Progesterone-receptive beta-endorphin and dynorphin B neurons in the arcuate nucleus project to regions of high gonadotropin-releasing hormone neuron density in the ovine preoptic area

Progesterone inhibits gonadotropin-releasing hormone (GnRH) secretion through interneuronal systems located in the mediobasal hypothalamus in ewes. Endogenous opioid peptides are implicated in this inhibition of GnRH secretion. The distributions of endogenous opioid peptides are known to overlap with progesterone receptors (PR) in the arcuate nucleus. We investigated whether PR is expressed by beta-endorphin and dynorphin B neurons in the arcuate nucleus and if a subset of double-labeled cells projects to the preoptic area where most GnRH neurons are detected. Injection of a retrograde tracer, Fluorogold, into the rostral preoptic area was performed in ovariectomized ewes pretreated with estrogen and progesterone. Brain sections were processed using double immunocytochemistry. Only brains of ewes with an injection site encompassing at least 80 GnRH neurons were processed for PR and then either beta-endorphin or dynorphin B immunocytochemistry. Antigen retrieval is essential for PR detection but causes Fluorogold to fade. Thus, quantitative analysis was performed on photographs taken before and after antigen retrieval. We found that 25-30% of PR-containing neurons, 20% of beta-endorphin cells and 22% of dynorphin B neurons in the arcuate nucleus project toward the preoptic area. From the PR/beta-endorphin double-labeled cells that represent 25 and 36% of PR and beta-endorphin cells, respectively, 35% were labeled with Fluorogold. From the PR/dynorphin B double-labeled cells that account for 39 and 62% of PR and dynorphin B neurons, respectively, 26% contained Fluorogold. These data strongly support the hypothesis that progesterone acts in the arcuate nucleus through beta-endorphin and dynorphin B neurons to affect preoptic area GnRH neurons.     

Dynamics of steroid regulation of GnRH secretion during the oestrus cycle of the ewe.

The oestrus cycle of the ewe is characterised by a long luteal phase followed by a short follicular phase and these periods are related to the production by the ovary of two major steroids: progesterone and oestrogen. Progesterone exerts a strong inhibitory effect on GnRH secretion during the luteal phase by a mechanism which is still unknown. Using an oestrogen-free ovine model and the portal blood collection technique we have obtained new insights into this mechanism. While progesterone removal induces a rapid increase in GnRH pulse frequency, progesterone reinsertion inhibits GnRH release even faster: less than 50 minutes. This action of progesterone is specific to the gonadotrophic axis and is mediated through an action on the nuclear receptor. Interestingly, this rapid mechanism is also strongly dependent of prior exposure to both progesterone and oestradiol. During the follicular phase, the rise in circulating oestradiol induces a robust preovulatory GnRH surge. In the ewe, this positive feedback effect is mainly exerted by an action of oestradiol on the mediobasal hypothalamus. Finally, we have also obtained evidence that progesterone priming is important for the full expression of the positive feedback action of oestradiol on GnRH secretion. In summary, progesterone and oestradiol sequentially exert opposite feedback effects on GnRH secretion during the oestrus cycle of the ewe but there is also clear evidence that the systems affected by these steroids are intimately linked.     

Demonstration of progesterone receptor-mediated gonadotrophin suppression in the human male

OBJECTIVE: Synthetic gestogens in combination with testosterone have potential as a male hormonal contraceptive, predominantly acting by augmenting suppression of gonadotrophin secretion. Little is known, however, of the effects of gestogens in the male. Gestogens have affinity for both androgen and progesterone receptors but the relative contribution of action at these two receptors in gonadotrophin suppression remains unclear. In this study the effects of progesterone, with no significant androgen-receptor affinity are compared to desogestrel, a synthetic gestogen with relatively low affinity for the androgen receptor, on gonadotrophin secretion in normal men. DESIGN: Subjects received either 50 mg progesterone intramuscularly (i.m.) or 300 micro g desogestrel orally daily for 7 days. Frequent blood sampling over 12 h was undertaken before and after drug administration. GnRH [100 micro g intravenously (i.v.)] was administered 2 h before the end of the frequent sampling period. SUBJECTS: Twenty healthy men were randomly allocated to the two treatment groups. RESULTS: Both progesterone and desogestrel administration resulted in decreases in the concentration of both LH and FSH secretion, as well as testosterone. Analysis of the pulsatile nature of LH secretion indicated that both treatments reduced LH pulse amplitude, and that progesterone reduced LH pulse frequency. Progesterone, but not desogestrel, treatment also reduced the increase in LH secretion in response to GnRH. CONCLUSIONS: The effects of progesterone were at least as marked as those of a maximally effective dose of desogestrel. As progesterone has negligible affinity for the androgen receptor, these results are compatible with the suppressive effects of synthetic 19-norgestogens on gonadotrophin secretion in the male being mediated via the progesterone receptor, with its androgenicity contributing minimally to gonadotrophin suppression.     

Progesterone increases dynorphin a concentrations in cerebrospinal fluid and preprodynorphin messenger ribonucleic Acid levels in a subset of dynorphin neurons in the sheep

Recent studies suggest that the endogenous opioid peptide, dynorphin, is an important mediator of progesterone negative feedback on GnRH pulse frequency in the ewe. These experiments tested this hypothesis by examining the effects of progesterone on dynorphin A concentrations in cerebrospinal fluid (CSF) collected from the third ventricle and expression of preprodynorphin (PPD) mRNA in hypothalamic nuclei. CSF was collected every 10 min for 5 h in three groups of ewes: 1) ovary-intact ewes during the luteal phase (d 6-7 of estrous cycle); 2) ewes 6-7 d after ovariectomy (OVX); and 3) OVX ewes treated for 6-7 d with implants that produced luteal-phase progesterone levels. Diencephalic tissue from these ewes was then collected and processed for in situ hybridization using an ovine cDNA probe against PPD. Progesterone treatment increased dynorphin A concentrations in CSF over that observed in untreated OVX ewes; CSF dynorphin A concentrations in ovary-intact ewes were midway between the other groups. OVX significantly decreased the number of PPD mRNA-expressing cells in the preoptic area (POA), anterior hypothalamic area (AHA), and arcuate nucleus (ARC), with no change seen in any other PPD-expressing nuclei. Progesterone treatment of OVX ewes restored PPD expression in the POA and AHA to levels seen in luteal-phase animals but had no effect on PPD expression in the ARC. These results are consistent with the hypothesis that progesterone acts via dynorphin neurons to inhibit pulsatile GnRH secretion and point to dynorphin neurons in the POA, AHA, and ARC as potential mediators of this action during the luteal phase.     

Progesterone-receptive dopaminergic and neuropeptide Y neurons project from the arcuate nucleus to gonadotropin-releasing hormone-rich regions of the ovine preoptic area

Progesterone inhibits gonadotropin-releasing hormone (GnRH) secretion in sheep through an interneuronal system located in the mediobasal hypothalamus. This study focused on known inhibitors of GnRH secretion in sheep, dopamine and neuropeptide Y (NPY). As the distributions of tyrosine hydroxylase (TH)- and NPY-immunoreactive neurons overlap with progesterone receptors (PR) in the arcuate nucleus, we hypothesized that, if these neurons mediate, at least partially, the inhibitory feedback signal of progesterone, then they should co-express PRs. Fluorogold (FG), a retrograde tracer, was injected into the preoptic area of ovariectomized ewes pretreated with estrogen and progesterone. When the FG injection site encompassed at least 80 GnRH neurons, sections from the arcuate nucleus were processed using dual immunocytochemistry for PR and either TH or NPY. We found that 30% of PR-immunoreactive, 12% of TH-containing and 21% of NPY-synthesizing neurons project toward this GnRH-rich region. Of the PR/TH dual-labeled cells, which represent 21% of PR and 31% of TH cells, respectively, 22% displayed FG labeling. Of the PR/NPY neurons, which account for 19% of PR and 67% of NPY neurons, respectively, 26% were FG fluorescent. This study suggests that subsets of arcuate nucleus dopaminergic and NPY neurons may transduce, at least in part, the progesterone-mediated inhibition of GnRH secretion.     

The timing of progesterone-induced ribonucleic acid and protein synthesis for augmentation of luteinizing hormone secretion.

Progesterone addition to pituitary cells pretreated with estradiol leads within 45 min to an unambiguous augmentation of pulsatile GnRH-stimulated LH secretion. To investigate this rapid action, we established the kinetics of early events through manipulation of RNA synthesis, protein synthesis, and progesterone-receptor binding. Female rat pituitary cells cultured in medium containing charcoal-treated serum plus 0.2 nM estradiol were changed to 0.1% BSA-medium +/- 200 nM progesterone at time 0; at 90 and 150 min the cells were challenged with 1 nM GnRH 15-min pulses. The 3-fold augmentation of GnRH-stimulated LH secretion induced by progesterone was inhibited completely by simultaneous addition of 1 microM actinomycin D or emetine as was GnRH self-priming. In another series, the ability of cycloheximide to completely block progesterone augmentation was gradually diminished with delay of addition, but even 90 min after progesterone (30 min before GnRH pulse) cycloheximide resulted in 50% blockade of augmentation. In contrast, inhibition of RNA synthesis 60-90 min after progesterone introduction had little or no effect on progesterone augmentation. The temporal profile of inhibition by the progesterone antagonist RU486 was indistinguishable from that resulting from blockade of RNA synthesis and suggests that continual activation of the receptor is required for continued RNA synthesis. In summary: 1) both RNA and protein synthesis are required for GnRH self-priming; and 2) progesterone augmentation of GnRH-stimulated LH secretion requires RNA synthesis and synthesis of protein(s) which appear to be turning over rapidly, accumulating slowly, or both.     

Progesterone treatment that either blocks or augments the estradiol-induced gonadotropin-releasing hormone surge is associated with different patterns of hypothalamic neural activation.

Progesterone can either augment or inhibit the surge of gonadotropin-releasing hormone (GnRH) that drives the preovulatory luteinizing hormone (LH) surge. This study investigated the central mechanisms through which progesterone might achieve these divergent effects by examining the effects of exogenous steroids on the activation of GnRH neurons and non-GnRH-immunopositive cells in the preoptic area/anterior hypothalamus of steroid-treated ovariectomized ewes. Fos expression (an index of cellular activation) was examined during the estradiol-induced GnRH surge in ewes treated with progesterone using regimes that have been reported to either augment (progesterone pretreatment) or inhibit (progesterone treatment at the time of the surge-inducing estradiol increment) the GnRH surge. Control groups received either no progesterone pretreatment or no surge-inducing estradiol increment. Induction of an LH surge was associated with a significant (p < 0.0001) increase in the proportion of activated GnRH neurons, irrespective of whether ewes received progesterone pretreatment. However, the number of non-GnRH-immunopositive cells activated during the surge was significantly (p < 0.0001) increased in ewes that received the progesterone pretreatment. By contrast, the proportion of GnRH neurons and non-GnRH-immunopositive cells that expressed Fos was significantly (p < 0.0001) reduced in ewes in which the surge was inhibited by progesterone compared to ewes in which a surge was stimulated. These data indicate that (1) progesterone pretreatment increases the activation of non-GnRH cells during the estradiol-induced surge, but does not affect the proportion of GnRH neurons activated and (2) when administered concurrently with a surge-inducing estradiol increment, progesterone prevents the activation of GnRH neurons and non-GnRH cells that is normally associated with the estradiol-induced surge. Therefore, progesterone does not appear to augment the GnRH surge by increasing the proportion of GnRH neurons that are activated by estradiol, whereas inhibition of the GnRH surge involves prevention of the activation of GnRH neurons. Thus, the augmentation and inhibition of the GnRH surge by progesterone appear to be regulated via different effects on the GnRH neurosecretory system.     

Direct regulation of postnatal GnRH neurons by the progesterone derivative allopregnanolone in the mouse

The mechanisms through which gonadal steroids exert critical feedback actions upon the activity of the GnRH neurons are not understood. We have examined here whether progesterone may modulate the electrical activity of the GnRH neurons following its rapid metabolism to the neuroactive steroid allopregnanolone within the brain. Using an acute brain slice preparation, whole-cell, patch-clamp recordings were made from GnRH neurons of juvenile (postnatal d 15-20) and adult (postnatal d 60-70) female mice in the presence of tetrodotoxin. Progesterone (1 microM) was not observed to have any actions (up to 5 min exposure) upon GnRH neurons. However, allopregnanolone (500 nM-1 microM) exerted rapid (<1 min) effects upon the baseline membrane potential of all GnRH neurons and also significantly (P < 0.01) enhanced their GABA responses by up to 4-fold. All GABA and allopregnanolone responses were abolished by the GABA(A) receptor antagonist bicuculline. No differences were detected in the allopregnanolone sensitivity of GnRH neurons recorded from juvenile and adult GnRH neurons. These results provide the first evidence for a direct action of the neurosteroid allopregnanolone on postnatal GnRH neurons and suggest a new mechanism through which fluctuating progesterone levels may influence the secretory activity of these important neurons in the female mouse.     

Evidence that the arcuate nucleus is an important site of progesterone negative feedback in the ewe

There is now considerable evidence that dynorphin neurons mediate the negative feedback actions of progesterone to inhibit GnRH and LH pulse frequency, but the specific neurons have yet to be identified. In ewes, dynorphin neurons in the arcuate nucleus (ARC) and preoptic area (POA) are likely candidates based on colocalization with progesterone receptors. These studies tested the hypothesis that progesterone negative feedback occurs in either the ARC or POA by determining whether microimplants of progesterone into either site would inhibit LH pulse frequency (study 1) and whether microimplants of the progesterone receptor antagonist, RU486, would disrupt the inhibitory effects of peripheral progesterone (study 2). Both studies were done in ovariectomized (OVX) and estradiol-treated OVX ewes. In study 1, no inhibitory effects of progesterone were observed during treatment in either area. In study 2, microimplants of RU486 into the ARC disrupted the negative-feedback actions of peripheral progesterone treatments on LH pulse frequency in both OVX and OVX+estradiol ewes. In contrast, microimplants of RU486 into the POA had no effect on the ability of systemic progesterone to inhibit LH pulse frequency. We thus conclude that the ARC is one important site of progesterone-negative feedback in the ewe. These data, which are the first evidence on the neural sites in which progesterone inhibits GnRH pulse frequency in any species, are consistent with the hypothesis that ARC dynorphin neurons mediate this action of progesterone.     

Estrogen receptor (ER) beta modulates ERalpha responses to estrogens in the developing rat ventromedial nucleus of the hypothalamus

The mechanisms by which estradiol exerts specific actions on neural function are unclear. In brain the actions of estrogen receptor (ER) alpha are well documented, whereas the functions of ERbeta are not yet fully elucidated. Here, we report that ERbeta inhibits the activity of ERalpha in an anatomically specific manner within the neonatal (postnatal d 7) brain. Using selective agonists we demonstrate that the selective activation of ERalpha in the relative absence of ERbeta activation induces progesterone receptor expression to a greater extent than estradiol alone in the ventromedial nucleus, but not the medial preoptic nucleus, despite high ERalpha expression. Selective activation of ERbeta attenuates the ERalpha-mediated increase in progesterone receptor expression in the ventromedial nucleus but has no effect in medial preoptic nucleus. These results suggest that ERalpha/ERbeta interactions may regulate the effects of estrogens on neural development and reveal the neonatal brain as a unique model in which to study the specificity of steroid-induced gene expression.     

Progesterone Directly and Rapidly Inhibits GnRH Neuronal Activity via Progesterone Receptor Membrane Component 1

GnRH neurons are essential for reproduction, being an integral component of the hypothalamic-pituitary-gonadal axis. Progesterone (P4), a steroid hormone, modulates reproductive behavior and is associated with rapid changes in GnRH secretion. However, a direct action of P4 on GnRH neurons has not been previously described. Receptors in the progestin/adipoQ receptor family (PAQR), as well as progesterone receptor membrane component 1 (PgRMC1) and its partner serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1) mRNA binding protein 1 (SERBP1), have been shown to mediate rapid progestin actions in various tissues, including the brain. This study shows that PgRMC1 and SERBP1, but not PAQR, are expressed in prenatal GnRH neurons. Expression of PgRMC1 and SERBP1 was verified in adult mouse GnRH neurons. To investigate the effect of P4 on GnRH neuronal activity, calcium imaging was used on primary GnRH neurons maintained in explants. Application of P4 significantly decreased the activity of GnRH neurons, independent of secretion of gamma-aminobutyric acidergic and glutamatergic input, suggesting a direct action of P4 on GnRH neurons. Inhibition was not blocked by RU486, an antagonist of the classic nuclear P4 receptor. Inhibition was also maintained after uncoupling of the inhibitory regulative G protein (G(i/o)), the signal transduction pathway used by PAQR. However, AG-205, a PgRMC1 ligand and inhibitor, blocked the rapid P4-mediated inhibition, and inhibition of protein kinase G, thought to be activated downstream of PgRMC1, also blocked the inhibitory activity of P4. These data show for the first time that P4 can act directly on GnRH neurons through PgRMC1 to inhibit neuronal activity.     

Role of endogenous opioid peptides in mediating progesterone-induced disruption of the activation and transmission stages of the GnRH surge induction process.

How progesterone blocks the E2-induced GnRH surge in females is not known. In this study we assessed whether the endogenous opioid peptides (EOPs) that mediate progesterone negative feedback on pulsatile GnRH secretion also mediate the blockade of the GnRH surge. We treated ovariectomized ewes with physiological levels of E2 and progesterone to stimulate and block the GnRH surge, respectively, using LH secretion as an index of GnRH release. A pilot study confirmed that blocking opioidergic neurotransmission with the opioid receptor antagonist, naloxone (NAL; 1 mg/kg.h, i.v.), could prevent the suppression of pulsatile LH secretion by progesterone in our model. By contrast, antagonizing EOP receptors with NAL did not restore LH surges in ewes in which the E2-induced GnRH surge was blocked by progesterone treatment during the E2-dependent activation stage (Exp 1) of the GnRH surge induction process. However, in ewes treated with progesterone during the E2-independent transmission stage (Exp 2), NAL partially restored blocked LH surges, as indicated by increased fluctuations in LH that, in some cases, resembled LH surges. We conclude, therefore, that the EOPs that mediate progesterone negative feedback on pulsatile GnRH secretion are not involved in blockade of activation of the E2-induced GnRH surge by progesterone, but do appear to be part of the mechanism by which progesterone disrupts the transmission stage.     

Dynamics and control of progesterone receptor synthesis in the fetal uterus of the guinea-pig in organ culture

Progesterone receptor concentrations increased in fetal guinea-pig uterus in organ culture to as high as 13 X 15 +/- 1 X 22 pmol/mg DNA without any added steroid, although cytosol and nuclear oestrogen receptor levels were very low (0 X 41-1 X 92 pmol/mg DNA). Even after a 3-day exposure to 5 X 10(-8) M-progesterone, which inhibits its own receptor (1 X 14 +/- 0 X 31 pmol/mg DNA), progesterone receptor levels rose to 8 X 58 +/- 1 X 39 pmol/mg DNA when progesterone was removed. This replenishment was inhibited by progesterone and 5 alpha-dihydroprogesterone but was not affected by oestradiol, tamoxifen or dexamethasone. The incorporation of [3H]thymidine into nucleic acids was not decreased by progesterone so that its inhibition of its own receptor in the explants was not due to an inhibition of cell replication. Fetal uterine explants from oestrogen-primed fetuses, after an initial decrease in progesterone receptor, also showed a rise to 7 pmol/mg DNA on day 2 which could be decreased by exposure to progesterone and replenished by removal of this hormone (6-8 pmol/mg DNA), the entire process occurring without apparent oestrogen stimulation. Progesterone rather than oestradiol appears to be a key regulator of progesterone receptor synthesis in the fetal guinea-pig uterus, although oestradiol, along with other factors, may also be involved.     

Galanin-like peptide as a possible link between metabolism and reproduction in the macaque

Galanin-like peptide (GALP) is a hypothalamic neuropeptide that has been implicated in the control of feeding, metabolism, and reproduction. The goal of this study was to examine the effects of central infusions of GALP on GnRH and LH secretion and to identify physiological factors that influence the expression of GALP mRNA in the brain of a primate species. Infusions of GALP into the lateral cerebroventricle of the macaque caused a significant increase in LH secretion, which was blocked by administration of the GnRH receptor antagonist acyline. However, the expression of GALP mRNA in the arcuate nucleus, as determined by in situ hybridization, was not regulated by either estradiol or progesterone. Compared with feeding ad libitum, fasting for 48 h produced a significant reduction in the hypothalamic expression of GALP mRNA. GALP neurons were found to express both neuropeptide Y Y1 receptor and serotonin 2C receptor by double-label in situ hybridization. Taken together, these results suggest that GALP neurons play a role of integrating metabolic signals, which are relayed to circuits controlling GnRH release in the macaque.     

Steroid secretion by granulosa cells isolated from a woman with 17 alpha-hydroxylase deficiency

Steroid synthesis in luteal/granulosa cells harvested after follicular aspiration in a patient with 17 alpha-hydroxylase deficiency was investigated. Follicular stimulation with purified FSH after the suppression of ACTH and gonadotropin secretion by corticoid analogs and superactive GnRH agonist permitted ovum pick-up and the study of steroid synthesis in the obtained granulosa cells. Progesterone synthesis was elevated while 17 alpha-hydroxy progesterone, testosterone, androstenedione, and estrogen production was minimal. Aromatase activity was retained in these cells, as demonstrated by the 100-fold increase in estrogen levels after the addition of androgens. Progesterone levels in follicular fluids were in the normal range, while estrogen, 17 alpha-hydroxy progesterone, and androgen levels were minimal. These results provide in vitro evidence of 17 alpha-hydroxylase deficiency in the granulosa cells of this patient.     

Hypophyseal actions of pulsatile gonadotropin-releasing hormone in the ewe: development and application of a new experimental model

Ovariectomy of ewes during seasonal anestrus and immediate replacement with subcutaneous Silastic progesterone implants which maintained a midluteal-phase level of circulating progesterone obliterated pulsatile luteinizing hormone (LH) secretion for up to 2 weeks without preventing a normal response of the pituitary to exogenous pulses of gonadotropin-releasing hormone (GnRH). Consideration was given to the possibility that such 'progesterone-suppressed ewes' would be useful as an animal model for isolating the pituitary from pulsatile GnRH secretion, and for testing the hypophyseotropic actions of exogenous GnRH. Two experiments were conducted using this progesterone-suppressed ewe as an animal model. In the first, the amplitude of LH pulses elicited by episodic delivery of GnRH was found to depend upon the frequency of exogenous GnRH pulses. Hourly frequency produced larger LH pulses than a 30-min frequency of GnRH. In the second experiment, LH surges were induced in progesterone-suppressed ewes by a combined treatment of estradiol and GnRH in patterns designed to approximate those secreted in the follicular phase of the estrous cycle. Our findings suggest that the progesterone-suppressed ewe is a suitable animal model for studying the hypophyseotropic actions of GnRH. Further, they are consistent with two hypotheses concerning the regulation of the tonic and surge modes of LH secretion. (1) The inverse relationship between LH pulse frequency and amplitude observed in a number of situations can be accounted for, at least in part, by a differential response of the pituitary to GnRH. (2) Progesterone can block the LH surge by an action on the brain and an inhibition of pulsatile GnRH release.     

Stimulation of gonadotropin-releasing hormone surges by estrogen. II. Role of cyclic adenosine 3'5'-monophosphate

Release of GnRH surges in female rats is directed by a daily neural signal and occurs only after exposure of the hypothalamus to sustained, elevated estrogen (E2) levels in serum. We have proposed that preovulatory E2 couples the daily neural signal to the circuitry governing GnRH release by a two-step process, which includes stimulation of neuronal progesterone receptors (PRs) by E2 and subsequent activation of PRs by the daily neural signal. In the preceding report we documented that PR activation is obligatory for the stimulation of GnRH surges by E2. In these studies we assess the validity of a second essential feature of this model, that neural signals can activate PRs and thereby prompt the release of GnRH and LH surges. Our efforts specifically focused on the role of cAMP in mediating neural PR trans-activation leading to GnRH surges. To assess whether cAMP may function as a daily neural signal, cAMP levels were examined via a competitive binding assay in anteroventral periventricular nucleus (AVPV) homogenates obtained at 0900, 1200, 1500, 1800, and 2100 h on all days of the estrous cycle. A significant rise in cAMP concentrations was observed at 1500 h on all estrous cycle days. A similar rise at the same time was observed in AVPV tissues of ovariectomized (OVX) rats regardless of steroid treatment. No significant increase in cAMP levels was observed at any time point in homogenates of ventromedial nucleus or cerebral cortex. In a second experiment, female rats were OVX on the afternoon of diestrous day 2 and simultaneously administered 30 microg estradiol benzoate or oil vehicle. On the following day of presumptive proestrus, rats received intracerebroventricular infusions of the cAMP analog, 8-bromo-cAMP, or saline vehicle at 0900 h. Rats treated with 8-bromo-cAMP exhibited LH surges that were advanced by 3 h compared with those in saline-treated controls. This advance did not occur in 8-bromo-cAMP-treated rats not primed with E2, or in E2-treated rats given the antiprogestin RU486. In a third experiment, OVX, estradiol benzoate-primed rats received intracerebroventricular infusions of saline vehicle or the adenylyl cyclase inhibitor SQ22536; although saline-treated rats exhibited normal LH surges, no surges were observed in the rats receiving SQ22536. In additional SQ22536-treated animals, however, LH surge release was rescued and greatly augmented by a pharmacological dose of progesterone. These results demonstrate that 1) cAMP levels in the AVPV are significantly elevated at 1500 h on a daily basis; 2) cAMP elevations in the AVPV can prematurely evoke LH surges by a mechanism that requires PR activation; 3) inhibition of adenylyl cyclase activity in the AVPV blocks LH surges, an action that can be reversed by progesterone; and 4) cAMP generation leads to PR transactivation in the AVPV. Our observations thus provide support for the hypothesis that an increase in intracellular cAMP in the AVPV acts as a component of the daily neural signal required to initiate GnRH and subsequent LH surges, and that transmission of this signal is mediated by cAMP-induced PR trans-activation in the AVPV.     

Progesterone effects on cell growth of U373 and D54 human astrocytoma cell lines

Astrocytomas are the most frequent primary brain tumors and constitute a leading cause of cancer-related deaths. We studied the effects of progesterone and its antagonist, RU486, on cell growth of two human astrocytoma cell lines with different evolution grade (U373, grade III; and D54, grade IV). Progesterone receptor expression was determined by Western blot. The effects of different doses of progesterone and RU486 on cell number, cell cycle, and apoptosis were analyzed for five consecutive days. Progesterone (10 nM) significantly increased the number of D54 cells from the second day of culture, and the number of U373 cells on days 3–5. RU486 (10 μM) blocked progesterone effects in both astrocytoma cell lines. Interestingly, RU486 administered without progesterone significantly reduced the number of cells from the second day of culture in both cell lines. Progesterone increased S phase of cell cycle in U373 cells (61%, on day 5). RU486 blocked the effects of progesterone on cell cycle but administered alone did not significantly change cell cycle profile. DNA fragmentation (TUNEL) assay showed that the diminution in the number of astrocytoma cells produced by RU486 was not by apoptosis. Progesterone receptor isoforms were detected in both cell lines. Our data suggest that progesterone induces cell growth of human astrocytoma cell lines through the interaction with its nuclear receptor.