Does cortisol inhibit pulsatile luteinizing hormone secretion at the hypothalamic or pituitary level?

Elevations in glucocorticoids suppress pulsatile LH secretion in sheep, but the neuroendocrine sites and mechanisms of this disruption remain unclear. Here, we conducted two experiments in ovariectomized ewes to determine whether an acute increase in plasma cortisol inhibits pulsatile LH secretion by suppressing GnRH release into pituitary portal blood or by inhibiting pituitary responsiveness to GnRH. First, we sampled pituitary portal and peripheral blood after administration of cortisol to mimic the elevation stimulated by an immune/inflammatory stress. Within 1 h, cortisol inhibited LH pulse amplitude. LH pulse frequency, however, was unaffected. In contrast, cortisol did not suppress either parameter of GnRH secretion. Next, we assessed the effect of cortisol on pituitary responsiveness to exogenous GnRH pulses of fixed amplitude, duration, and frequency. Hourly pulses of GnRH were delivered to ewes in which endogenous GnRH secretion was blocked by estradiol. Cortisol, again, rapidly and robustly suppressed the amplitude of GnRH-induced LH pulses. We conclude that, in the ovariectomized ewe, cortisol suppresses pulsatile LH secretion by inhibiting pituitary responsiveness to GnRH rather than by suppressing hypothalamic GnRH release.     

Insight into the neuroendocrine site and cellular mechanism by which cortisol suppresses pituitary responsiveness to gonadotropin-releasing hormone

Stress-like elevations in plasma glucocorticoids rapidly inhibit pulsatile LH secretion in ovariectomized sheep by reducing pituitary responsiveness to GnRH. This effect can be blocked by a nonspecific antagonist of the type II glucocorticoid receptor (GR) RU486. A series of experiments was conducted to strengthen the evidence for a mediatory role of the type II GR and to investigate the neuroendocrine site and cellular mechanism underlying this inhibitory effect of cortisol. First, we demonstrated that a specific agonist of the type II GR, dexamethasone, mimics the suppressive action of cortisol on pituitary responsiveness to GnRH pulses in ovariectomized ewes. This effect, which became evident within 30 min, documents mediation via the type II GR. We next determined that exposure of cultured ovine pituitary cells to cortisol reduced the LH response to pulse-like delivery of GnRH by 50% within 30 min, indicating a pituitary site of action. Finally, we tested the hypothesis that suppression of pituitary responsiveness to GnRH in ovariectomized ewes is due to reduced tissue concentrations of GnRH receptor. Although cortisol blunted the amplitude of GnRH-induced LH pulses within 1-2 h, the amount of GnRH receptor mRNA or protein was not affected over this time frame. Collectively, these observations provide evidence that cortisol acts via the type II GR within the pituitary gland to elicit a rapid decrease in responsiveness to GnRH, independent of changes in expression of the GnRH receptor.     

Effects of glucocorticoids on responsiveness of luteinizing hormone and follicle-stimulating hormone to gonadotropin-releasing hormone by male rat pituitary cells in vitro

To determine if the inhibitory effects of glucocorticoids on GnRH-stimulated secretion of LH observed in male rats in vivo are exerted directly on the pituitary, dispersed pituitary cells from adult male rats were treated with 60 or 600 ng/ml cortisol (F) or corticosterone (B) during one or two 48-h incubations. Control cells received no glucocorticoids. During the second 48 h, some cells from each group were treated with GnRH (2.4 X 10(-11)-6.2 X 10(-8) M). Concentrations of LH and FSH in media and cells were measured by RIA. Treatment with steroids had no effect on basal secretion or maximal GnRH-stimulated secretion of LH, or on maximal secretion of FSH. Treatment with 600 ng/ml B for 96 h increased basal secretion of FSH relative to controls. All treatments with glucocorticoids increased the slopes of the GnRH dose-response curves for both LH and FSH, cell content of LH, total (cells + medium) LH, and total FSH. Incubation with 6 micrograms/ml F or B or 60 ng/ml dexamethasone gave similar results. Decreasing the time period of the second incubation to 6 h results in no significant differences between control cells and cells treated with B or F. These results show that glucocorticoids have different effects in vivo and in vitro, suggesting that inhibitory effects of glucocorticoids on secretion of LH in vivo may not be exerted directly on the pituitary but are exerted elsewhere, perhaps by altered hypothalamic secretion of GnRH. Also, these results show that male and female pituitaries in vitro respond differently to glucocorticoids.     

Inhibitory actions of keoxifene on luteinizing hormone secretion in pituitary gonadotrophs

The effect of keoxifene (LY 156 758) on GnRH-stimulated LH release and its ability to antagonize estrogen actions were investigated in rat anterior pituitary cells. Estrogens exert either stimulatory or inhibitory effects on GnRH-induced LH secretion in rat pituitary cells depending on the incubation time with the steroid. When pituitary cells were treated for 24 h with 10(-9) M estradiol, the LH response to GnRH was clearly enhanced, and this effect was completely inhibited by 300 nM keoxifene. Short term treatment (4 h) of pituitary cells with 10(-9) M estradiol inhibits GnRH-stimulated LH release, and this effect was also blocked by keoxifene in a dose-dependent manner. In the absence of exogenous estrogen the treatment of pituitary cells for 4 h with increasing concentrations of keoxifene reduced the LH response to 10(-9) M GnRH only at very high concentrations (10(-5) M) of the antiestrogen. After treatment for 24 h, the inhibitory effect of keoxifene was evident at concentrations greater than or equal to 10(-8) M, with a reduction of GnRH-induced LH release by up to 60%. The effects of the antiestrogen were also analyzed in a dynamic culture system, in which pituitary cells grown on microcarrier beads were continuously perifused with medium and stimulated with GnRH in a pulsatile fashion. The LH response to a 2 min pulse of 10(-9) M GnRH was reduced in magnitude after 40 min of perifusion with 10(-9) M estradiol. When keoxifene (300 nM) was present at the same time, the LH response was identical to that observed in vehicle-treated cells. At the concentration of 300 nM, keoxifene per se did not change the responsiveness of the pituitary cells to the GnRH stimulus. These findings show that keoxifene is a potent antagonist of both positive and negative estrogen actions in the pituitary gonadotroph. In addition, after short term treatment with high concentrations or after long term treatment, keoxifene itself exerts an inhibitory effect on GnRH-induced LH secretion.     

Effects of glucocorticoids on secretion of luteinizing hormone and follicle-stimulating hormone by female rat pituitary cells in vitro

To determine if the divergent effects of glucocorticoids on the circulating levels of LH and FSH in female rats are exerted directly on the pituitary, adult female pituitary cells were treated either with no glucocorticoids or with 60 or 600 ng/ml cortisol or corticosterone during one or two 48-h incubations. During the second 48 h, some cells from each group were treated with GnRH (1.7 X 10(-12) - 4.6 X 10(-9) M). Concentrations of LH and FSH in media and cells were measured by RIA. Basal secretion of LH was inhibited 38-43% by different glucocorticoid treatment during the first 48 h and 21% by 600 ng/ml corticosterone during the second 48 h. In contrast, basal secretion of FSH was enhanced 22-64% during the first 48 h and 25-124% during the second 48 h. Secretion of LH in response to maximal stimulation with GnRH was unaffected by glucocorticoids, but maximal secretion of FSH was increased 68%. The responsiveness of the cells to GnRH, as determined from the slope of the GnRH dose-response curve for LH, was increased 43-50% by cortisol. The slope of the dose-response curve for FSH was unaffected, but the mean concentration of FSH as a function of the log dose of GnRH was increased 45-79%. Glucocorticoids had no effect on cell content of LH or total LH per dish, either under basal or maximal GnRH-stimulated conditions. Glucocorticoids increased basal cell content of FSH 41-82%, basal total FSH 35-93%, and maximal GnRH-stimulated total FSH 40-84%. These results suggest that the only negative effect of glucocorticoids on reproduction exerted at the level of the pituitary is a slight suppression of basal LH secretion, that glucocorticoids affect the pituitary directly by increasing FSH synthesis, and that the divergent effects of glucocorticoids on LH and FSH provide a novel model for differential regulation of the gonadotropins.     

Effects of inhibin from primate Sertoli cells on follicle-stimulating hormone and luteinizing hormone release by perifused rat pituitary cells

We used a pituitary cell perifusion system to investigate the time course and selectivity of the inhibin effect on pulsatile GnRH-stimulated LH and FSH release. Dispersed pituitary cells from 7- to 8-week-old male rats were perifused on a Cytodex bead matrix and stimulated with 10 nM GnRH for 2 min every hour for 8-11 h. The addition of a preparation of inhibin partially purified from primate Sertoli cells reduced pulsatile FSH release within 2 h. After removal of inhibin from the perifusion medium, the effect was reversed within 3 h. GnRH-stimulated LH release was also influenced by inhibin, although the decline in LH was less than that in FSH (80 +/- 3% vs. 68 +/- 4% of control; P less than 0.025). Smaller doses of inhibin suppressed GnRH-induced FSH secretion, but had no effect on LH release. Further, prolonged incubation of pituitary cells with inhibin at the higher dose reduced its FSH inhibitory effect and eliminated the effect on LH. These results indicate that inhibin can reduce both LH and FSH secretion in vitro, although the specificity and magnitude of the effect are a function of both the dose and duration of inhibin treatment. Further, the actions of inhibin and GnRH on the pituitary may be interrelated.     

Phytoestrogens and gonadotropin-releasing hormone pulse generator activity and pituitary luteinizing hormone release in the rat

Phytoestrogens can produce inhibitory effects on gonadotropin secretion in both animals and humans. The aims of this study were 2-fold: 1) to determine in vivo whether genistein and coumestrol act on the GnRH pulse generator to suppress hypothalamic multiunit electrical activity volleys and associated LH pulses and/or on the pituitary to suppress the LH response to GnRH; and 2) to examine the effect of these phytoestrogens on GnRH-induced pituitary LH release in vitro and to determine whether estrogen receptors are involved. Wistar rats were ovariectomized and chronically implanted with recording electrodes and/or indwelling cardiac catheters, and blood samples were taken every 5 min for 7--11 h. Intravenous infusion of coumestrol (1.6-mg bolus followed by 2.4 mg/h for 8.5 h) resulted in a profound inhibition of pulsatile LH secretion, a 50% reduction in the frequency of hypothalamic multiunit electrical activity volleys, and a complete suppression of the LH response to exogenous GnRH. In contrast, both genistein (1.6-mg bolus followed by 2.4 mg/h for 8.5 h) and vehicle were without effect on pulsatile LH secretion. Coumestrol (10(-5) M; over 2 or 4 h) suppressed GnRH-induced pituitary LH release in vitro, an effect blocked by the antiestrogen ICI 182,780. It is concluded that coumestrol acts centrally to reduce the frequency of the hypothalamic GnRH pulse generator. In addition, the inhibitory effects of coumestrol on LH pulses occur at the level of the pituitary by reducing responsiveness to GnRH via an estrogen receptor-mediated process.     

Interactions of insulin-like growth factor-I, insulin and estradiol with GnRH-stimulated luteinizing hormone release from female rat gonadotrophs

BACKGROUND: It is well established that ovarian steroids modulate gonadotropin secretion from anterior pituitary cells. It has been speculated that insulin and IGF-I might influence gonadotropin secretion. OBJECTIVE: To investigate the effects of IGF-I and estradiol alone, or combinations of IGF-I with insulin and estradiol on GnRH-stimulated LH release from female rat pituitary cells in serum-supplemented and serum-free culture conditions. METHODS: Pituitary cells were incubated for 24 h or 48 h with a series of increasing concentrations of IGF-I or estradiol and stimulated with 1 nmol/l GnRH for 3 h. To determine the interaction of IGF-I and estradiol on GnRH-stimulated LH secretion, cells were exposed to increasing concentrations of IGF-I and 100 pmol/l estradiol for 24 h. We also investigated the effects of combined treatment with IGF-I and insulin on GnRH-stimulated LH secretion. RESULTS: Our findings indicate that long-term IGF-I treatment (24 h) alone has a significant augmenting effect on GnRH-stimulated LH release in serum-free medium only, with a maximum at low concentrations (10 and 100 pmol/l). Estradiol significantly increased GnRH-induced LH release in a dose-dependent manner. The extent of GnRH-stimulated LH secretion by long-term estradiol treatment (24 h) was significantly greater in serum-supplemented (+42%) medium than in serum-free medium. Estradiol facilitated IGF-I-primed LH responses to GnRH in serum-free medium. In contrast, in serum-supplemented medium, the facilitating potential of estradiol was lower. We also found that, in GnRH-stimulated cells, LH release was augmented by insulin treatment, in contrast to quiescent cells that had been pretreated with 100 pmol/l IGF-I alone and 1 nmol/l insulin alone. CONCLUSIONS: IGF-I and to a lesser extent insulin stimulate GnRH-induced LH secretion from pituitary gonadotrophs. This action is enhanced by estradiol treatment of the cells. However, the well known stimulatory action of estradiol on LH secretion is dependent on the presence of growth factors.     

Gonadal steroid modulation of signal transduction and luteinizing hormone release in cultured chicken pituitary cells

The mechanism whereby gonadal steroids modulate GnRH-stimulated LH secretion by primary cultures of chicken pituitary cells was investigated. Estradiol (10(-8) M), testosterone (10(-7) M), and progesterone (10(-7) M) inhibited LH release stimulated by GnRH (10(-7) M) by 56%, 61%, and 53%, respectively, and the inhibitory effects required prolonged preincubation (24-48 h) with the steroids. The steroids inhibited the spike (0-3 min) and plateau (9-30 min) phases of LH release to a similar degree. The ED50 values of estradiol, testosterone, and progesterone for inhibition of GnRH-stimulated LH release were 7 x 10(-11), 2 x 10(-9), and 1 x 10(-9) M, respectively. Estradiol, testosterone, and progesterone inhibited the maximal LH response to GnRH, but the ED50 of GnRH (4 x 10(-9) M) was not altered by steroid pretreatment. Steroid pretreatment did not cause a change in cellular LH content, suggesting that the steroids do not inhibit LH synthesis. Combinations of two or three of the steroids were not additive, suggesting that all three steroids affect GnRH-stimulated LH release via the same mechanism. In experiments investigating their mechanism of action, the steroids inhibited LH release stimulated by GnRH and Ca2+ ionophore A23187, but generally had no effect on the responses to phorbol ester (12-O-tetradecanoylphorbol-13-acetate), forskolin, K+, Bay K8644, or veratridine. Estradiol inhibited GnRH-stimulated 45Ca2+ efflux, but its inhibitory effect on GnRH-induced inositol phosphate production was not significant. Estradiol had no effect on binding of 125I-[His5,D-Tyr6]GnRH to a pituitary cell preparation. These findings suggest that the site of steroid modulation of GnRH action is distal to binding of GnRH to its receptor, and that the inhibitory effects are exerted at two intracellular sites: 1) the coupling events linking receptor activation to mobilization of Ca2+, and 2) a site distal to Ca2+ mobilization.     

Melatonin inhibits gonadotropin-releasing hormone-induced elevation of intracellular Ca2+ in neonatal rat pituitary cells.

GnRH stimulates LH release by increasing intracellular Ca2+ ([Ca2+]i). Melatonin is known to inhibit GnRH-stimulated LH release from neonatal rat pituitary cells. In the present report, the issue of whether melatonin acts through [Ca2+]i was addressed. [Ca2+]i was studied in cells in suspension, using Fluo-3 as a fluorescent indicator. In neonatal rat pituitary cells, melatonin inhibited the GnRH-induced [Ca2+]i increase in a dose-dependent manner; the GnRH-induced increase in [Ca2+]i was inhibited 40% by 100 nM melatonin. The relative potencies of several indoles as inhibitors of the GnRH stimulation of [Ca2+]i in neonatal pituitary cells (2-iodo-melatonin greater than melatonin greater than 6-hydroxymelatonin) correlate with their known potencies to inhibit LH release and with their binding affinity to high affinity melatonin receptors, which indicates that these receptors probably mediate the effects of melatonin. Further support for this interpretation comes from the observation that melatonin does not inhibit the GnRH effect on [Ca2+]i in cells obtained from adolescent rat pituitary glands, which lack melatonin receptors and are insensitive to melatonin as an inhibitor of GnRH-stimulated LH release. The possible involvement of an inhibitory G-protein was also investigated by studying the effects of pertussis toxin. Pretreatment with pertussis toxin antagonized the effects of melatonin on [Ca2+]i and LH release. This indicates that melatonin may inhibit the GnRH-induced increase in [Ca2+]i through a mechanism involving a pertussis toxin-sensitive G-protein. To examine the role of extracellular Ca2+ in this effect, the effects of melatonin were examined in a low Ca2+ medium. Under these conditions, the effect of melatonin was markedly reduced, which indicates that melatonin may act by inhibiting Ca2+ influx. These observations indicate that melatonin inhibits GnRH stimulation of [Ca2+]i in neonatal rat gonadotrophs, and this probably explains the inhibitory action of melatonin on GnRH stimulation of LH release.     

Inhibition of luteinizing hormone release by morphine and endogenous opiates in cultured pituitary cells

Morphine sulfate was found to have a direct inhibitory effect on both basal and GnRH-stimulated LH release by cultured rat pituitary cells. The inhibitory effect of morphine on LH release was prevented by the opiate antagonist naltrexone, and treatment of cells with naltrexone or beta-endorphin antiserum significantly increased basal LH release. Also, incubation of pituitary cells with CRF caused a significant decrease in basal LH release, an effect that was reversed by naltrexone. Saturable opiate-binding sites were demonstrated in enriched gonadotrophs by [3H]etorphine binding studies. The ability of morphine to inhibit gonadotropin secretion through a direct action on pituitary opiate receptors suggests that long term exposure to exogenous opiates may suppress reproductive function at the hypophyseal level. In addition, the converse effects of CRF and naltrexone or beta-endorphin antiserum on LH release indicate that intrapituitary opioid peptides could exert a paracrine inhibitory action on the gonadotroph.     

The effect of gonadotrophin surge-inhibiting factor on the self-priming action of gonadotrophin-releasing hormone in female rats in vitro.

The present study was designed to explore further the functional antagonism between gonadotrophin-releasing hormone (GnRH) and the ovarian factor, gonadotrophin surge-inhibiting factor (GnSIF). In all experiments, pituitary tissue was exposed to various amounts of GnSIF, after which the self-priming action of GnRH was studied. GnSIF was increased in vivo by FSH treatment and increased in vitro by adding various amounts of follicular fluid (FF) to cultured pituitary cells. Treatment with 3 or 10 IU FSH suppressed the initial LH response and delayed the maximally primed LH response to GnRH. Treatment with FSH was only effective in intact rats on days 1 and 2 of dioestrus. There was no difference in the rate of maximal LH release irrespective of treatment with either FSH or saline. Since FSH treatment was ineffective in long-term ovariectomized rats, it was concluded that the initial suppressive effect of FSH on LH release was mediated by GnSIF. Cycloheximide prevented the self-priming action of GnRH by inhibiting GnRH-induced protein synthesis. The initial protein synthesis-independent GnRH-stimulated LH release, which was already suppressed by FSH treatment, remained suppressed in the presence of cycloheximide. Pretreatment with GnRH in vivo increased the protein synthesis-independent GnRH-induced LH release during subsequent incubation of the glands. This increase did not occur after FSH treatment. Pituitary cells, cultured for 20 h in medium only, failed to elicit the self-priming effect of GnRH. Preincubation with FF maintained the self-priming effect. This was independent of the concomitant presence of various amounts of oestradiol. Preincubation with bovine FF suppressed the initial GnRH-stimulated LH release dose-dependently. Porcine FF, human FF and testicular extract suppressed the release of LH in a similar way. It was concluded that GnSIF suppresses the initial LH response to continuous GnRH stimulation. Increased levels of GnSIF caused by FSH treatment also delayed the primed LH release. The mechanism of functional antagonism between GnSIF and GnRH could give rise to the occurrence of the phenomenon of GnRH self-priming.     

Acute effects of oestradiol and progesterone on melittin- and gonadotrophin-releasing hormone-induced LH secretion.

It is well established that oestradiol and progesterone modulate gonadotrophin-releasing hormone (GnRH)-induced LH secretion from cultured rat pituitary cells. Short-term oestradiol and long-term progesterone treatment exert inhibition, while short-term progesterone and long-term oestradiol treatment lead to enhancement of GnRH-stimulated LH secretion. There are several lines of evidence to suggest that the steroid effects might be mediated via a mechanism involving modulation of the GnRH signal-transduction system. To evaluate the role of arachidonic acid, which serves as an intracellular signal transducer by itself or its lipoxygenase metabolites, in the mediation of oestradiol and progesterone actions, we examined their effects on melittin (activator of phospholipase (A2)-stimulated LH secretion. When pituitary cells from adult female rats were treated for 48 h with 1 nmol oestradiol/l or 1 nmol oestradiol/l plus 100 nmol progesterone/l, GnRH (1 nmol/l)-induced LH secretion was stimulated or inhibited respectively. However, melittin (10-300 nmol/l)-stimulated LH secretion remained unaffected after such treatment. Short-term treatment with oestradiol inhibited GnRH-induced LH secretion while progesterone treatment of oestradiol-primed cells led to a stimulatory effect. Interestingly, melittin-stimulated LH secretion was influenced in the same way after the short treatment paradigm. Perifusion studies were performed to assess the kinetics of these acute steroid actions further. Four separate perifusion chambers were continuously perifused with medium and stimulated for 2 min with 1 nmol GnRH/l or 1 mumol melittin/l every 50 min in a pulsatile fashion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypophysiotropic role of RFamide-related peptide-3 in the inhibition of LH secretion in female rats

Gonadotropin-inhibitory hormone (GnIH), a newly discovered hypothalamic RFamide peptide, inhibits reproductive activity by decreasing gonadotropin synthesis and release in birds. The gene of the mammalian RFamide-related peptides (RFRP) is orthologous to the GnIH gene. This Rfrp gene gives rise to the two biologically active peptides RFRP-1 (NPSF) and RFRP-3 (NPVF), and i.c.v. injections of RFRP-3 suppress LH secretion in several mammalian species. In this study, we show whether RFRP-3 affects LH secretion at the pituitary level and/or via the release of GnRH at the hypothalamus in mammals. To investigate the suppressive effects of RFRP-3 on the mean level of LH secretion and the frequency of pulsatile LH secretion in vivo, ovariectomized (OVX) mature rats were administered RFRP-3 using either i.c.v. or i.v. injections. Furthermore, the effect of RFRP-3 on LH secretion was also investigated using cultured female rat pituitary cells. With i.v. administrations, RFRP-3 significantly reduced plasma LH concentrations when compared with the physiological saline group. However, after i.c.v. RFRP-3 injections, neither the mean level of LH concentrations nor the frequency of the pulsatile LH secretion was affected. When using cultured pituitary cells, in the absence of GnRH, the suppressive effect of RFRP-3 on LH secretion was not clear, but when GnRH was present, RFRP-3 significantly suppressed LH secretion. These results suggest that RFRP-3 does not affect LH secretion via the release of GnRH, and that RFRP-3 directly acts upon the pituitary to suppress GnRH-stimulated LH secretion in female rats.     

Stimulation of luteinizing hormone release and cyclic nucleotide production by arachidonic acid in cultured pituitary gonadotrophs

Gonadotropin-releasing hormone (GnRH) stimulates luteinizing hormone (LH) release and cyclic guanosine 3',5-cyclic monophosphate (cGMP) production in rat anterior pituitary cells through a calcium-dependent activation mechanism that involves increased phospholipid turnover and liberation of arachidonic acid. In enriched pituitary gonadotrophs, LH release was stimulated by arachidonic acid and its oxygenated metabolite, 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), in a dose-dependent manner. The prominent LH responses of purified gonadotrophs to arachidonic acid suggest that the secretory actions of arachidonate are exerted primarily on the gonadotroph and do not involve the participation of other pituitary cell types. Preincubation of pituitary cells with stimulatory concentrations of arachidonic acid for up to 120 min did not alter the subsequent LH responses elicited by GnRH, indicating that the secretory mechanism was unimpaired by arachidonate treatment and that no cross-desensitization occurs during sequential exposure of gonadotrophs to the two stimuli of LH release. Cyclic adenosine 3',5-monophosphate (cAMP) production was stimulated by 10 microM arachidonic acid to the same degree (about 2-fold) as by GnRH, but did not parallel the progressive LH response to higher arachidonate concentrations. cGMP production was initially stimulated by addition of arachidonic acid but returned to the control value after 5 min, whereas GnRH typically elicited a prolonged cGMP response. In contrast to the calcium-independent action of arachidonic acid, the stimulatory effect of 5-HETE on LH release required the presence of extracellular Ca2+, as previously observed for GnRH. These findings demonstrate that arachidonic acid and its metabolite, 5-HETE, partially reproduce the actions of GnRH upon LH release and cyclic nucleotide production.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of ovarian steroids with pituitary adenylate cyclase-activating polypeptide and GnRH in anterior pituitary cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) releases LH and FSH from anterior pituitary cells. Although this effect is relatively weak, it has a strong sensitizing action on GnRH-induced gonadotropin secretion. Here we investigated the possibility that ovarian steroids, which are well-known modulators of LH secretion, interact with PACAP and GnRH in pituitary gonadotrophs. Rat pituitary cells were treated for 48 h with vehicle, 1 nmol/l estradiol, 1 nmol/l estradiol + 100 nmol/l progesterone or 48 h with 1 nmol/l estradiol and 4 h with 100 nmol/l progesterone. The cells were stimulated for 3 h with 1 nmol/l GnRH or 100 nmol/l PACAP. Estradiol treatment alone enhanced basal as well as GnRH- or PACAP-stimulated LH secretion. LH release was facilitated by additional short-term progesterone treatment. Long-term treatment with estradiol and progesterone led to reduced LH responses to GnRH and PACAP. Neither treatment paradigms affected cAMP production. However, estradiol treatment led to enhanced cAMP accumulation in quiescent or GnRH-stimulated cells. PACAP-induced increases of cAMP production were inhibited by estradiol treatment. After 7-h preincubation with 10 nmol/l PACAP, cells responded with enhanced LH secretion to GnRH stimulation. When steroid pretreatment was performed the responsiveness of gonadotrophs to low concentrations of GnRH was still increased. In contrast, at high concentrations of GnRH the sensitizing action of PACAP on agonist-induced LH secretion was lost in steroid-treated cells. There were no significant differences between the steroid treatment paradigms. It is concluded that estradiol but not progesterone acts as a modulator of adenylyl cyclase in gonadotrophs. The stimulatory effect of estradiol is thought to be involved in its sensitizing action on agonist-induced LH secretion. The inhibitory effect of estradiol on PACAP-stimulated adenylyl cyclase activities seems to be responsible for the loss of its action to sensitize LH secretory responses to GnRH.     

Effects of gonadal steroids on gonadotropin secretion in males: studies with perifused rat pituitary cells

The feedback effects of testosterone (T) and estradiol (E2) on FSH and LH secretion were compared in dispersed pituitary cells from adult male rats perifused with pulses of GnRH. Cells were stimulated with 10 nM GnRH for 2 min every 1 h. T (10 nM) pretreatment for 24 h reduced the amplitude of FSH and LH pulses to 77 +/- 4% (mean +/- SE) and 47 +/- 3% of control values, respectively (P less than 0.01), whereas 6-h T treatment was without effect. By contrast, interpulse secretion of FSH was increased after 24 h T to 184 +/- 7% of the control value (P less than 0.01), but interpulse LH release was unchanged (104 +/- 5%). E2 (0.075 nM) treatment of pituitary cells reduced GnRH-stimulated FSH and LH release within 2 h to 75 +/- 2% and 73 +/- 3% of control values, respectively (P less than 0.01). E2 pretreatment for 24 h stimulated (P less than 0.025) GnRH-induced FSH (136 +/- 10%) and LH (145 +/- 8%) release and also increased (P less than 0.01) interpulse FSH (127 +/- 5%) and LH (145 +/- 8%) secretion. These data indicate that the suppression of FSH and LH secretion by T in males is due in part to a direct effect on the pituitary. The findings that T suppresses GnRH-stimulated FSH less than LH, and that T stimulates interpulse FSH, but not LH, provide evidence for differential regulation of FSH and LH secretion by T. The dissimilar actions of T on GnRH-stimulated pulses and interpulse gonadotropin secretion suggest that interpulse secretion is unrelated to stimulation by GnRH, although its physiological significance is unknown. Since E2, in physiological levels for males, increased pituitary FSH and LH secretion, the suppression of gonadotropin secretion by E2 in vivo in males may result from an effect on the hypothalamic pulse generator; however, additional studies are needed before extending these conclusions to higher mammals and men.     

In vitro effect of ethanol exposure on basal and GnRH-stimulated LH secretion from pituitary cells

The question of whether ethanol's (ETOH's) known suppressive effect on serum luteinizing hormone (LH) could be mediated directly at the anterior pituitary level was addressed by examining the effects of ETOH in vitro on release of LH from cultured male rat pituitary cells. The impact of added ethanol concentrations ranging from 50 to 400 mg% on LH release was examined in the basal state and after stimulation by gonadotropin-releasing hormone (GnRH) at a dose of 5 x 10(-10) M. While ETOH did not significantly suppress basal LH release, secretion stimulated with GnRH was noted to be attenuated with higher doses of ETOH (greater than or equal to 100 mg%) compared to stimulated control cells. It is concluded that ETOH exposure in vitro alters stimulated LH secretion by acting directly on pituitary gonadotropes.     

Differential effects of in vitro glucocorticoids on luteinizing hormone and follicle-stimulating hormone secretion: dependence on sex of pituitary donor

We have used a dynamic perifusion system to determine whether glucocorticoids exert a direct effect on the secretion of LH and FSH from rat anterior pituitaries. Anterior pituitary fragments from male, proestrous female, or metestrous female rats were perifused for 8 h in either the absence (basal secretion rate) or presence of pulsatile GnRH administration (50 ng/ml peak concentration). Perifusions used medium containing 0.05% ethanol (vehicle), 600 ng/ml corticosterone, or 600 ng/ml cortisol. GnRH-stimulated secretion of FSH was enhanced in pituitaries from both male and female rats after in vitro incubation with either corticosterone or cortisol. The basal secretion rate of FSH was also elevated in proestrous females after glucocorticoid treatment. The GnRH-stimulated secretion rate for LH was significantly decreased in pituitaries from male rats treated with either glucocorticoid. In contrast, pituitaries from proestrous rats responded to either cortisol or corticosterone with an increase in LH secretion. Metestrous pituitaries showed divergent effects of the glucocorticoids on LH secretion; corticosterone enhanced secretion rates, and cortisol effected a decrease. Our data demonstrate that 1) glucocorticoids exert a direct effect on the secretion of LH and FSH from male and female rat pituitaries; 2) glucocorticoids elicit different effects on the secretion of LH and FSH, suggesting that they act at separate sites to regulate LH and FSH secretion; and 3) the effect of in vitro glucocorticoid treatment on gonadotropin secretion is dependent on sex and cycle stage of the pituitary donor and may be linked to prior in vivo concentrations of estrogen.