Modulatory effect of steroid hormones on GnRH-induced LH secretion by cultured rat pituitary cells.

The purpose of the present experiments was to examine the short- and long-term effects of estradiol-17 beta (E2), progesterone (P), and 5 alpha-dihydrotestosterone (DHT), alone and in combination, on the gonadotrophin-releasing hormone (GnRH)-induced luteinizing hormone (LH) secretion, using an ovariectomized rat pituitary cells culture model. After 72 h in steroid-free medium, pituitary cells were further cultured for 24 h in medium with or without E2 (1 nM), P (100 nM), or DHT (10 nM). Cultures were then incubated for 5 h in the absence or presence of 1 nM GnRH with or without steroids. LH was measured in the medium and cell extract by radioimmunoassay. The results show that the steroid hormones exert opposite effects on the release of LH induced by GnRH, which seems to be dependent upon the length of time the pituitary cells have been exposed to the steroids. In fact, short-term (5 h) action of E2 resulted in a partial inhibition (64% of control) of LH release in response to GnRH, while long-term (24 h) exposure enhanced (158%) GnRH-induced LH release. Similar results were obtained with DHT, although the magnitude of the effect was lower than with E2. Conversely, P caused an acute stimulatory action (118%) on the LH released in response to GnRH and a slightly inhibitory effect (90%) after chronic treatment. GnRH-stimulated LH biosynthesis was also influenced by steroid treatment. Significant increases in total (cells plus medium) LH were observed in pituitary cells treated with E2 or DHT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulatory actions of estradiol and progesterone on phorbol ester-stimulated LH secretion from cultured rat pituitary cells.

We compared the ability of estradiol and progesterone to modulate gonadotropin-releasing hormone (GnRH) and protein kinase C (PKC)-mediated luteinizing hormone (LH) secretion. Long-term (48 h) treatment of rat pituitary cells with 1 nM estradiol enhanced GnRH and phorbol ester (TPA)-stimulated LH secretion. This positive effect was facilitated by additional short-term (4 h) treatment with progesterone (100 nM). However, long-term progesterone treatment, which inhibited GnRH-stimulated LH secretion, did not influence TPA-stimulated gonadotropin release. These steroid actions occurred without an effect on the total amount of LH in the cell cultures (total LH = LH secreted + LH remaining in the cell) and neither the secretagogues nor the steroids altered total LH. Since GnRH or TPA-induced LH secretion depends on Ca2+ influx into the gonadotroph, we also analyzed the effects of estradiol and progesterone under physiological extracellular Ca2+ concentrations and in the absence of extracellular Ca2+. The steroids were able to influence GnRH or TPA-induced LH secretion under both conditions. However, when TPA was used as stimulus in Ca(2+)-deficient medium the relative changes induced by estradiol and progesterone were more pronounced, possibly indicating that the extracellular Ca(2+)-independent component of PKC-mediated LH secretion is more important for the regulation of the steroid effects. It is concluded that estradiol and progesterone might mediate their modulatory actions on GnRH-stimulated LH secretion via an influence on PKC. This effect can occur independently from de novo synthesis of LH and Ca2+ influx into gonadotrophs.     

Differential actions of corticosterone on luteinizing hormone and follicle-stimulating hormone biosynthesis and release in cultured rat anterior pituitary cells: interactions with estradiol

Previous in vivo studies from our laboratory suggested that glucocorticoids antagonize estrogen-dependent actions on LH secretion. This study investigated whether corticosterone (B) may have similar actions on gonadotropin biosynthesis and secretion in vitro. Enzymatically dispersed anterior pituitary cells from adult female rats were cultured for 48 h in alpha-modified Eagle's medium containing 10% steroid-free horse serum with or without 0.5 nM estradiol (E2). The cells were then cultured for 24 h with or without B in the presence or absence of E2. To evaluate hormone release, 5 x 10(5) cells were incubated with varying doses of GnRH (0, 10(-11)-10(-7) M) or pulsatile GnRH (10(-9) M; 20 min/h) for 4 h. Cell and medium LH and FSH were measured by RIA. To evaluate LH biosynthesis, 5 x 10(6) cells were incubated for an additional 24 h with 10(-10) M GnRH, 60 microCi 3H-glucosamine (3H-Gln), 20 microCi 35S-methionine (35S-Met), and the appropriate steroid hormones. Radiolabeled precursor incorporation into LH subunits was determined by immunoprecipitation, followed by SDS-PAGE. Continuous exposure to GnRH stimulated LH release in a dose-dependent manner, and this response was enhanced by E2. B by itself had no effect on LH release, but inhibited LH secretion in E2-primed cells at low concentrations of GnRH (10(-10) M or less). Total LH content was not altered by GnRH or steroid treatment. Similar effects of B were observed in cells that were given a pulsatile GnRH stimulus. In contrast to LH, E2 or B enhanced GnRH-stimulated FSH release at the higher doses of GnRH, while the combination of E2 and B increased basal and further augmented GnRH-stimulated release. Total FSH content was also increased in the presence of B, but not E2 alone, and was further augmented in cells treated with both steroids. There were no effects of the steroids on the magnitude of FSH release in response to GnRH pulses, but the cumulative release of FSH was greater in the E2 + B group compared to controls, indicating an increased basal release. Independent of E2, B suppressed the incorporation of 3H-Gln into LH by more than 50% of control, with only subtle effects on the incorporation of 35S-Met.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hormone-induced redistribution of calcium-activated phospholipid-dependent protein kinase in pituitary gonadotrophs

The distribution of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) between cytosol and membrane fractions was analyzed in cultured pituitary gonadotrophs during treatment with gonadotropin-releasing hormone (GnRH). In pituitary cells purified by centrifugal elutriation, the extent of protein kinase C redistribution during GnRH stimulation was correlated with the enrichment of gonadotrophs. GnRH-stimulated release of luteinizing hormone (LH) from gonadotroph-enriched cells was accompanied by a rapid and dose-dependent decrease in cytosolic protein kinase C and by a corresponding increase in protein kinase C activity in the particulate fraction. Retinal directly inhibited the activity of cytosolic protein kinase C and also attenuated the release of LH from GnRH-stimulated gonadotrophs. These findings, and the ability of GnRH to cause rapid translocation of cytosolic protein kinase C to a membrane-associated form, suggest that hormonal activation of protein kinase C is an intermediate step in the stimulation of pituitary LH secretion by GnRH.     

Staurosporine enhances gonadotrophin-releasing hormone-stimulated luteinizing hormone secretion

In intact sheep gonadotropes, the protein kinase inhibitor, staurosporine, inhibited the stimulatory effect of phorbol 12-myristate 13-acetate (PMA) on luteinizing hormone (LH) secretion. Under the same conditions staurosporine enhanced gonadotrophin-releasing hormone (GnRH)-stimulated LH exocytosis without altering the EC50 of GnRH and without affecting basal LH exocytosis. These results suggest that PKC does not play a major role in mediating acute GnRH-stimulated LH exocytosis. Furthermore, they demonstrate that staurosporine enhances GnRH stimulus-secretion coupling. Both extracellular Ca2(+)-dependent and Ca2(+)-independent components of GnRH-stimulated LH secretion were enhanced by the drug. Staurosporine had no effect on GnRH stimulation of cAMP and inositol phosphate synthesis. In permeabilized cells staurosporine did not enhance Ca2(+)- and cAMP-stimulated LH exocytosis. Based on these results we hypothesize that staurosporine inhibits a protein kinase which is activated by GnRH and which negatively modulates GnRH stimulus-secretion coupling.     

Dependence of secretory responses to gonadotropin-releasing hormone on diacylglycerol metabolism. Studies with a diacylglycerol lipase inhibitor, RHC 80267

The role of diacylglycerol (DG) as a source of arachidonic acid during gonadotropin-releasing hormone (GnRH) stimulation of gonadotropin secretion was analyzed in primary cultures of rat anterior pituitary cells. An inhibitor of DG lipase (RHC 80267, RHC) caused dose-dependent blockade of GnRH-stimulated luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion. The DG lipase inhibitor did not alter gonadotropin responses to arachidonic acid, and addition of arachidonic acid reversed its inhibition of GnRH-stimulated LH and FSH release. In [3H]arachidonic acid-prelabeled cells, incubation with RHC increased the accumulation of [3H]DG. These results suggest that DG lipase participates in GnRH action and that arachidonic acid mobilization from DG is involved in the mechanism of gonadotropin release. Gonadotropin responses to tetradecanoyl phorbol acetate and dioctanoyl glycerol were not altered by RHC, and the addition of these activators of protein kinase C (Ca2+- and phospholipid-dependent enzyme) did not prevent the inhibition of GnRH-induced gonadotropin release by RHC. Activation of phospholipase A2 by melittin increased LH and FSH secretion, whereas blockade of this enzyme by quinacrine reduced GnRH-stimulated hormone release. However, RHC did not diminish the gonadotropin response to melittin. The inhibitory actions of RHC and quinacrine were additive and were reversed by concomitant treatment with arachidonic acid. Ionomycin also increased LH and FSH release, and the gonadotropin responses to the ionophore were unaltered by RHC but were reduced by quinacrine. Incubation of cells in Ca2+-depleted (+/- [ethylenebis(oxyethylenenitrilo)]tetraacetic acid) medium reduced but did not abolish the LH and FSH releasing activity of GnRH. Treatment with RHC also reduced the gonadotropin responses to GnRH under Ca2+-depleted conditions. These observations indicate that RHC inhibition of GnRH action is not due to nonspecific actions on Ca2+ entry, protein kinase C activation and actions, nor phospholipase A2 enzyme activity. The results of this study provide further evidence for an extracellular Ca2+-independent mechanism of GnRH action, and suggest that GnRH causes mobilization of arachidonic acid by two distinct lipases, namely, phospholipase A2 and DG lipase, during stimulation of gonadotropin secretion.     

Effect of phorbol ester on stimulus-secretion coupling mechanisms in gonadotropin releasing hormone-stimulated pituitary gonadotrophs

The feedback regulatory control mechanism exerted by activated Ca2+/phospholipid-dependent protein C kinase upon gonadotropin releasing hormone (GnRH) binding, stimulation of phosphoinositide turnover and gonadotropin secretion was investigated in cultured pituitary cells. Addition of the tumor promoter phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), at concentrations which activate pituitary protein C kinase, to cultured pituitary cells resulted in up-regulation of GnRH receptors (155% at 4 h). The stimulatory effect of GnRH on [3H]inositol phosphates (Ins-P) production in myo-[2-3H]inositol prelabeled pituitary cells was not inhibited by prior treatment of the cells with TPA (10(-9)-10(-7) M). Higher concentrations of TPA (10(-6)-10(-5) M) inhibited the effect of GnRH on [3H]Ins-P production. Increasing concentrations of TPA or the permeable analog of diacylglycerol 1-oleoyl-2-acetylglycerol (OAG) stimulated luteinizing hormone (LH) release from cultured pituitary cells with ED50 values of 5 x 10(-9) M and 10 micrograms/ml, respectively. No consistent inhibition or additivity of LH release was observed when increasing doses of TPA or OAG were added with a submaximal dose of GnRH. These results suggest that protein C kinase might mediate the known homologous up-regulation of GnRH receptors during the reproductive cycle. Protein C kinase is positively involved in mediating the process of gonadotropin secretion. Unlike many other systems, activation of protein C kinase in pituitary gonadotrophs is not involved in negative feed-back regulation of stimulus-secretion-coupling mechanisms in GnRH-stimulated gonadotrophs.     

Preparation and characterization of photoreactive derivatives of GnRH. Persistent activation of function by photoaffinity labeling

A photoreactive derivative of the highly potent gonadotropin releasing hormone (GnRH) agonist, D-Lys6-GnRH(1-9)-ethylamide, was prepared by selective modification of the epsilon-amino group with 2-nitro-4-azidophenyl sulfenyl chloride (2,4-NAPS C1). The modified peptide [D-Lys(NAPS)]6-GnRH-(1-9)-ethylamide was found to be a full agonist of LH release from rat pituitary cells with a relative potency 23 compared to GnRH. Covalent attachment of the photoreactive analog to rat pituitary cells resulted in prolonged activation of LH secretion which could not be inhibited by a potent GnRH antagonist. Persistent stimulation of pituitary gonadotrophs caused by covalently bound hormone led to desensitization of the LH releasing mechanism.     

GnRH stimulates LH release directly via inositol phosphate and indirectly via cAMP in African catfish

In African catfish, two gonadotropin-releasing hormone (GnRH) peptides have been identified: chicken GnRH (cGnRH)-II and catfish GnRH (cfGnRH). The GnRH receptors on pituitary cells producing gonadotropic hormone signal through inositol phosphate (IP) elevation followed by increases in intracellular calcium concentration (?Ca(2+)(i)). In primary pituitary cell cultures of male African catfish, both cGnRH-II and cfGnRH dose dependently elevated IP accumulation, ?Ca(2+)(i), and the release of the luteinizing hormone (LH)-like gonadotropin. In all cases, cGnRH-II was more potent than cfGnRH. The GnRH-stimulated LH release was not associated with elevated cAMP levels, and forskolin-induced cAMP elevation had no effect on LH release. With the use of pituitary tissue fragments, however, cAMP was elevated by GnRH, and forskolin was able to stimulate LH secretion. Incubating these fragments with antibodies against cfGnRH abolished the forskolin-induced LH release but did not compromise the forskolin-induced cAMP elevation. This suggests that cfGnRH-containing nerve terminals are present in pituitary tissue fragments and release cfGnRH via cAMP signaling on GnRH stimulation, whereas the GnRH receptors on gonadotrophs use IP/?Ca(2+)(i) to stimulate the release of LH.     

Testosterone-dependent effects of galanin on pituitary luteinizing hormone secretion in male rats

Galanin is a 29-amino-acid peptide that colocalizes with GnRH in hypothalamic neurons. High concentrations of galanin are present in portal vessel blood of both male and female rats, and galanin receptors are present on gonadotropes in both sexes. Results from studies of female rats indicate that galanin acts at the level of the pituitary to directly stimulate LH secretion and also to enhance GnRH-stimulated LH secretion. The effects of galanin on pituitary LH secretion in male rats are relatively uncharacterized; thus, the present in vivo study was conducted 1). to examine the ability of galanin to affect basal or GnRH-stimulated LH secretion in male rats and 2). to determine whether the effects of galanin on LH secretion in male rats are testosterone-dependent. All three doses of galanin used (1, 5, and 10 micro g/pulse) significantly enhanced GnRH-stimulated LH secretion in intact male rats. Only the highest dose of galanin directly stimulated LH secretion (without GnRH coadministration) in intact males. Galanin did not directly stimulate LH secretion or enhance GnRH-stimulated LH secretion in castrated male rats. In fact, the highest dose of galanin inhibited GnRH-stimulated LH secretion in castrated males. Upon testosterone replacement, the ability of galanin to directly stimulate LH secretion and to enhance GnRH-stimulated LH secretion was restored in castrated males. These results suggest a role for galanin in the regulation of LH release in male rats and demonstrate that testosterone upregulates the ability of the pituitary to respond to the stimulatory effects of galanin.     

An in vitro study of LH release, synthesis and heterogeneity in pituitaries from proestrous and short-term ovariectomized rats

It is known that acute ovariectomy (OVX) greatly attenuates the pituitary luteinizing hormone (LH) response to gonadotropin-releasing hormone (GnRH) in vitro. The present study evaluated possible quantitative and/or qualitative differences in the biosynthesis and secretion of LH in pituitaries from proestrous and acutely (72 h) OVX rats. Paired anterior pituitary glands were incubated for 4 h in a medium containing +/- 10 nM GnRH. Pituitary and secreted LH were measured by radioimmunoassay with differences in total LH (tissue plus medium) +/- GnRH being indicative of GnRH-stimulated LH synthesis. Qualitative changes in LH were evaluated by isoelectrofocusing (IEF). The results show that the major form of LH stored in and released from the pituitaries consisted of LH molecules with an isoelectric point (pI) in the alkaline pH range (alkaline LH), and a lesser amount (approximately 30%) of LH molecules in the acidic pH range (acidic LH). The ratio of alkaline/acidic LH observed in the pituitary and medium was similar in the proestrous and OVX groups, although the amount of alkaline and acidic LH release in response to GnRH was 2-3 times greater in the proestrous group. In both groups, the alkaline/acidic LH ratio of secreted LH was higher in the presence of GnRH than in its absence. Alkaline LH synthesis was increased by GnRH in both groups, with the response being greater in the proestrous than in the OVX group; GnRH-stimulated acidic LH synthesis was observed only in the proestrous group. In both groups, the amount of LH synthesized was about 60% of the amount released, which suggests that LH synthesis does not fully account for differences in GnRH-stimulated LH release. Treatment of pituitary extracts with neuraminidase decreased acidic LH, and proportionately increased alkaline LH. These results suggest that the quality of LH stored in and secreted from pituitaries of proestrous and OVX rats is similar, and that there is a preferential release of the major alkaline LH isoform in response to GnRH. The ovarian steroid environment, presumably estradiol, proportionately increases the amount of alkaline and acidic LH released, and differentially affects the amounts of the various isoforms synthesized in response to GnRH. The charge heterogeneity of alkaline and acidic LH may be related to the sialic acid content of the LH molecule.     

Effects of gonadal steroids on follicle-stimulating hormone and luteinizing hormone secretion by pituitary cells from castrated and intact male rats

The effectiveness of androgens in suppressing gonadotropin secretion declines with time following orchidectomy; however, the mechanism for this acquired resistance to androgen action is unknown. The role of the pituitary was studied by use of perifused rat pituitary cells and cells in monolayer culture. Pituitary cells from 7-wk-old intact male rats and rats that had been castrated 2 wk previously were treated with 10 nM testosterone (T) for 24 h; cells were then packed into perifusion chambers and stimulated with 2.5 nM GnRH for 2 min every hour for 8 h during which time T treatment was continued. T suppressed GnRH-stimulated LH secretion and LH pulse amplitude equally in both groups to approximately 60% of control values. Interpulse LH secretion was unchanged by T in either group. GnRH-stimulated FSH release was suppressed more (p less than 0.05) by T with cells from castrated rats than with cells from intact rats (76 +/- 4% vs. 90 +/- 2% of control; mean +/- SEM). By contrast, the action of T to increase interpulse basal FSH secretion was less (p less than 0.05) with cells from castrated rats (115 +/- 10% of control) than with cells from intact rats (146 +/- 6% of control). T treatment for 72 h also increased basal FSH secretion by pituitary cells in monolayer culture to a lesser extent with cells from castrated rats than with cells from intact rats (151 +/- 14% vs. 191 +/- 16% of control, p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Catecholamine inhibition of luteinizing hormone secretion in isolated pig pituitary cells

This study investigated the direct effect of catecholamines, epinephrine (EPI), and norepinephrine (NE) on basal and gonadotropin-releasing hormone (GnRH)-stimulated secretion of luteinizing hormone (LH) from dispersed pig pituitary cells in vitro. Pig pituitaries were dispersed into cells with collagenase and DNAase and then cultured in McCoy's 5a medium containing horse serum (10%) and fetal calf serum (2.5%) pretreated with dextran-coated charcoal for 3 days. EPI and NE did not affect basal LH secretion after 4 h of incubation. When pituitary cells were incubated with EPI or NE (1 microgram/ml) for longer than 30 min, GnRH-stimulated LH secretion was reduced. The degree of this reduction was dependent on EPI and NE, and a concentration of EPI and NE higher than 1 ng/ml and 100 ng/ml, respectively, was needed. L-isoproterenol, a nonselective beta-agonist, also inhibited the LH response to GnRH. Propranolol, a beta-antagonist, blocked the inhibitory effect of EPI, whereas phentolamine, an alpha-antagonist, had no effect. These results suggest that catecholamines, acting by a beta 2-adrenergic receptor, may play a role in the control of the porcine pituitary gonadotrope's response to GnRH.     

Effects of hyperprolactinemia on the control of luteinizing hormone and follicle-stimulating hormone secretion in the male rat

Experiments were conducted to determine the effects of acute hyperprolactinemia (hyperPRL) on the control of luteinizing hormone and follicle-stimulating hormone secretion in male rats. Exposure to elevated levels of prolactin from the time of castration (1 mg ovine prolactin 2 X daily) greatly attenuated the post-castration rise in LH observed 3 days after castration. By 7 days after castration, LH concentrations in the prolactin-treated animals approached the levels observed in control animals. HyperPRL had no effect on the postcastration rise in FSH. Pituitary responsiveness to gonadotropin hormone-releasing hormone (GnRH), as assessed by LH responses to an i.v. bolus of 25 ng GnRH, was only minimally effected by hperPRL at 3 and 7 days postcastration. LH responses were similar at all time points after GnRH in control and prolactin-treated animals, except for the peak LH responses, which were significantly smaller in the prolactin-treated animals. The effects of hyperPRL were examined further by exposing hemipituitaries in vitro from male rats to 6-min pulses of GnRH (5 ng/ml) every 30 min for 4 h. HyperPRL had no effect on basal LH release in vitro, on GnRH-stimulated LH release, or on pituitary LH concentrations in hemipituitaries from animals that were intact, 3 days postcastration, or 7 days postcastration. However, net GnRH-stimulated release of FSH was significantly higher by pituitaries from hyperprolactinemic, castrated males. To assess indirectly the effects of hyperPRL on GnRH release, males were subjected to electrical stimulation of the arcuate nucleus/median eminence (ARC/ME) 3 days postcastration. The presence of elevated levels of prolactin not only suppressed basal LH secretion but reduced the LH responses to electrical stimulation by 50% when compared to the LH responses in control castrated males. These results suggest that acute hyperPRL suppresses LH secretion but not FSH secretion. Although pituitary responsiveness is somewhat attenuated in hyperprolactinemic males, as assessed in vivo, it is normal when pituitaries are exposed to adequate amounts of GnRH in vitro. Thus, the effects of hyperPRL on pituitary responsiveness appear to be minimal, especially if the pituitary is exposed to an adequate GnRH stimulus. The suppression of basal LH secretion in vivo most likely reflects inadequate endogenous GnRH secretion. The greatly reduced LH responses after electrical stimulation in hyperprolactinemic males exposed to prolactin suggest further that hyperPRL suppresses GnRH secretion.     

Direct pituitary effects of estradiol and progesterone on luteinizing hormone release, stores, and subunit messenger ribonucleic acids

To determine the direct, chronic actions of progesterone (P4) and estrogen (estradiol, E2) on anterior pituitary synthesis and release of LH, 24 western range ewes underwent hypothalamic-pituitary disconnection (HPD) and ovariectomy (OVX) during the breeding season and were pulsed with exogenous GnRH with or without steroid replacement. Sequential blood samples were collected before infusion of GnRH and on Days 7 and 14 of GnRH infusion. Silastic capsules of P4 and/or E2 were implanted s.c. on Day 7 and remained in place throughout the experiment. Control ewes received only GnRH infusion. Blood sampling was centered around three exogenous GnRH pulses. After the final blood sampling, pituitaries were collected and stored at -70 degrees C. Concentrations of LH in serum and pituitaries were determined by RIA. Relative concentrations of LH subunit mRNAs were determined by Fast Blot analysis. Simultaneous implantation of P4 and E2 lowered LH pulse amplitude 70% and mean serum levels 30% compared with controls. Neither steroid alone affected LH release. E2 alone or in combination with P4 lowered LH-beta subunit mRNA concentrations 40% compared with controls while alpha-subunit levels were unchanged. Only E2 alone altered the pituitary content of LH, causing a 60% decrease. We conclude that the combination of P4 and E2 is necessary for inhibition of GnRH-stimulated LH secretion. E2 inhibits GnRH-stimulated LH-beta subunit mRNA concentrations but does not affect alpha-subunit mRNA concentrations. The control of pituitary LH content by P4 and E2 is the result of changes in both LH-beta subunit mRNA concentrations and LH secretion.     

Stimulatory and inhibitory effects of progesterone on FSH secretion by the anterior pituitary.

The purpose of this study was to investigate whether progesterone exerted progesterone receptor mediated direct effects on the anterior pituitary in the secretion of FSH and whether such effects were mediated through the 5 alpha-reduction of progesterone. Treatment of anterior pituitary dispersed cells for 48 h with 0.5 nM estradiol reduced the ED50 for gonadotropin releasing hormone (GnRH)-stimulated FSH release from 0.58 to 0.36 ng/ml and the ED50 for GnRH-induced LH release from 0.54 to 0.19 ng/ml. When dispersed pituitary cells were treated with 0.5 nM estradiol and exposed to various doses of progesterone for 1 to 6 h, the most consistent rise in basal and GnRH-stimulated FSH release was observed with the 50 nM dose of progesterone with a 3-h exposure period. All three doses of progesterone elevated basal LH and GnRH-stimulated LH was increased by the 50 and 100 nM doses of progesterone during the 3-h period of treatment. Using the 50 nM dose of progesterone, basal and GnRH-stimulated LH was increased after 2, 3 and 6 h of progesterone treatment. When the period of exposure of progesterone was extended to 12, 36 or 48 h, there was a significant inhibition of GnRH-stimulated FSH release. GnRH-stimulated LH release was inhibited at 36 and 48 but not 12 h after progesterone treatment. These studies showed that the effect of progesterone administered for periods of 1 to 6 h enhanced the secretion of LH and FSH whereas progesterone administered for periods beyond 12 h inhibited FSH and LH release by dispersed pituitary cells in culture. These results are similar to those observed in vivo after progesterone treatment. Furthermore estrogen priming of the dispersed pituitary cells was necessary to observe the effects of progesterone. The progesterone antagonist RU486 prevented the progesterone-induced rise in GnRH-stimulated FSH release. Furthermore the 5 alpha-reductase inhibitor N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane- 17 beta-carboxamide also prevented the progesterone-induced rise in GnRH-stimulated FSH release in estrogen-treated dispersed pituitary cells. These results indicate that the anterior pituitary is a major site of action of progesterone in the release of FSH and that 5 alpha-reduction of progesterone plays an important role in FSH release.     

Long-term superfusion of rat pituitary cells: interaction of 17 beta-estradiol with pulses of gonadotropin-releasing hormone on luteinizing hormone release

In a series of four experiments, the temporal development of acute inhibitory and delayed stimulatory effects of 17 beta-estradiol (E) on luteinizing hormone (LH) release by superfused rat anterior pituitary cells pulsed with gonadotropin-releasing hormone (GnRH) was studied. Dispersed anterior pituitary cells from ovariectomized rats were cultured on Bio-Beads for 3 days and then placed in columns and superfused for up to 24 hr. During superfusion, the cells were exposed to GnRH pulses (3 X 10(-9) M, one 6-min pulse/hr). Cells treated with E (3 X 10(-10) M) either before (only 24 hr prior to superfusion) or before and during superfusion released significantly (P less than 0.05) more LH in response to the first few pulses of GnRH than cells treated with diluent. In contrast, cells treated with E only during superfusion initially released less GnRH-induced LH than cells treated with diluent. In a subsequent experiment, the inhibitory effect of E reached a maximum by 1.5 hr (P less than 0.01), and then gradually disappeared after 4.5 hr. Cells superfused simultaneously with E and fixed "low"-dose GnRH (5 X 10(-10) M) pulses did not exhibit enhanced LH responses with time to that dose of GnRH. However, E-superfused cells responded more than diluent-superfused cells to subsequent stimulation with a higher-dose GnRH pulse. Superfusion of cells with E for 16.5 hr in the absence of GnRH pulses also did not increase release of LH to low-dose (5 X 10(-10) M) pulses of GnRH, yet did cause a transitory increase to subsequent high-dose (10(-8) M) GnRH pulses. In conclusion, these results demonstrate the direct biphasic inhibitory then stimulatory effects of E on GnRH-induced LH release by superfused rat anterior pituitary cells. Expression of the stimulatory effect of E is related to the dose of GnRH.     

Independent actions of gonadotropin releasing hormone upon cyclic GMP production and luteinizing hormone release

Gonadotropin releasing hormone (GnRH) and its potent analog [D-Ser(tBu)6]des-Gly10-GnRH N-ethylamide elevate pituitary cyclic GMP levels while stimulating gonadotropin release in cultured pituitary cells. Addition of mycophenolic acid to pituitary cell cultures decreased basal and GnRH-induced cGMP production to undetectable levels, but did not reduce basal or GnRH-stimulated luteinizing hormone (LH) release. Elevation of endogenous cGMP levels by sodium nitroprusside, or addition of cGMP or its potent derivatives, was also without effect on basal or GnRH-stimulated LH release. These findings demonstrate that the elevation of intracellular cGMP during GnRH action does not mediate the release of LH by pituitary cells.     

Steroidal modulation of in vitro gonadotropin secretion in prepubertal and adult male Siberian hamsters

The effects of exogenous gonadal steroids, testosterone (T), and 17beta-estradiol (E(2)) upon the hypothalamo-pituitary-gonadal axis were reported to be different between prepubertal and adult Siberian hamsters. Utilizing an in vitro static culture system, we investigated if age-related differences in steroid responsiveness occurs at the pituitary. Prepubertal (20 days old) or adult (140 days old) male Siberian hamsters were implanted with 1 mm silastic capsules containing undiluted T, E(2) or cholesterol (Ch, control). After 15 days, pituitaries were removed, incubated in vitro, and subjected to the following treatments: two baseline measurements, one challenge with 10ng/ml of D-Lys(6)-gonadotropin-releasing hormone (GnRH), and three post-challenge washes. Fractions were collected every 30 minutes and measured for follicle-stimulating hormone (FSH) and luteinizing hormone (LH). T and E(2 )reduced basal secretion of LH and FSH in juveniles but not adults. In juveniles, E(2) increased GnRH-induced FSH and LH secretion, while T augmented GnRH-induced FSH secretion but attenuated GnRH-induced LH secretion. Steroid treatment had no effect on GnRH-stimulated LH or FSH release in adults. The only effect of steroid hormones upon adult pituitaries was the more rapid return of gonadotropin secretion to baseline levels following a GnRH challenge. These data suggest both basal and GnRH-induced gonadotropin secretion are more sensitive to steroid treatment in juvenile hamsters than adults. Further, differential steroidal regulation of FSH and LH at the level of the pituitary in juveniles might be a mechanism for the change in sensitivity to the negative effects of steroid hormones that occurs during the pubertal transition.     

Contraceptive progestins and gonadotropin secretion in vitro

In an in vitro bioassay using rat pituitary cell cultures the effect of contraceptive progestins was tested on basal and gonadotropin-releasing hormone (GnRH)-induced luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion in vitro. Progestins diminished gonadotropin release in pituitary cells stimulated with GnRH, but did not alter basal values. This inhibitory effect was dose dependent in a range of 10(-10)-10(-5) M tested and the inhibitory action of most of the progestins examined was more potent than that of progesterone. The maximal reduction of LH and FSH values was by 60% of GnRH-induced control levels. Progestins also caused a shift in sensitivity of cells to GnRh (10(-12)-10(-6) M). When time dependence was investigated, some progestins potentiated GnRH effect on gonadotropins in pituitary cell cultures pre-incubated for a short time (4 h) with steroids. More prolonged pre-incubations from 23 to 71 h resulted in a progressive suppression of LH and FSH response to GnRH (10(-7) M). In order to examine intracellular effects, cells were pretreated with progestins and inositol phosphate metabolism was investigated. The data obtained in pituitary cells give evidence that polyphosphoinositide breakdown is potentially an early step in the action of GnRH on gonadotropin secretion by providing diacylglycerol and inositol phosphates. Addition of gonadotropin-releasing hormone to myo-2[3H]inositol-prelabeled rat pituitary cells in primary culture evoked a dose-dependent increase of the accumulation of [3H]inositol phosphates with a rise of inositol triphosphate, inositol diphosphate and inositol monophosphate within 1 min. Using one contraceptive progestin, gestoden, inositol phosphate production was inhibited by 80% compared to controls of GnRH-treated cells without the addition of steroids. The data obtained in this study suggest that this in vitro bioassay using rat pituitary cells is a useful tool in testing progestational compounds regarding their potency on gonadotropin release. In addition, these results show that one possible site of interference of progestins with GnRH-induced gonadotropin secretion may involve polyphosphoinositide breakdown.