neurons and/or through inhibition of excitatory VMH–ARH pathways to proopiomelanocortin neurons. It is unclear whether OFQ/N activates the VMH output motor pathways directly or via the deactivation of MPN MOP. In Exp II we tested whether ORL-1 activation is necessary for estradiol-only or estradiol + progesterone lordosis facilitation. Blocking ORL-1 with UFP-101 inhibited estradiol-only lordosis and MPN MOP deactivation but had no effect on estradiol + progesterone facilitation of lordosis and MOP deactivation. In conclusion, steroid facilitation of lordosis inhibits ARH β-END neurons to deactivate MPN MOP, but estradiol-only and estradiol + progesterone treatments appear to use different neurotransmitter systems to inhibit ARH-MPN signaling.     

Central peptidergic neurons as targets for glucocorticoid action. Evidence for the presence of glucocorticoid receptor immunoreactivity in various types of classes of peptidergic neurons.

By means of double immunolabeling procedures it has been possible to demonstrate glucocorticoid receptor (GR) immunoreactivity (IR) in large numbers of various peptidergic neurons of the brain including neurons containing gastrointestinal peptides, opioid peptides, and peptides with a hypothalamic hormone function. For each peptide system, however, marked heterogeneities exist among brain regions. Thus, in the neocortex and the hippocampal formation most of the brain peptide neurons lack GR IR, while the same types of peptide neurons in the arcuate and paraventricular nucleus [e.g. neuropeptide Y (NPY), somatostatin (SRIF) and the cholecystokinin (CCK) neurons] possess strong GR IR. Furthermore, in the arcuate, parvocellular part of the paraventricular nuclei and the central amygdaloid nucleus practically all the peptidergic neurons are strongly GR IR, while in the lateral hypothalamus, mainly the neurotensin (NT) and galanin (GAL) IR neurons are GR IR. These marked differences among areas probably reflect functional differences dependent upon their participation in stress regulated circuits. All the paraventricular NT, corticotropin-releasing factor (CRF), growth hormone-releasing factor (GRF), thyrotropin-releasing hormone (TRH) and SRIF IR neurons appear to contain GR IR, while the luteinizing hormone-releasing hormone (LHRH) IR neurons lack GR IR, underlying the importance of glucocorticoids (GC) in controlling endocrine function. Finally, the GC may influence pain and mood control mainly via effects on enkephalin (ENK) neurons especially in the basal ganglia (mood) and on all beta-endorphin (beta-END) neurons of the arcuate nucleus, while most of the dynorphin neurons are not directly controlled by GC.