Sexual dimorphism in the content of progesterone and estrogen receptors, and their cofactors in the lung of adult rats

Progesterone and estradiol play an important role in the regulation of lung functions such as ventilation and vasoconstriction. The genomic actions of progesterone and estradiol are mediated by their nuclear receptors: progesterone receptors (PR) and estrogen receptors (ER). These actions are linked to interactions between steroid receptors and some cofactors that function as coactivators or corepressors. In this work we determined the content of PR isoforms, ER-beta, one coactivator (SRC-1), and one corepressor (SMRT) in the lung of both female rats during the estrous cycle and intact males by Western blot. The rat lung presented a higher content of PR-A than that of PR-B during the estrous cycle. The highest content of both PR isoforms was observed on the day of proestrus whereas the lowest one was found on the day of estrus. In contrast, the content of ER-beta was the lowest on the day of proestrus and it increased at estrus. The content of SRC-1 and SMRT increased on the day of diestrus. SRC-1 content decreased at proestrus and estrus, while SMRT content decreased at proestrus and increased again at estrus. In the lung of adult male rats the content of PR isoforms, ER-beta and SMRT was lower than in that of females, whereas the content of SRC-1 was similar in both sexes. Our results suggest a sexual dimorphism in the content of PR, ER-beta, and SMRT in the rat lung as well as a relation of their content to the physiological levels of progesterone and estradiol.     

Prenatal exposure to bisphenol A promotes angiogenesis and alters steroid-mediated responses in the mammary glands of cycling rats

Prenatal exposure to BPA disturbs mammary gland histoarchitecture and increases the carcinogenic susceptibility to chemical challenges administered long after BPA exposure. Our aim was to assess the effect of prenatal BPA exposure on mammary gland angiogenesis and steroid hormone pathways in virgin cycling rats. Pregnant Wistar rats were exposed to either 25 or 250 g/kg/day (25 and 250 BPA, respectively) or to vehicle. Female offspring were autopsied on postnatal day (PND) 50 or 110. Ovarian steroid serum levels, the expression of steroid receptors and their co-regulators SRC-3 and SMRT in the mammary gland, and angiogenesis were evaluated. At PND 50, all BPA-treated animals had lower serum levels of progesterone, while estradiol levels remained unchanged. The higher dose of BPA increased mammary ERα and decreased SRC-3 expression at PND 50 and PND 110. SMRT protein levels were similar among groups at PND 50, whereas at PND 110, animals exposed to 250 BPA showed a lower SMRT expression. Interestingly, in the control and 25 BPA groups, SMRT increased from PND 50 to PND 110. At PND 50, an increased vascular area associated with higher VEGF expression was observed in the 250 BPA-treated rats. At PND 110, the vascular area was still increased, but VEGF expression was similar to that of control rats. The present results demonstrate that prenatal exposure to BPA alters the endocrine environment of the mammary gland and its angiogenic process. Increased angiogenesis and altered steroid hormone signals could explain the higher frequency of pre-neoplastic lesions found later in life. This article is part of a Special Issue entitled 'Endocrine disruptors'.     

Evidence of sex hormone binding globulin binding sites in the medial preoptic area and hypothalamus.

We have demonstrated a high density of both radiolabeled progesterone and estradiol conjugated to bovine serum albumin binding sites in the medial preoptic area and hypothalamus. Infusions of sex hormone binding globulin into the medial preoptic area of rats increased their female sexual receptivity similarly to the effect of estradiol conjugated to bovine serum albumin, suggesting sex hormone binding globulin acts at binding sites for estradiol conjugated to bovine serum albumin. In this study sex hormone binding globulin was used to displace radiolabeled progesterone conjugated to bovine serum albumin from plasma membrane fractions from the medial preoptic area-anterior hypothalamus and medial basal hypothalamus of ovariectomized rats injected with either 5 microg estradiol benzoate or sesame oil vehicle. We found that sex hormone binding displaced radiolabeled progesterone conjugated to bovine serum albumin in both areas and that in vivo estradiol treatment greatly increased the relative displacement by sex hormone binding globulin in the medial preoptic area-anterior hypothalamus. We interpret these data as indicating the presence of sex hormone binding globulin receptors in brain plasma membranes and further suggest that endogenous steroid conditions may alter these receptors.     

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.     

Characterization of the androgen receptors in the hypothalamus, preoptic area and brain cortex of the rat.

The specific androgen receptors for testosterone (T) (1) and 5alpha-dihydrotestosterone (DHT) in the cytosol fraction of the hypothalamus, preoptic area and brain cortex of the rat have been characterized using electrophoresis and isoelectric focusing in polyacrylamide gels. After labeling of the cytosol fractions in vivo and in vitro we were able to demonstrate androgen-receptor complexes moving with an electrophoretic mobility (R(f) of 0.5 in 3.25% acrylamide gels containing 0.5% agarose and 10% glycerol. Polyacrylamide gel electrophoresis was used as a quantitative assay for androgen receptors in the tissues. The hypothalamus, preoptic area and brain cortex were found to possess a single class of high affinity binding sites for androgens and the dissociation constants (K(D) were estimated to be 3.4, 4.3 and 2.6 X 10 (-10M) respectively. The binding capacities were 3.7 (hypothalamus), 3.5 (preoptic area) and 1.8 X 10 (-15) (brain cortex) moles of high affinity binding sites per mg protein. Like other androgen-receptor complexes, the testosterone-receptor complexes of the hypothalamus, preoptic area and brain cortex were temperature labile, sulfhydryl dependent and revealed a very slow rate of dissociation at o degrees C (t1/2 greater than 36 hr). The receptors in all the tissues had an isoelectric point of 5.8. The steroid specificity of the cytoplasmic androgen receptors was tested in vitro by the competing efficiency of different unlabeled steroids for (3H)-testosterone binding. In the three tissues in investigation the following order of affinity was found: DHT greater than T greater than Cyproterone acetate greater than progesterone greater than androstenedione greater than 17beta-estradiol. Cortisol did not effect androgen binding significantly. Thus, the physiochemical characteristics of the cytoplasmic androgen receptors of the hypothalamus, preoptic area and brain cortex are very similar, if not identical, to those of the androgen receptors described in the anterior pituitary, ventral prostate, epididymis and testis.     

Regulation of the female rat estrous cycle by a neural cell-specific epidermal growth factor-like repeat domain containing protein,NELL2

NELL2, a protein containing epidermal growth factor-like repeat domains, is predominantly expressed in the nervous system. In the mammalian brain, NELL2 expression is mostly neuronal. Previously we found that NELL2 is involved in the onset of female puberty by regulating the release of gonadotropin-releasing hormone (GnRH), and in normal male sexual behavior by controlling the development of the sexually dimorphic nucleus of the preoptic area (POA). In this study we investigated the effect of NELL2 on the female rat estrous cycle. NELL2 expression in the POA was highest during the proestrous phase. NELL2 mRNA levels in the POA were increased by estrogen treatment in ovariectomized female rats. Blocking NELL2 synthesis in the female rat hypothalamus decreased the expression of kisspeptin 1, an important regulator of the GnRH neuronal apparatus, and resulted in disruption of the estrous cycle at the diestrous phase. These results indicate that NELL2 is involved in the maintenance of the normal female reproductive cycle in mammals.     

Steroid receptors in the brain: Topography and some functional implications

The anatomical localization of brain cells which concentrate steroid hormones or their metabolites was carried out by radioautographic procedures. Ovariectomized or adrenalectomized animals were injected with the appropriate tritiated hormones, and brain tissue was processed through procedures which minimize the removal or displacement of steroids. Target cells were characterized by the concentration and retention of radioactive hormone in their nuclei. For each mammalian steroid hormone, nuclear binding sites exist in populations of cells with a specific regional localization in the brain and in the pituitary. The distribution of estrogen target cells was remarkably similar in the brains of rodents and primates although some minor species differences existed. Heavily labeled cells were present in the preoptic region, the septum, the amygdala and the mediobasal hypothalamus. The localization of progestagen-concentrating cells in the rodent and galago brain was limited to two hypothalamic areas: the preoptic region and the mediobasal hypothalamus. Corticosterone target cells were situated in extrahypothalamic regions of the rat central nervous system such as the hippocampus, the septum, the amygdala and certain regions of the brain cortex. However, the synthetic glucocorticosteroid, dexamethasone, was mainly found in the pituitary cells and in some neurons and glial cells of the mediobasal hypothalamus. The distribution pattern of steroid-sensitive cells within the brain and the pituitary gland corresponds to sites which are involved in the neuroendocrine processes regulating reproduction, including gonadotropin secretion and sexual behavior.     

Changes in the content and distribution of microtubule associated protein 2 in the hippocampus of the rat during the estrous cycle

The molecular mechanisms involved in the regulation of synaptic plasticity in the hippocampus during the estrous cycle of the rat are not completely understood. Because this process implicates changes in neuronal cytoskeleton organization, we analyzed the content of microtubule associated protein 2 (MAP2) and Tau in the hippocampus and the frontal cortex of the rat by Western blot, as well as the hippocampal distribution of MAP2 during the estrous cycle by immunohistochemistry. In the hippocampus the lowest content of MAP2 was found on diestrus day, and it significantly increased at proestrus. This increase was maintained on estrus and metestrus days. In the frontal cortex MAP2 content did not significantly change during the estrous cycle. In contrast, the content of Tau did not vary during the estrous cycle in either the hippocampus or the frontal cortex. The immunohistochemical analysis showed an increase in dendrite thickness and in dendritic branching in the CA1 region on proestrus day, as well as an aggregation of MAP2 in apical dendrites near to pyramidal somata on this day in comparison with diestrus. We suggest that changes in the content and neuronal distribution of MAP2 are involved in the structural changes that occur in the hippocampus of the rat during the estrous cycle, and that these variations are related to changes in estradiol and progesterone levels.     

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.     

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)     

Expression of nuclear hormone receptors, their coregulators and type I iodothyronine 5'-deiodinase gene in mammary tissue of nonlactating and postlactating rats

The aim of the study was to test the hypothesis that expression of retinoid receptors (RARalpha, RARbeta, RARgamma), rexinoid receptors (RXRalpha, RXRbeta), thyroid hormone receptors (TRalpha, TRbeta), estrogen receptors (ERalpha, ERbeta), nuclear receptor coregulators (N-CoR, SRC-1, SMRT), and in addition type I iodothyronine 5'-deiodinase (5'-DI), EGFR and erb-B2/neu would be different in mammary postlactating tissue in comparison with that of nonlactating mammary gland. Using RT-PCR, we have shown that expression of RARalpha, RXRalpha,TRalpha, ERalpha,ERbeta,N-CoR, SRC-1, SMRT and EGFR in rat was significantly increased in postlactating mammary gland when compared to that of nonlactating mammary tissue. Postlactating mammary glands were found to express all RAR and RXR subtypes studied when compared to nonlactating mammary tissues that express exclusively RARalpha and RXRalpha subtypes. Enhanced expression of a number of nuclear hormone receptors, their coregulators in mammary tissue of postlactating rats in comparison with nonlactating animals identify a potential role for retinoid, thyroid and estrogen signalling pathways also after lactation period.     

Changes in the pituitary and hypothalamic content of methionine-enkephalin during the estrous cycle of rats

Met-Enkephalin content of the anterior hypothalamic-preoptic area, medial basal hypothalamus, anterior pituitary, intermediate and posterior pituitary was measured using a specific radioimmunoassay. Met-Enkephalin content of the anterior hypothalamic-preoptic area, medial basal hypothalamus and anterior pituitary was very high on the morning of proestrus but decreased on the afternoon of proestrus and on estrus. The content of met-Enkephalin was more variable in the anterior pituitary than in the anterior hypothalamic-preoptic area and medial basal hypothalamus during the estrous cycle. The results suggest that the met-Enkephalin may be involved in regulating the hypothalamo-hypophyseal function during estrous cycle in the rat.     

The effect of the estrous cycle and estrogen on the release of immunoreactive alpha-melanocyte-stimulating hormone

Circulating levels and tissue content of alpha-MSH were measured on the morning of various days of the estrous cycle, and on the afternoon of proestrus in freely moving conscious rats. No surges of alpha-MSH were detected by RIA in the morning of various days of the cycle. The neurointermediate lobe content of alpha-MSH was slightly elevated on diestrus 1 as compared to the levels on diestrus 11 and proestrus but not to estrous levels. No changes in alpha-MSH content were detected in the anterior pituitary, the median eminence, mediobasal hypothalamus and the preoptic area at various stages of the estrous cycle. Plasma alpha-MSH levels were slightly elevated at 1500 hr of proestrus which was followed three hours later by a decline. This profile of plasma alpha-MSH on the afternoon of proestrus was reproduced by the SC administration of estradiol benzoate to long-term ovariectomized rats. These data suggest that, contrary to the results obtained by bioassay of alpha-MSH no surges of alpha-MSH occur on any day of the cycle, although a slight elevation on the afternoon of proestrus was detected. The altered pattern of release of this peptide on the afternoon of proestrus may be induced by estrogen.     

Sex differences and synchronous development of steroid receptor coactivator-1 and synaptic proteins in the hippocampus of postnatal female and male C57BL/6 mice

The structure and function including synaptic plasticity of the hippocampus are deeply affected by steroids in a sex-dependant manner, these processes are believed to be mediated by steroid receptors though their coactivators. Our previous studies have reported the developmental profiles of steroid receptor coactivator-1 (SRC-1) and PSD-95 in the hippocampus of postnatal female rats and the sex-differences of SRC-1 immunoreactivities in the brain of adult mice. However, whether there are any sex differences about postnatal development of SRC-1 and synaptic proteins in the hippocampus remain unclear. In this study, we investigated the postnatal profile of SRC-1 and key synaptic protein synaptophysin (SYN), PSD-95 and GluR1 in the hippocampus of female and male mice using immunohistochemistry and Western blot. The results showed that in the female hippocampus, the highest levels of SRC-1 were detected at P14, SYN and GluR1 at P30 and PSD-95 at P60; while in the males, the highest levels of SRC-1, SYN and GluR1 were detected at P30, and PSD-95 at P60. Female hippocampus tended to have higher levels of SRC-1, SYN and GluR1 before P30 and PSD-95 before P14; while male hippocampus have higher levels of PSD-95 at P14, P60 and GluR1 at P0. Correlation analysis showed the profiles of SRC-1 were highly correlated with each synaptic protein. The above results showed that in the hippocampus, except some minor sex differences detected at some time-point examined, females and males shared similar postnatal developmental profile and SRC-1 may be deeply involved in the regulation of hippocampal synaptogenesis.     

AAV-mediated leptin receptor installation improves energy balance and the reproductive status of obese female Koletsky rats

Leptin is a hormone secreted primarily by white adipocytes that regulates energy homeostasis and reproduction via CNS receptors. Koletsky (f/f) rats with a leptin receptor (OB-Rb) gene mutation are obese, diabetic and infertile. We employed recombinant adeno-associated viral (rAAV) vectors to transfer the human OB-Rb gene into the brains of female Koletsky rats to identify sites of leptin action in the brain. rAAV-OB-Rb was microinjected into the medial preoptic area (MPOA), the paraventricular nucleus (PVN), the ventromedial hypothalamus, the arcuate nucleus (ARC), or the dorsal vagal complex in the brainstem. Food intake and body weight were monitored bi-weekly for 55 days. Vaginal cytology was examined daily to assess estrous cyclicity. After sacrifice, uncoupling protein-1 (UCP-1) mRNA in brown adipose tissue and serum concentrations of leptin, insulin, glucose, estradiol and progesterone were measured. Expression of OB-Rb was documented by RT-PCR and site specificity of microinjection was verified by immunohistochemical detection of green fluorescent protein following a control microinjection of rAAV-GFP. OB-Rb installation in the ARC reduced food intake, however, energy expenditure, assessed by UCP-1 mRNA expression, was increased by OB-Rb installation in all sites except the PVN. When injected into the MPOA and ARC, rAAV-OB-Rb stimulated the reproductive axis as evidenced by normalization of estrous cycle length and increased luteinizing hormone releasing hormone concentrations in the hypothalamus. These studies show that long-term installation of a functional leptin receptor in the CNS is achievable using rAAV vectors and further show that leptin acts on specific sites in the brain to produce differential effects on food intake, energy expenditure and reproduction.     

Neurotransmitter-controlled steroid hormone receptors in the central nervous system

Results are discussed indicating that neurotransmitters affect steroid hormone activity not only by controlling via neuroendocrine events the hypophysial-gonadal and hypophysial-adrenal axes, but also by modulating cell responsiveness to steroids in target cells. Hyper- or hypoactivity of pineal nerves result in enhancement or impairment of estradiol and testosterone effects on pineal metabolism in vivo and in vitro. Pineal cytoplasmic and nuclear estrogen and androgen receptors are modulated by norepinephrine released from nerve endings at the pinealocyte level. Neural activity affects the cycle of depletion-replenishment of pineal estrogen receptors following estradiol administration. Another site of modulation of steroid effects on the pinealocytes is the intracellular metabolism of testosterone and progesterone; nerve activity has a positive effect on testosterone aromatization and a negative effect on testosterone and progesterone 5α-reduction. NE activity on the pineal cells is mediated via β-adrenoceptors and cAMP. In the central nervous system information on the neurotransmitter modulation of steroid hormone action includes the following observations: (a) hypothalamic deafferentation depresses estrogen receptor levels in rat medial basal hypothalamus; (b) changes in noradrenergic transmission affect, via α-adrenoceptors, the estradiol-induced increase of cytosol progestin receptor concentration in guinea pig hypothalamus; (c) cAMP increases testosterone aromatization in cultured neurons from turtle brain; (d) electrical stimulation of dorsal hippocampus augments, and reserpine or 6-hydroxydopamine treatment decrease, corticoid binding in cat hypothalamus. In the adenohypophysis changes in dopaminergic input after median eminence lesions or bromocriptine treatment of rats result in opposite modifications of pituitary estrogen receptor levels. Therefore all these observations support the view that neurotransmitters can modulate the attachment of steroid hormones to their receptors in target cells.     

Sex and estrous cycle-dependent differences in glial fibrillary acidic protein immunoreactivity in the adult rat hippocampus

Sex differences in the morphology and function of the hippocampus have been reported in several species, but it is unknown whether a sexual dimorphism exists in glial fibrillary acidic protein (GFAP) expression in the rat hippocampus. We analyzed GFAP immunoreactivity in the hippocampus of intact adult male rats as well as in females during diestrus and proestrus phases of the estrous cycle. We found that in CA1, CA3, and dentate gyrus, GFAP immunoreactivity was higher in proestrus females as compared with males and diestrus females. In CA1, a similar GFAP immunoreactivity was found in males and in diestrus females, but in dentate gyrus, males presented the lowest GFAP content. Interestingly, differences in astrocyte morphology were also found. Rounded cells with numerous and short processes were mainly observed in the hippocampus during proestrus whereas cells with stellate shape with few and long processes were present in the hippocampus of males and diestrus females. The marked sex and estrous cycle-dependent differences in GFAP immunoreactivity density and in astrocyte number and morphology found in the rat hippocampus, suggest the involvement of sex steroid hormones in the sexually dimorphic functions of the hippocampus, and in the change in its activity during the estrous cycle.