Facilitation or inhibition of the oestradiol-induced gonadotrophin surge in the immature female rat by progesterone: effects on pituitary responsiveness to gonadotrophin-releasing hormone (GnRH), GnRH self-priming and pituitary mRNAs for the progesterone receptor A and B isoforms.

Progesterone can either facilitate or inhibit the oestradiol (E(2))-induced gonadotrophin surge. We have previously developed immature female rat models to characterise and investigate the mechanisms of progesterone inhibition or facilitation. The aim of the present study was to determine the role of pituitary responsiveness to gonadotrophin-releasing hormone (GnRH) and GnRH self-priming under conditions of progesterone-facilitation and progesterone-inhibition, and whether the underlying mechanisms reflect changes in mRNAs encoding the A and B isoforms of the progesterone receptor (PR) in the pituitary gland. Pituitary responsiveness to GnRH, determined by measuring the luteinising hormone (LH) response to one i.v. injection of GnRH, was decreased by 60-80% (P < 0.001) in the progesterone-inhibition model. GnRH self-priming, estimated as the increment in the LH response to the second of two injections of GnRH separated by 60 min, was also significantly reduced (P < 0.05) in this model. In the progesterone-facilitation model, the LH response to GnRH injection was increased 2.5-3-fold (P < 0.05), an effect suppressed by the progesterone receptor antagonist, mifepristone. Progesterone-facilitation of LH release and increased pituitary responsiveness to GnRH were blocked by sheep anti-GnRH serum injected i.v. immediately after insertion of progesterone implants. The PR-B mRNA isoform, measured by solution hybridisation/RNase protection assay, was the predominant form in the pituitary of the immature female rat. PR-B was increased by E(2) and decreased by progesterone in both models. Thus, in immature female rats, progesterone-inhibition of the E(2)-induced LH surge is due to significant reduction in pituitary responsiveness to GnRH as well as in the magnitude of GnRH self-priming. Progesterone-facilitation of the E(2)-induced LH surge is due to increased pituitary responsiveness to GnRH, which is mediated by PR, and depends on endogenous GnRH release. The differences between progesterone-facilitation and progesterone-inhibition are not due to differences in regulation of pituitary PR-B mRNA.

Modulation of IL-18 production in the adrenal cortex following acute ACTH or chronic corticosterone treatment.

Interleukin (IL)-18 is a proinflammatory cytokine and a stimulator of cell-mediated immune responses. We have previously reported that acute stress stimulates the production of IL-18 mRNA in the glucocorticoid (GC)-producing cells of the adrenal cortex. In order to investigate the mechanisms governing the expression of IL-18 in the adrenal cortex, the effects of acute ACTH or chronic corticosterone treatment on the levels of IL-18 mRNA and protein were examined by in situ hybridization and Northern and Western blot assays. Adult male Sprague-Dawley rats received a subcutaneous injection of ACTH or subcutaneous implantation of slow-release corticosterone pellets followed by an injection of saline or ACTH. After 4 h, ACTH induced a 4-fold increase in IL-18 mRNA levels and elevated the content of pro-IL-18 peptide. Six days of chronic corticosterone treatment did not alter the basal levels of IL-18 mRNA and reduced those of pro-IL-18. Finally, ACTH treatment of animals under the corticosterone regimen induced a 2-fold increase in IL-18 mRNA and elevated the levels of the pro-IL-18 protein. The levels of the precursor, p45, and the active subunit p10 peptides of the IL-18-processing enzyme, IL-1beta-converting enzyme (ICE), showed no substantial differences in all the conditions tested. IL-1beta was not detected under these experimental conditions. These data demonstrate that the production of IL-18 in the adrenal cortex is stimulated by ACTH treatment and is not inhibited by the direct action of corticosterone. In contrast to the anti-inflammatory action of GCs, IL-18 may have an immunostimulatory role during acute stress.

Transcriptional regulation of the glycoprotein hormone alpha-subunit gene by pituitary adenylate cyclase-activating polypeptide (PACAP) in alphaT3-1 cells.

We showed previously that pituitary adenylate cyclase-activating polypeptide (PACAP) increases glycoprotein hormone alpha-subunit gene expression and secretion in alphaT3-1 cells. We have now used 5'-flanking deletion and clustered point mutations of the mouse alpha-subunit promoter fused to the luciferase (LUC) reporter gene in transient transfection assays to further characterize the cell signaling pathways and sequences involved in responsiveness to PACAP. PACAP stimulated LUC activity at a lower concentration than VIP, supporting the notion that PACAP acts through type 1 receptors. The effect of PACAP on LUC activity was observed by 2 h, peaked at 4-12 h, and persisted until at least 20 h. alphaT3-1 cells were transfected with mouse alpha-LUC constructs truncated at -507, -424, -288, -205, -146, and -133, and treated with PACAP, a cell-permeable cAMP analog (8Br-cAMP), phorbol myristate acetate (PMA), or control medium. Transcriptional activation by PACAP was highest with the -288 and -205 mouse alpha-LUC vectors (7-8-fold stimulation) and decreased significantly with truncation of the 5'-flanking region to -146 or -133. The pattern of alpha-subunit stimulation by cAMP closely paralleled that of PACAP. With PMA, stepwise decrements in LUC activity were observed between -507 and -424 and, especially, -424 and -288, and there was no further loss of activity with deletion to -205, -146, or -133. Clustered point mutations in the pituitary glycoprotein hormone basal element (-337 to -330) or the gonadotropin-releasing hormone response element (GnRH-RE)(-406 to -399) of the -507 to +46 mouse alpha-promoter significantly (P < 0.05) increased and decreased, respectively, PACAP's effect on transcriptional activity. These results indicate that there are several regions of the mouse alpha-subunit promoter that mediate responsiveness to PACAP. The co-localization of PACAP and cAMP responsiveness as well as the results of studies involving specific inhibitors of protein kinase A (H-89) or protein kinase C (PKC) (bisindolylmaleimide) suggests that the action of PACAP on alpha-subunit transcription is mediated primarily by the protein kinase A (PKA) pathway.

Mathematical exploration of pulsatility in cultured gonadotropin-releasing hormone neurons.

Pulsatile gonadotropin-releasing hormone (GnRH) release has been demonstrated in cultures of an immortalized line of GnRH expressing neurons (GT1 cells) in experiments by four different research groups. Pulsatile release is known to play a crucial role in GnRH-mediated signaling in vivo, and thus deserves theoretical and quantitative consideration, especially as GT1 cells are presumably genetically homogeneous. Here we have modeled idealized GT1 cells with a differential equation/logic based modeling program, Stella II. We have created a network of 'neurons', with randomized (within the same preset limits for each neuron) thresholds, number and weight of connections to other neurons, and build-up of signal; as well as continuous decay of stored signal. Surprisingly, we found that with this minimal set of assumptions, without any sort of predefined pacemaking cells, it is possible to create pulsatility similar to that observed in the laboratory. A variety of different parameter sets was found to produce these pulses. Network behaviors similar to those of GT1 cells depended on the degree of interconnection between neurons and their functioning within a critical range of network excitability. These findings allow for a clearer consideration of the critical elements of such networks as well as experimental predictions regarding the production of pulsatile behavior.

Facilitation or inhibition of the estradiol-induced gonadotropin surge in the immature rat by progesterone: regulation of GnRH and LH messenger RNAs and activation of GnRH neurons.

UNLABELLED: We have developed and extensively characterized immature female rat models to demonstrate inhibition or facilitation of the estradiol (E2)-induced gonadotropin surge by progesterone (P). We show here that the surge of free alpha-subunit is regulated similarly by P in these models. To investigate the possibility that P alters the biosynthesis of GnRH and/or LH, we measured levels of LH subunit mRNAs by Northern blot hybridization and GnRH mRNA by a solution hybridization-RNase protection assay. In the P inhibition model, alpha-subunit mRNA was significantly decreased when P was administered together with E2 for 32 or 48 h, and LHbeta, at 29 h. In the facilitation model, neither alpha-subunit nor LHbeta mRNA increased with premature and enhanced release of LH and free alpha-subunit. Levels of GnRH mRNA in E2-treated rats were significantly higher on the afternoon of the LH surge than on that or the following morning. There was no effect of P on GnRH mRNA levels, however, before, during, or after the LH surge in either paradigm. The time course of activation of GnRH neurons in P-facilitated rats was determined by double-label immunocytochemistry for GnRH and cFos. When serum LH concentrations were basal there was no expression of cFos in GnRH neurons. LH secretion in P-facilitated rats was initiated at 14.00 h and remained elevated until at least 19.00 h. During this time 63-78% of GnRH neurons were cFos positive. Both serum LH concentrations and the percentage of cFos-activated GnRH neurons were significantly lower in control rats treated with E2 alone than in those treated also with P. IN CONCLUSION: 1) suppression of LH and free alpha-subunit secretion by P can be accounted for at least partly by suppression of alpha-subunit mRNA levels; 2) P facilitation is not associated with changes in LH subunit or GnRH mRNA levels; 3) the large proportion of cFos-positive GnRH neurons in P-facilitated rats closely parallels increases in serum LH concentrations but is not accompanied by changes in GnRH mRNA levels. It is likely, therefore, that P acts in the facilitation model to trigger release of pre-existing GnRH stores by altering synthesis or activity of neuro-transmitters/neuropeptides involved in GnRH regulation and/or release of LH stores by altering, for example, pituitary responsiveness to GnRH (including self-priming) and components of the LH secretory apparatus. Similar possibilities may also obtain for the blockade of the gonadotropin surge in the inhibition model.

Glucocorticoid repression of gonadotropin-releasing hormone gene expression and secretion in morphologically distinct subpopulations of GT1-7 cells.

Two morphologically distinct subpopulations of GT1-7 cells have been characterized and examined for their responsiveness to glucocorticoids. Type I cells have a neuronal phenotype, extending many lengthy processes, and express neuronal, but not glial, markers. Type II cells show weaker or negative immunostaining for neuronal markers and exhibit fewer processes. The effect of glucocorticoids on gonadotropin-releasing hormone (GnRH) secretion and gene expression was compared in type I and type II GT1-7 cells. For secretion studies, cells were attached to Cytodex beads and perifused with control medium or medium containing dexamethasone (dex). The high level of GnRH secreted by type I cells was slightly enhanced in the presence of dex, whereas dex rapidly and profoundly decreased the already low level of GnRH secreted by type II cells. Immunocytochemistry for GnRH showed dark reaction product in the cell bodies and processes of type I cells and little or no immunoreactivity in type II cells. Both the endogenous mouse GnRH mRNA and the transcriptional activity of a mouse GnRH promoter luciferase reporter gene plasmid were suppressed to a greater extent in type II cells than in type I. In electrophoretic mobility shift assays, there was no difference between type I and type II nuclear extracts in the pattern of protein-DNA complexes formed on two previously identified negative glucocorticoid response elements located at -237 to -201 and -184 to -150 bp of the mouse promoter. Both cell types contained glucocorticoid receptors (GR) by Western blot analysis. Cytosols from type I or type II cells were incubated with [3H]dex to obtain GR binding parameters. Binding data were consistent with a one-site model for dex binding in each case. Small differences in Kd (1.7 nM, type I; 3.1 nM, type II) or Bmax (approximately 3600 sites/cell, type I; approximately 1800 sites/cell, type II) were not likely to account for the differential sensitivity to dex treatment. In conclusion, nuclear alterations in type II cells leading to greater transcriptional susceptibility to dex, coupled with low GnRH storage levels, may be reflected in exquisite sensitivity of GnRH secretion to glucocorticoid repression. This represents the first example of a steroid hormone acting directly on GnRH-producing cells to alter GnRH secretion.

Differential LHRH secretion, dye coupling, and protein expression in two morphologically distinct cell types identified in GT1-7 cultures.

The immortalized neuronal cell line, GT1-7, has been shown to secrete LHRH in a pulsatile manner and to possess many other characteristics of hypothalamic LHRH neurons in vivo, and thus provides a potential model system for studying biochemical and physiological mechanisms regulating LHRH secretion. In the present study, two morphologically and functionally distinct types of cells have been identified in GT1-7 cultures and each type purified to over 95% homogeneity. One type (N cells) appeared more neuronal with extended neurites and somewhat rounded cell perikarya, while the other type (G cells) had flatter cell perikarya that contained filopodia but no neurites. Growth properties of the two cell types also differed. The doubling time for proliferation of N cells was nearly two-fold shorter than that for G cells and N cells displayed 'piling up' whereas G cells exhibited contact inhibition. Functionally, N cells, but not G cells, were dye-coupled as measured by a fluorescence photobleaching assay. While both cell types expressed LHRH, N cells released significantly higher levels of LHRH into the culture media and exhibited more intense LHRH immunostaining. The two cell types also showed differences in immunostaining for other proteins. N cells, unlike G cells, immunostained positive for neuron-specific enolase (NSE), whereas G cells, unlike N cells, stained immunopositive for vimentin. Both cell types expressed SV-40 T antigen protein, indicating that they were derived from the same transgenic mouse hypothalamic tumour. The physiological significance of these two cell types in GT1-7 cultures remains to be determined, but elucidation of their morphological and biochemical properties is intended to contribute to better understanding and application of this experimentally important neuroendocrine cell line.

Progesterone blockade of a luteinizing hormone surge blocks luteinizing hormone-releasing hormone Fos activation and activation of its preoptic area afferents.

Progesterone is capable of facilitating or blocking the luteinizing hormone (LH) surge, depending on the timing of its administration. However, the precise targets of progesterone's actions are unknown. Since recent studies described the presence of a periventricular preoptic area (pePOA) neuron population afferent to LH-releasing hormone (LHRH) neurons that is co-activated to express c-Fos with LHRH neurons at the time of the LH surge, the present study was designed to determine if the pePOA neurons contain progesterone receptors (PRs) and whether progesterone inhibition is manifested by a failure of LHRH and pePOA neurons to become activated at the time of an LH surge. For progesterone facilitation, a group of immature rats each received a silastic capsule (1.57 mm i.d., 3.18 mm o.d., 1.5 cm long) containing estradiol-17beta (E2) in peanut oil (150 microg/ml) at 09.00 h on postnatal day 28 followed 24 h later by a progesterone implant (crystalline, 1.57 mm i.d., 3.18 mm o.d., 1.5 cm long). For progesterone inhibition, a second group of rats received the estrogen capsule and a progesterone capsule (3.35 mm i.d., 4.65 mm o.d., 3.0 cm long) together at 09.00 h on day 28, and 24 h later received only a blank capsule. On the afternoon of postnatal day 29, all animals were anesthetized and perfused for localization of c-Fos and LHRH, PRs alone, or c-Fos and PRs. The present studies determined that following a progesterone-inhibition paradigm, along with blockade of the LH surge, both activation of LHRH and pePOA neurons was low or absent. Staining of PRs in progesterone-facilitated and progesterone-inhibited rats indicated that the pePOA neurons contained PRs in similar patterns. Double labeling of c-Fos and PRs in progesterone-facilitated rats indicated that nearly all the c-Fos-positive neurons of the pePOA (80 +/- 4.2%) co-expressed PRs; in progesterone-inhibited rats, only 32 +/- 12% of few c-Fos-positive neurons also contained PRs. In no instance were LHRH neurons found to contain PRs. Taken together, these data suggest that both progesterone facilitation and inhibition likely involve direct actions of progesterone on the pePOA neurons, and are consistent with a role for the pePOA neurons in transducing steroid effects on LHRH neurons.

Glucocorticoid repression of the mouse gonadotropin-releasing hormone gene is mediated by promoter elements that are recognized by heteromeric complexes containing glucocorticoid receptor.

We have identified two regions of the mouse gonadotropin-releasing hormone (GnRH) promoter, one between -237 and -201 (distal element) and the other between -184 and -150 (proximal element), which are required for glucocorticoid repression in transiently transfected GT1-7 cells. These sequences show no similarity to known positive or negative glucocorticoid response elements (nGREs) and do not function when placed upstream of heterologous viral promoters. The glucocorticoid receptor (GR) does not bind directly to the distal or proximal promoter elements but may participate in glucocorticoid repression of GnRH gene transcription by virtue of its association within multiprotein complexes at these nGREs. Electrophoretic mobility shift assays with GT1-7 nuclear extract demonstrate the presence of GR-containing protein complexes on GnRH nGREs. One protein that co-occupies the distal nGRE in vitro along with GR is the POU domain transcription factor Oct-1. Thus, the tethering of GR to the GnRH distal nGRE, by virtue of a direct or indirect association with DNA-bound Oct-1, could play a role in hormone-dependent transcriptional repression of the GnRH gene. In contrast, Oct-1 does not appear to be a component of the GR-containing protein complex that is bound to the proximal nGRE.

Repression of glycoprotein hormone alpha-subunit gene expression and secretion by activin in alpha T3-1 cells.

The alpha T3-1 cell line, a GnRH-responsive gonadotroph cell line developed by targeted oncogenesis in transgenic mice, was used to study regulation of the glycoprotein hormone alpha-subunit by activin. Transient transfection assays established that activin suppressed transcription of both the human and mouse alpha-subunit genes. Initial studies demonstrated that activin decreased transcription of -846 and -180 human alpha-subunit-luciferase constructs by about 30%, but that inhibin and follistatin were without effect. Subsequent studies to localize sequences mediating responses to activin were carried out using a series of 5'-deletions (-507 to -133) of the mouse alpha-subunit promoter fused to luciferase. The luciferase activity of the -507-base pair construct was decreased by 60-70% in the presence of activin, and follistatin prevented this decrease. There were significant stepwise losses of activin responsiveness when sequences between -507 and -424, -424, and -288, and -288 and -205 base pairs were eliminated. Clustered point mutations of the mouse alpha-subunit gene, shown previously to reduce basal expression and GnRH responsiveness, were tested to further identify sequences mediating activin repression. Constructs containing a mutated (-337 to -330) pituitary glycoprotein hormone basal element (PGBE) showed significant loss of activin responsiveness in the context of both the native promoter (-507 to +46) and a minimal promoter downstream of the -507 to -205 region of the mouse alpha-subunit gene, whereas mutation of sequences (-406 to -399) in the GnRH-response element had no effect. Multimers of the PGBE element (-344 to -300) were insufficient to mediate a full activin response when linked to a minimal promoter. When added together with GnRH to transfected cells, activin abolished the stimulatory effect of GnRH on alpha-transcription. Secretion of free alpha-subunit by alpha T3-1 cells decreased 10-50% after exposure to activin for approximately 20 h, and steady state levels of alpha-subunit messenger RNA (mRNA) decreased by about 20-25% after 24-72 h. As changes in activin sensitivity could modulate its action, activin receptor II mRNA levels were measured by Northern blot hybridization at various times after activin (or inhibin) treatment. The three species of ActRII mRNA present in alpha T3-1 cells (approximately 6, 3, and 0.5 kilobases) were unaffected up to 72 h by these treatments. These observations provide the first demonstration that activin regulates a gonadotropin subunit gene at the level of transcription. Suppression of transcription of the mouse alpha-subunit gene by activin appears to involve several segments of the alpha-promoter, one of which is in the region of the PGBE. Thus, alpha T3-1 cells may provide a favorable system to further identify the DNA sequences and nuclear factors through which activin acts to alter transcription.

Regulation of alpha-subunit mRNA transcripts by pituitary adenylate cyclase-activating polypeptide (PACAP) in pituitary cell cultures and alpha T3-1 cells.

Pituitary adenylate cyclase activating polypeptide (PACAP) increases glycoprotein hormone alpha-subunit mRNA levels suggesting a role for PACAP in maintaining the high levels of alpha-subunit protein characteristic of the pituitary. The present study used primary pituitary cell cultures and the alpha T3-1 pituitary cell line to investigate how PACAP affects alpha-subunit mRNA transcripts. Stimulation of cultured pituitary cells with 10 nM PACAP38, 10 nM GnRH, or the combination, for 24 h increased alpha-subunit mRNA levels 1.5-fold, whereas GnRH more effectively (P<0.01) stimulated alpha-subunit protein release than did PACAP38 (3.2- vs. 2.0-fold). alpha-Subunit mRNA levels in alphaT3-1 cells were also increased by PACAP38 and by GnRH to maximum values at 12 h (P<0.05), and alpha-subunit protein secretion rose proportionately and in parallel with alpha-subunit mRNA levels. PACAP38 was a 100-fold more potent stimulator of alpha-subunit mRNA than was VIP, and a VIP-antagonist failed to block the stimulatory effect of PACAP38, suggesting an effect via type PACAP 1 receptors. Type I receptor mRNA transcripts were identified by Northern analysis in alphaT3-1 cells. Depletion of PCK activity by PMA failed to block the stimulatory effect of PACAP38, but prevented GnRH from increasing alpha-subunit mRNA levels and alpha-subunit secretion. PACAP38, like 8Br-cAMP and forskolin, stimulated (P<0.05) luciferase (LUC) activity in alphaT3-1 cells transfected with a plasmid containing the first 846 of 180 base pairs of the 5'-flanking region of the human alpha-subunit gene linked upstream to a LUC reporter gene. Finally, experiments using the transcription inhibitor DRB reveal that PACAP does not appreciably change alpha-subunit mRNA half-life. These findings are consistent with the proposal that PACAP contributes to the high levels of alpha-subunit protein characteristic of the pituitary by activating Type I receptors and stimulating alpha-subunit gene transcription in part by the cAMP/PKA pathway.

Replacement with recombinant human inhibin immediately after orchidectomy in the hypophysiotropically clamped male rhesus monkey (Macaca mulatta) maintains follicle-stimulating hormone (FSH) secretion and FSH beta messenger ribonucleic acid levels at precastration values.

This study directly tested the inhibin hypothesis by examining the ability of replacement with recombinant human (rh-) inhibin, either alone or in combination with testosterone (T), to maintain FSH secretion and FSH beta messenger RNA (mRNA) at intact levels after orchidectomy in the hypophysiotropically clamped juvenile rhesus monkey. Thirteen male monkeys (11-21 months of age) received an intermittent i.v. infusion of GnRH (0.1 microgram/min for 3 min every 3 h). After 4-6 weeks of GnRH stimulation, 10 animals were orchidectomized, and 3 monkeys were sham castrated. Hormone replacement was initiated at castration and maintained for 4 days. Three monkeys received a combination of inhibin and T replacement, 4 monkeys received replacement with inhibin alone, and 3 monkeys received T replacement alone. A continuous i.v. infusion of rh-inhibin (832 ng/h.kg) was used to replace the testicular protein, whereas SILASTIC capsules were implanted sc for T replacement. The FSH response to castration and hormone replacement was determined by measuring circulating concentrations of this gonadotropin before a GnRH pulse and for 3 h thereafter on the day before surgery and on days 2 and 4 postcastration. Circulating immunoactive inhibin was measured by a RIA that recognizes the free alpha-subunit of inhibin as well as inhibin dimers. At the end of the study, anterior pituitaries were collected for analysis of steady state levels of FSH beta, LH beta, and alpha-subunit mRNAs. Steroid replacement alone, which produced circulating T concentrations in the upper physiological range, failed to prevent the postcastration increases in circulating FSH concentrations and pituitary FSH beta mRNA levels. In contrast, when circulating immunoactive inhibin in T-replaced monkeys was maintained at precastration levels (approximately 2 ng/ml) by infusion of rh-inhibin, FSH secretion and synthesis were held at control values. When T was omitted from combined replacement, the FSH-suppressing action of the recombinant hormone was not compromised. These results demonstrate that rh-inhibin is biologically active in the monkey, and the action of inhibin to suppress FSH synthesis and secretion does not require a concomitant action of T. Moreover, because the hypophysiotropic drive to the pituitary-testicular axis was clamped, the FSH-suppressing action of rh-inhibin must be at the pituitary.

Glucocorticoid receptor-mediated repression of gonadotropin-releasing hormone promoter activity in GT1 hypothalamic cell lines.

The synthesis and release of GnRH within a specific subset of neurons in the hypothalamus, which serves as the primary drive to the hypothalamic-pituitary-gonadal (HPG) axis, is subject to various levels of control. Although a number of direct synaptic connections to GnRH-containing neurons have been identified, which presumably provide some regulatory inputs, the mechanisms responsible for hormonal regulation of GnRH synthesis and release mediated by either cell surface or intracellular receptors remain controversial. The recent demonstration that a subset of GnRH-containing neurons in the rat hypothalamus possesses immunoreactive glucocorticoid receptors (GR) implies that this class of steroid hormones could exert a direct effect to regulate the functioning of these neurons and perhaps the HPG axis. We used the GT1-3 and GT1-7 cell lines of immortalized GnRH-secreting hypothalamic neurons as a model to study the direct effects of glucocorticoids on GnRH gene expression. We demonstrated that these cell lines possess GR that bind the synthetic glucocorticoid, dexamethasone, in vitro with high affinity (Kd = 2-3 nM). These receptors are functional, as indicated by their ability to activate transcription from exogenously introduced heterologous glucocorticoid-responsive promoters. Furthermore, dexamethasone represses both the endogenous mouse GnRH gene, decreasing steady state levels of GnRH mRNA, and the transcriptional activity of transfected rat GnRH promoter-reporter gene vectors. Glucocorticoid repression of rat GnRH promoter activity appears to be mediated by sequences contained within the promoter proximal 459 basepairs and not be influenced by the relative basal activity of the GnRH promoter. Thus, our results provide the first direct demonstration of glucocorticoid repression of transcription in a hypothalamic cell line and suggest that GR acting directly within GnRH neurons could be at least partly responsible for negative regulation of the HPG axis by glucocorticoids.

The chronic intracerebroventricular infusion of interleukin-1 beta alters the activity of the hypothalamic-pituitary-gonadal axis of cycling rats. I. Effect on LHRH and gonadotropin biosynthesis and secretion.

We have previously reported that the acute injection of interleukin-1 beta (IL-1 beta) into the brain ventricles of intact female rats promptly decreases LHRH release and inhibits gene expression of this peptide in the medial preoptic area (MPOA). The present studies were therefore designed to determine whether continuous exposure to the cytokine would disrupt the estrous cycle. IL-1 beta was injected intracerebroventricularly for 4-6 days at a rate of 4 ng/h. Daily vaginal smears were obtained to follow the cycle; pituitary LH and FSH secretion were measured at regular intervals. Steady state levels of LH and FSH messenger RNA (mRNA) in the pituitary, and LHRH gene expression in the MPOA, were measured at the end of the treatment. Infusion of IL-1 beta caused a total disruption of the estrous cycle, characterized by persistent smears indicative of the diestrus stage. When compared to animals treated with the vehicle, rats infused with IL-1 beta showed a significant decrease in circulating LH concentrations, which was accompanied by lowered mRNA levels in the pituitary. This statistical difference (P < 0.01) persisted even when treated rats were compared to control in a similar stage of the cycle (i.e. diestrus). Plasma FSH levels remained low at all times after IL-1 beta infusion but showed the expected cyclic changes in control animals. At the end of treatment, LHRH gene expression was also markedly suppressed in LHRH neurons distributed between the rostral preoptic area/organum vasculosum of the lamina terminalis and the MPOA of these animals. These results indicate that prolonged infusion of IL-1 beta into brain ventricles disrupts the estrous cycle, an event accompanied by decreased biosynthesis/release of LHRH and gonadotropins. We report in a related study that IL-1-treated rats also show increased plasma progesterone levels. However, it is improbable that this change was responsible for the interruption of the cycle described here; indeed we have previously observed that the central administration of IL-1 beta to intact rats resulted in an immediate blockade of the spontaneous activity of LHRH perikarya during the afternoon of proestrus and significantly decreased LHRH mRNA levels in gonadectomized animals. Taken together, these data suggest that the primary effect of IL-1 beta is at the level of LHRH perikarya, and that the resulting interruption of the cycle is caused by altered LHRH neuronal activity and blunted gonadotropin secretion.

The follicle stimulating hormone-inhibin feedback loop in male primates.

This paper reviews our current knowledge of the role of testicular inhibin in the regulation of follicle stimulating hormone (FSH) secretion in the rhesus monkey. Species differences between monkey and rat are described, and evidence for and against the hypothesis that control of FSH secretion in the human male is similar to that for the monkey is presented.

Decay of follicle-stimulating hormone-beta messenger RNA in the presence of transcriptional inhibitors and/or inhibin, activin, or follistatin.

In primary cultures of rat pituitary cells, inhibin and follistatin reduce steady state levels of FSH beta mRNA to less than 10% of control within 4-6 h, while activin increases this mRNA 2- to 3-fold after 2-4 h of treatment. The effects of these three gonadal polypeptide hormones on the LH beta and common alpha-subunit mRNAs are more gradual and of lesser magnitude. The present study was designed to determine whether inhibin, activin, and/or follistatin act at the posttranscriptional level by altering the stability of the gonadotropin subunit mRNAs. To determine the decay rates of FSH beta, LH beta, and alpha-subunit mRNAs, primary pituitary cell cultures were treated for 1-24 h with either of two transcriptional inhibitors, actinomycin-D or 5,6-dichloro-1-beta-ribofuranosyl benzimidazole (DRB), in the presence or absence of recombinant human inhibin-A, recombinant human activin-A, or purified bovine follistatin. The decay of preexisting gonadotropin subunit mRNAs was followed by Northern blot analysis. Levels of LH beta and alpha-subunit mRNAs remained constant or increased during the 24-h exposure to transcriptional inhibitors; therefore, it was not possible to calculate their half-lives. The stability of these mRNAs was not altered by inhibin, activin, or follistatin. In contrast, FSH beta mRNA turned over rapidly: the estimated half-life was 2.6 +/- 0.19 h (mean +/- SEM of eight determinations) after actinomycin-D treatment and 1.9 +/- 0.14 h (mean +/- SEM of 12 determinations) after DRB treatment. When new RNA synthesis was blocked by either actinomycin-D or DRB, there were no significant effects of inhibin, activin, or follistatin on the stability of FSH beta mRNA (n = 2-4 for each hormone). The decay of FSH beta mRNA in the presence of inhibin or follistatin alone, however, was even more rapid than that determined after the administration of transcriptional inhibitors (P < 0.005). After an initial lag of 1-2 h, the half-life of FSH beta mRNA was 0.88 +/- 0.15 h (n = 4) or 0.62 +/- 0.11 h (n = 3), in the presence of inhibin or follistatin, respectively. The most likely interpretation of these results is that inhibin/follistatin reduces steady state levels of FSH beta mRNA by inducing a labile protein that accelerates the degradation of this mRNA species, and the synthesis of this protein is blocked by actinomycin-D or DRB treatment. It is not clear at present whether inhibin, follistatin, and activin have additional effects on transcription of the gonadotropin subunit genes.

Glucocorticoid induced up-regulation of a pituitary K+ channel mRNA in vitro and in vivo.

Hormones might produce long-term changes in cell excitability by regulating K+ channel gene expression. Recently, we found that dexamethasone increases expression of Kv1.5 K+ channel mRNA in GH3 rat pituitary tumor cells. We wished to test if this effect is specific for the Kv1.5 gene, if it is mediated by activation of glucocorticoid receptors, and whether it occurs in normal pituitary cells. Here we report that dexamethasone treatment of GH3 cells for 3 hours increases Kv1.5 mRNA without affecting Kv1.4 or Kv2.1 K+ channel mRNAs or D Ca2+ channel mRNA. Treatment with sex steroids fails to alter Kv1.5 mRNA levels, while natural glucocorticoids increase expression of the channel mRNA. RU38486, a competitive inhibitor of glucocorticoid receptors, inhibits the response to dexamethasone. We then tested whether Kv1.5 mRNA is induced by dexamethasone in normal rat pituitary cells. To study in vivo effects, channel mRNA levels in pituitaries from adrenalectomized rats were measured with RNAse protection assays. One day following dexamethasone injection Kv1.5 mRNA was increased 8-fold. Dexamethasone induction of Kv1.5 mRNA was also found in primary cultured anterior pituitary cells. We conclude that activated glucocorticoid receptors specifically induce Kv1.5 K+ channel mRNA expression in normal and clonal anterior pituitary cells.

Effects of orchidectomy on gonadotropin and inhibin subunit messenger ribonucleic acids in the pituitary of the rhesus monkey (Macaca mulatta).

Our research programs required the preparation of hypophysectomized and orchidectomized rhesus monkeys. This afforded us the possibility to characterize and compare levels of the gonadotropin and inhibin subunit mRNAs in pituitaries from intact and castrate monkeys. Eighteen adult male monkeys, four of which had been bilaterally orchidectomized 5-9 months previously, were used in this study. Plasma concentrations of LH and FSH were, respectively, 188.5 +/- 5.3 and 246.8 +/- 25.2 ng/ml in the castrate monkeys and 25.8 +/- 4.5 and 4.1 +/- 1.1 ng/ml (mean +/- SEM) in the intact animals. Total pituitary RNA was hybridized to cDNA probes for cynomolgus monkey gonadotropin subunits (FSH beta, LH beta, and the common alpha-subunit) and for human inhibin subunits (alpha, beta B, and beta A) by Northern blot analysis, and mRNA levels were normalized by subsequent hybridization to cyclophilin. Each of the gonadotropin subunit probes hybridized to a single RNA species with the approximate sizes of 1.6 kilobases (kb; FSH beta), 0.7 kb (LH beta), and 0.8 kb (alpha). Levels of LH beta and alpha-subunit mRNAs in pituitaries from castrate monkeys were about 5- and 2-fold higher, respectively, than those in pituitaries from intact monkeys. FSH beta mRNA, on the other hand, was elevated about 27-fold in castrate monkeys [mean +/- SEM, 3176 +/- 408 cpm bound (n = 4 castrate) and 116 +/- 30 cpm bound (n = 8 intact]). Inhibin beta B-subunit mRNA was present in the monkey pituitary as a doublet of about 5 kb, and it was approximately twice as abundant in intact pituitaries as in castrate pituitaries. Hybridizations involving inhibin beta A cDNA revealed a faint band in the region expected for monkey beta A mRNA (6.5 kb) in three of six RNA samples from intact monkeys and a 0.3- to 0.4-kb mRNA species. mRNA encoding the inhibin alpha-subunit was undetectable by Northern blot hybridization. These results indicate that the postpubertal testis imposes an inhibition on the expression of the genes encoding FSH beta, LH beta, and glycoprotein hormone alpha-subunit and that this suppression of the FSH beta gene in the monkey is much greater than that in the rat. In addition, the monkey pituitary may be a source of activin, which may act locally to modulate FSH gene expression and secretion.