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.     

Dependence of GnRH action on Na+, K+, and Ca2+ in the frog, Rana pipiens, pituitary

The roles of K+, Ca2+, and Na+ ions in the mechanism of gonadotropin releasing hormone (GnRH) action on frog (Rana pipiens) hemipituitaries were studied using an in vitro superfusion system. The effects of elevated K+ alone or in combination with Ca2+-depleted medium, tetrodotoxin (TTX), or with 100 ng/ml GnRH were examined. The involvement of K+ was also studied indirectly through the use of tetraethyl ammonium chloride (TEA). The importance of Ca2+ was established by the loss of responsiveness to GnRH in Ca2+-depleted medium, or in the presence of the Ca2+ competitor CoCl2. The absence of a major dependence of GnRH on Na+ was revealed by the continued gonadotropin secretion after addition of 1 microM TTX to medium containing GnRH or 36.3 mM KCl, or by replacement of NaCL with choline chloride. High (10 X normal) KCl (36.3 mM) stimulated gonadotropin--both LH and FSH--secretion, but the response was more gradual than for GnRH. The inclusion of TEA (to block K+ efflux) in medium with GnRH accentuated the effect of GnRH, and the effects of elevated (36.3 mM) KCl and 100 ng/ml GnRH (a relatively high dose) were additive. Responses to high K+, like GnRH, were abolished by removal of Ca2+ from the medium. Overall, the roles of K+, Ca2+, and Na+ ions in the mechanism of GnRH action are very similar between mammals and frogs; Ca2+ apparently serves a critical function in the mechanism of GnRH action, while Na+ appears not to be involved. K+ can induce gonadotropin secretion, but it is not clear that it plays a direct role in the mediation of the action of GnRH.     

Mechanisms of secretory responses to gonadotropin-releasing hormone and phorbol esters in cultured pituitary cells. Participation of protein kinase C and extracellular calcium mobilization

The role of protein kinase C in luteinizing hormone (LH) release was analyzed in studies on the actions of gonadotropin releasing hormone (GnRH) and phorbol esters in cultured pituitary cells. During incubation in normal medium, GnRH stimulated LH release with an ED50 of 0.35 nM. Incubation in Ca2+-deficient medium (Ca2+-free, 10 microM) substantially decreased but did not abolish the LH responses to GnRH. The extracellular Ca2+-dependent component of GnRH action could be mimicked by high K+ concentrations, consistent with the presence of voltage-sensitive calcium channels (VSCC) in pituitary gonadotrophs. Ca2+ channel agonist (Bay K 8644) and antagonist (nifedipine) analogs, respectively, enhanced or partially inhibited LH responses to GnRH and also to K+, the latter confirming the participation of two types of VSCC (dihydropyridine-sensitive and -insensitive) in K+-induced secretion. Phorbol esters, including 12-O-tetradecanoylphorbol-13-acetate (TPA), 4 beta-phorbol-12,13-dibenzoate, and 4 beta-phorbol-12,13-diacetate, stimulated LH release with ED50s of 5, 10, and 1000 nM, respectively, and with about 70% of the efficacy of GnRH. Phorbol ester-stimulated LH secretion was decreased but not abolished by progressive reduction of [Ca2+]e in the incubation medium, and the residual LH response was identical with that elicited by GnRH in Ca2+-deficient medium. TPA increased [Ca2+]i to a peak after 20 s in normal medium but not in the absence of extracellular Ca2+, indicating that protein kinase C (Ca2+/phospholipid-dependent enzyme) promotes calcium entry but can also mediate secretory responses without changes in calcium influx and [Ca2+]i. The extracellular Ca2+-dependent action of TPA on LH release was blocked by Co2+. However, nifedipine did not alter TPA action on [Ca2+]i and LH release. These observations indicate that protein kinase C can participate in GnRH-induced LH release that is independent of Ca2+ entry, but also promotes the influx of extracellular Ca2+ through dihydropyridine-insensitive Ca2+-channels.     

Evidence for a role of protein kinase C in luteinizing hormone synthesis and secretion. Impaired responses to gonadotropin-releasing hormone in protein kinase C-depleted pituitary cells

The role of protein kinase C in luteinizing hormone (LH) release was analyzed in studies on the actions of phorbol esters and gonadotropin-releasing hormone (GnRH) in normal and protein kinase C (Ca2+/phospholipid-dependent enzyme)-depleted pituitary cell cultures. LH secretory responses of normal pituitary cells to GnRH were reduced but not abolished in Ca2+-deficient medium, consistent with the existence of extracellular Ca2+-dependent and -independent components of GnRH action. Both of these components could be elicited by treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). The LH secretory responses to TPA and GnRH were additive only at low doses and converged to a common maximum at high concentrations of the agonists in the presence or absence of extracellular Ca2+. The release of stored LH by GnRH and TPA was accompanied by secretion of newly synthesized LH from 2 to 5 h during stimulation by either of the agonists. LH synthesis was increased in a progressive and dose-dependent manner by GnRH and TPA, and the ratio between newly synthesized and released hormone was near 1:2. TPA caused rapid and complete translocation of cytosolic protein kinase C to the particulate fraction of pituitary cells, followed by a progressive decrease in total enzyme content to approximately 10% after 6 h. Partial recovery of the cytosolic enzyme (to 20%) occurred after washing and reincubation for 15 h. Such kinase C-depleted cells showed prominent, dose-dependent reductions in the actions of GnRH and TPA on LH release and synthesis in both normal and Ca2+-deficient media. These observations support the hypothesis that protein kinase C participates in LH biosynthesis and secretion in pituitary gonadotrophs and is involved in the actions of GnRH upon these processes.     

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.     

Feedback effects of ovarian steroids on the hypothalamic-hypophyseal axis in the rabbit

The feedback effects of two ovarian steroids, estradiol-17 beta (E2) and 20 alpha-hydroxypregn-4-en-3-one (20 alpha OH), were examined in both intact (INT) and ovariectomized (OVEX) does. We measured steroid-induced alterations in endogenous gonadotropin-releasing hormone (GnRH) from sequential 10-min samples of hypothalamic perfusates, simultaneous changes in peripheral plasma luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and the modification of pituitary responsiveness, i.e., increments in plasma LH (delta LH) and plasma FSH (delta FSH), after 50 ng, 250 ng, and 1 microgram of exogenous GnRH in individual does of 6 treatment groups. The groups were: INT does, OVEX does, OVEX does receiving either one (1 E2) or two (2 E2) E2-filled Silastic capsules, OVEX does receiving a 20 alpha OH-filled capsule (20 alpha OH), and OVEX does receiving both capsules of E2 and 20 alpha OH (1 E2 + 20 alpha OH). Ovariectomy enhanced the pulsatile release of hypothalamic GnRH and pituitary LH and FSH, and increased the LH response (delta LH) to exogenous GnRH (OVEX vs. INT, p less than 0.05). Replacement of E2 at the time of ovariectomy prevented the increased GnRH and gonadotropin secretion as well as the enhanced delta LH that were observed in untreated OVEX does. The release of hypothalamic GnRH in the 20 alpha OH group was lower (p less than 0.05) than that in the OVEX group and not different from that in the INT group. The release of pituitary LH and FSH and the delta LH in the 20 alpha OH group was not different from that in the OVEX group, but these parameters were greater (p less than 0.05) than those in the INT group. The hypothalamic GnRH pulse frequency in the 1 E2 + 20 alpha OH group was lower (p less than 0.05) than that in either the 1 E2 or the 20 alpha OH group, but the delta LH in the 1 E2 + 20 alpha OH group was not different from that in either the 1 E2 or the 20 alpha OH group. The highest dose (1 microgram) of exogenous GnRH stimulated a modest increase in FSH in the OVEX, 20 alpha OH, 1 E2 + 20 alpha OH, and 1 E2 groups; but a steroid effect on delta FSH among these 4 groups was not apparent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biphasic activation of cytosolic free calcium and LH responses by gonadotropin-releasing hormone

Gonadotropin-releasing hormone (GnRH) stimulates rapid peak increases in [Ca2+]i and LH release, followed by lower but sustained elevations of both [Ca2+]i and hormone secretion. Omission of extracellular Ca2+ only slightly decreased the peak of [Ca2+]i, but reduced the peak LH response by 40% and prevented the prolonged increases in [Ca2+]i and LH release. Dihydropyridine calcium antagonists did not affect the peak [Ca2+]i and LH responses, but reduced the sustained increases by up to 50%. Whereas GnRH-induced mobilization of intracellular calcium initiates the LH peak, and Ca2+ entry through dihydropyridine-insensitive channels contributes to the peak and plateau phases of LH release, dihydropyridine-sensitive L-type Ca2+ channels participate only in the sustained phase of gonadotropin secretion.     

A Mathematical Model for the Actions of Activin,Inhibin, and Follistatin on Pituitary Gonadotrophs

The timed secretion of the luteinizing hormone (LH) and follicle stimulating hormone (FSH) from pituitary gonadotrophs during the estrous cycle is crucial for normal reproductive functioning. The release of LH and FSH is stimulated by gonadotropin releasing hormone (GnRH) secreted by hypothalamic GnRH neurons. It is controlled by the frequency of the GnRH signal that varies during the estrous cycle. Curiously, the secretion of LH and FSH is differentially regulated by the frequency of GnRH pulses. LH secretion increases as the frequency increases within a physiological range, and FSH secretion shows a biphasic response, with a peak at a lower frequency. There is considerable experimental evidence that one key factor in these differential responses is the autocrine/paracrine actions of the pituitary polypeptides activin and follistatin. Based on these data, we develop a mathematical model that incorporates the dynamics of these polypeptides. We show that a model that incorporates the actions of activin and follistatin is sufficient to generate the differential responses of LH and FSH secretion to changes in the frequency of GnRH pulses. In addition, it shows that the actions of these polypeptides, along with the ovarian polypeptide inhibin and the estrogen-mediated variations in the frequency of GnRH pulses, are sufficient to account for the time courses of LH and FSH plasma levels during the rat estrous cycle. That is, a single peak of LH on the afternoon of proestrus and a double peak of FSH on proestrus and early estrus. We also use the model to identify which regulation pathways are indispensable for the differential regulation of LH and FSH and their time courses during the estrous cycle. We conclude that the actions of activin, inhibin, and follistatin are consistent with LH/FSH secretion patterns, and likely complement other factors in the production of the characteristic secretion patterns in female rats.     

Calcium mobilization and influx during the biphasic cytosolic calcium and secretory responses in agonist-stimulated pituitary gonadotrophs

Stimulation of enriched pituitary gonadotrophs by gonadotropin-releasing hormone (GnRH) elicits dose-dependent biphasic elevations of cytosolic calcium ([Ca2+]i) and luteinizing hormone (LH) release, with rapid initial peaks followed by sustained plateaus during continued exposure to the agonist. A potent GnRH-antagonist, [N-acetyl-D-p-Cl-Phe1,2,D-Trp3,D-Lys6,D-Ala10]GnRH, prevented the biphasic [Ca2+]i and LH responses when added before GnRH, and rapidly abolished both responses to GnRH when added during the plateau phase. In low Ca2+ medium the LH peak responses to GnRH were reduced and the subsequent sustained responses were almost completely abolished; reduction of extracellular Ca2+ during exposure to GnRH caused a prompt decline of LH release. The initial [Ca2+]i peak is derived largely from intracellular calcium mobilization with a partial contribution from calcium influx, while the sustained phase is dependent on the entry of extracellular Ca2+ through both L-type and dihydropyridine-insensitive channels. The presence of L-type voltage-sensitive calcium channels (VSCC) in pituitary gonadotrophs was indicated by the ability of elevated extracellular [K+] to stimulate calcium influx and LH release, and the sensitivity of these responses to dihydropyridine agonist and antagonist analogs. In cells pretreated with high [K+], the peak [Ca2+]i response to GnRH was enhanced but the subsequent plateau phase was markedly attenuated. This divergent effect of sustained membrane depolarization on the biphasic [Ca2+]i response suggests that calcium entry through VSCC initially potentiates agonist-induced mobilization of Ca2+ from intracellular storage sites. However, established Ca2+ entry through depolarization-activated VSCC cannot be further increased by agonist stimulation because both processes operate through the same channels, probably by changes in their activation-inactivation kinetics. Finally, the reciprocal potentiation by the dihydropyridine agonist, BK 8644, and GnRH of [Ca2+]i and LH responses confirms that both compounds act on the same type of channels, i.e., L-type VSCC, that participate in agonist-mediated calcium influx and gonadotropin secretion.     

Metformin decreases GnRH- and activin-induced gonadotropin secretion in rat pituitary cells: potential involvement of adenosine 5' monophosphate-activated protein kinase (PRKA)

Metformin is an insulin sensitizer molecule used for the treatment of infertility in women with polycystic ovary syndrome and insulin resistance. It modulates the reproductive axis, affecting the release of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH). However, metformin's mechanism of action in pituitary gonadotropin-secreting cells remains unclear. Adenosine 5' monophosphate-activated protein kinase (PRKA) is involved in metformin action in various cell types. Here, we investigated the effects of metformin on gonadotropin secretion in response to activin and GnRH in primary rat pituitary cells (PRP), and studied PRKA in rat pituitary. In PRP, metformin (10 mM) reduced LH and follicle-stimulating hormone (FSH) secretion induced by GnRH (10(-8) M, 3 h), FSH secretion, and mRNA FSHbeta subunit expression induced by activin (10(-8) M, 12 or 24 h). The different subunits of PRKA are expressed in pituitary. In particular, PRKAA1 is detected mainly in gonadotrophs and thyrotrophs, is less abundant in lactotrophs and somatotrophs, and is undetectable in corticotrophs. In PRP, metformin increased phosphorylation of both PRKA and acetyl-CoA carboxylase. Metformin decreased activin-induced SMAD2 phosphorylation and GnRH-induced mitogen-activated protein kinase (MAPK) 3/1 (ERK1/2) phosphorylation. The PRKA inhibitor compound C abolished the effects of metformin on gonadotropin release induced by GnRH and on FSH secretion and Fshb mRNA induced by activin. The adenovirus-mediated production of dominant negative PRKA abolished the effects of metformin on the FSHbeta subunit mRNA and SMAD2 phosphorylation induced by activin and on the MAPK3/1 phosphorylation induced by GnRH. Thus, in rat pituitary cells, metformin decreases gonadotropin secretion and MAPK3/1 phosphorylation induced by GnRH and FSH release, FSHbeta subunit expression, and SMAD2 phosphorylation induced by activin through PRKA activation.     

Active immunization of gilts against gonadotropin-releasing hormone: effects on secretion of gonadotropins, reproductive function, and responses to agonists of gonadotropin-releasing hormone

Sexually mature gilts were actively immunized against gonadotropin-releasing hormone (GnRH) by conjugating GnRH to bovine serum albumin, emulsifying the conjugate in Freund's adjuvant, and giving the emulsion as a primary immunization at Week 0 and as booster immunizations at Weeks 10 and 14. Antibody titers were evident by 2 wk after primary immunization and increased markedly in response to booster immunizations. Active immunization against GnRH caused gonadotropins to decline to nondetectable levels, gonadal steroids to decline to basal levels, and the gilts to become acyclic. Prolactin concentrations in peripheral circulation were unaffected by immunization against GnRH. The endocrine status of the hypothalamic-pituitary-ovarian axis was examined by giving GnRH and two agonists to GnRH and by ovariectomy. An i.v. injection of 100 micrograms GnRH caused release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in control animals, but not in gilts immunized against GnRH. In contrast, administration of 5 micrograms D-(Ala6, des-Gly-NH2(10] ethylamide or 5 micrograms D-(Ser-t-But6, des-Gly-NH2(10] ethylamide resulted in immediate release of LH and FSH in both control and GnRH-immunized gilts. Circulating concentrations of LH and FSH increased after ovariectomy in the controls, but remained at nondetectable levels in gilts immunized against GnRH. Prolactin concentrations did not change in response to ovariectomy. We conclude that cyclic gilts can be actively immunized against GnRH and that this causes cessation of estrous cycles and inhibits secretion of LH, FSH, and gonadal steroids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of gonadotropin-releasing hormone pulse-frequency modulation on luteinizing hormone, follicle-stimulating hormone and testosterone secretion in hypothalamo/pituitary-disconnected rams

The effects of changes in pulse frequency of exogenously infused gonadotropin-releasing hormone (GnRH) were investigated in 6 adult surgically hypothalamo/pituitary-disconnected (HPD) gonadal-intact rams. Ten-minute sampling in 16 normal animals prior to HPD showed endogenous luteinizing hormone (LH) pulses occurring every 2.3 h with a mean pulse amplitude of 1.11 +/- 0.06 (SEM) ng/ml. Mean testosterone and follicle-stimulating hormone (FSH) concentrations were 3.0 +/- 0.14 ng/ml and 0.85 +/- 0.10 ng/ml, respectively. Before HPD, increasing single doses of GnRH (50-500 ng) elicited a dose-dependent rise of LH, 50 ng producing a response of similar amplitude to those of spontaneous LH pulses. The effects of varying the pulse frequency of a 100-ng GnRH dose weekly was investigated in 6 HPD animals; the pulse intervals explored were those at 1, 2, and 4 h. The pulsatile GnRH treatment was commenced 2-6 days after HPD when plasma testosterone concentrations were in the castrate range (less than 0.5 ng/ml) in all animals. Pulsatile LH and testosterone secretion was reestablished in all animals in the first 7 days by 2-h GnRH pulses, but the maximal pulse amplitudes of both hormones were only 50 and 62%, respectively, of endogenous pulses in the pre-HPD state. The plasma FSH pattern was nonpulsatile and FSH concentrations gradually increased in the first 7 days, although not to the pre-HPD range. Increasing GnRH pulse frequency from 2- to 1-hour immediately increased the LH baseline and pulse amplitude. As testosterone concentrations increased, the LH responses declined in a reciprocal fashion between Days 2 and 7. FSH concentration decreased gradually over the 7 days at the 1-h pulse frequency. Slowing the GnRH pulse to a 4-h frequency produced a progressive fall in testosterone concentrations, even though LH baselines were unchanged and LH pulse amplitudes increased transiently. FSH concentrations were unaltered during the 4-h regime. These results show that 1) the pulsatile pattern of LH and testosterone secretion in HPD rams can be reestablished by exogenous GnRH, 2) the magnitude of LH, FSH, and testosterone secretion were not fully restored to pre-HPD levels by the GnRH dose of 100 ng per pulse, and 3) changes in GnRH pulse frequency alone can influence both gonadotropin and testosterone secretion in the HPD model.     

Actions of 17 beta-estradiol on gonadotropin release induced by drugs that activate intracellular signal transduction mechanisms in rat anterior pituitary cells

We studied the effects of 17 beta-estradiol (E2) on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release induced by drugs that activate different intracellular signal transduction mechanisms in rat anterior pituitary cells. Cells were pretreated with E2 (6 x 10(-10) M) or diluent for 24 h. Then, both E2- and diluent-pretreated cells were incubated for 4 h with E2 or diluent, respectively, with or without drugs, and in the presence or absence of gonadotropin-releasing hormone (GnRH). Media were assayed for LH and FSH by radioimmunoassays. E2 treatment had no effect on basal FSH release, but occasionally stimulated basal LH release. Phospholipase C (PLC), L-alpha-1,2-dioctanoyl glycerol (C8), veratridine, 8-bromo-cyclic adenosine 3',5'-monophosphate (8-Br-cAMP), melittin (a phospholipase A2 [PLA2] activator), arachidonic acid, PLA2, and GnRH all stimulated LH and FSH release in both E2- and diluent-treated cells. E2 treatment increased both LH and FSH release induced by GnRH, PLC, C8, veratridine, and 8-Br-cAMP, but not by melittin, arachidonic acid, and PLA2. Neither C8, PLA2, nor arachidonic acid in combination with a maximal dose of GnRH had additive effects on either LH or FSH release, whereas melittin increased the maximal response to GnRH in both E2- and diluent-treated cells. The effects of veratridine and 8-Br-cAMP depended on dose of GnRH and presence or absence of E2. These results suggest that E2 augments stimulus-coupled gonadotropin release by interacting with the Ca2+-, and/or diacylglycerol-, and cAMP-activated pathways, but not with the arachidonic acid-activated pathway.     

Effect of treatment with cortisol in vivo on secretion of gonadotropins in vitro

To investigate the site of action of glucocorticoids in modulating secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from pituitaries of male rats, we implanted intact male rats with 250-mg pellets of cortisol (F) or cholesterol (C). Four days later, we collected and enzymatically dispersed the pituitaries. After the dispersed pituitaries had been in culture for 2 days, we treated the cells with gonadotropin-releasing hormone (GnRH) (0-150 nM) and determined the concentrations of LH and FSH in the medium after 6 h of incubation. Cells from donor animals pretreated with F secreted 30-60% more LH approximately 75% more FSH than cells from donor animals pretreated with C. This increase occurred regardless of the presence of F or C in the incubation medium in vitro. The slopes and ED50s of the GnRH dose-response curves were not altered. These data show that glucocorticoids have stimulatory effects on both LH and FSH. The inhibitory effects observed in vivo must be exerted by some mechanism that is not carried over to the in vitro model, and perhaps involve sites of action in addition to the pituitary.     

Similar effects of inhibin and cycloheximide on gonadotropin release in superfused pituitary cell cultures

The actions of two inhibin preparations and cycloheximide on gonadotropin release were investigated in superfused pituitary cell cultures. Pituitary cells isolated from 18-day-old male rats were grown in Matrigel-coated superfusion chambers in chemically defined medium. After stationary culture for 4 days, the cell monolayers were superfused at a constant speed (0.25 ml/min) and were intermittently stimulated (6 min/h) with 10 nM gonadotropin-releasing hormone (GnRH). Groups of cultures were exposed to the test substances for varying time periods during stationary culture and/or during superfusion. Inhibitory effects of both inhibin preparations on the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in response to GnRH pulses were observed after 2 h of exposure and became maximal after about 6 h. Basal secretion of FSH between GnRH pulses was also suppressed, whereas the basal interpulse secretion of LH was not changed. When exposure to inhibin was discontinued, the secretion of both FSH and LH progressively increased and returned to control values by approximately 6 h. Cycloheximide (500 ng/ml) affected gonadotropin release with dynamics similar to those observed for the inhibin preparation. These data support the hypothesis that inhibition of gonadotropin synthesis may be an important step in the molecular mechanism of action by which inhibin regulates gonadotropin release.     

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.     

Phorbol ester-induced LH release in pituitary gonadotrophs: effects of antagonists of calmodulin and GnRH.

The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent activator of Ca(2+)- and phospholipid-dependent protein kinase (C kinase), stimulates luteinizing hormone (LH) release from rat pituitary cells. The actions of TPA upon LH release were compared with those of the GnRH superagonist [D-Ala6] des-Gly10-GnRH N-ethylamide (GnRHa) in cultured pituitary cells. LH release was stimulated by 0.1 nM TPA and the maximum response at 10 nM TPA was 50% of the LH response to GnRHa. The ED50 values for TPA and GnRHa were 1.2 and 0.037 nM, respectively, and the maximum stimulatory effects of TPA and GnRHa on LH release were not additive. GnRHa-stimulated LH release was decreased by calmodulin (CaM) antagonists including pimozide, trifluoperazine, W5 and W7, being most effectively reduced (by 70%) by 10 microM pimozide. In contrast to their inhibition of GnRH action, these antagonists enhanced TPA-stimulated LH release, so that 10 microM pimozide and W7 doubled the maximum LH response. The potent GnRH antagonist [Ac-D-p-Cl-Phe1.2, D-Trp3, D-Lys6, D-Ala10]GnRH, which completely inhibited GnRHa-stimulated LH release with ID50 of 6.8 nM, also reduced maximum TPA-stimulated LH release by about 50%. These results suggest that both Ca2+/CaM and C kinase pathways are involved in the LH release mechanism, and indicate that C kinase plays a major role in the action of GnRH upon gonadotropin secretion. The synergism between CaM antagonists and TPA suggests that blockade of CaM-mediated processes leads to enhanced activation of the C kinase pathway, possibly by removal of an inhibitory influence. Furthermore, the partial inhibition of TPA-stimulated LH release by a GnRH antagonist suggests that the pathway(s), specifically connected with LH release in the diverse effects of C kinase, might be locked by the continuous receptor inactivation by antagonist and indicates the complicated pathways which diverge from the receptor and converge into specific cellular response.     

Gonadotropin secretion in ovariectomized ewes: effect of passive immunization against gonadotropin-releasing hormone (GnRH) and infusion of a GnRH agonist and estradiol

Gonadotropin secretion was examined in ovariectomized sheep after passive immunization against gonadotropin-releasing hormone (GnRH). Infusion of ovine anti-GnRH serum, but not control antiserum, rapidly depressed serum concentrations of luteinizing hormone (LH). The anti-GnRH-induced reduction in serum LH was reversed by circhoral (hourly) administration of a GnRH agonist that did not cross-react with the anti-GnRH serum. In contrast, passive immunization against GnRH led to only a modest reduction in serum concentrations of follicle-stimulating hormone (FSH). Pulsatile delivery of the GnRH agonist did not influence serum concentrations of FSH. Continuous infusion of estradiol inhibited and then stimulated gonadotropin secretion in animals passively immunized against GnRH, with gonadotrope function driven by GnRH agonist. However, the magnitude of the positive feedback response was only 10% of the response noted in controls. These data indicate that the estradiol-induced surge of LH secretion in ovariectomized sheep is the product of estrogenic action at both hypothalamic and pituitary loci. Replacement of the endogenous GnRH pulse generator with an exogenous generator of GnRH-like pulses that were invariant in frequency and amplitude could not fully reestablish the preovulatory-like surge of LH induced by estradiol.     

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.     

Evidence that gonadotropin-releasing hormone (GnRH) II stimulates luteinizing hormone and follicle-stimulating hormone secretion from monkey pituitary cultures by activating the GnRH I receptor

Mammalian gonadotropin-releasing hormone (GnRH) I is the neuropeptide that regulates reproduction. In recent years, a second isoform of GnRH, GnRH II, and its highly selective type II GnRH receptor were cloned and identified in monkey brain, but its physiological function remains unknown. We sought to determine whether GnRH II stimulates LH and FSH secretion by activating specific receptors in primary pituitary cultures from male monkeys. Dispersed pituitary cells were maintained in steroid-depleted media and stimulated with GnRH I and/or GnRH II for 6 h. Cells were also treated with Antide (Bachem, King of Prussia, PA), a GnRH I antagonist, to block gonadotropin secretion. In monkey as well as rat pituitary cultures, GnRH II was a less effective stimulator of LH and FSH secretion than was GnRH I. In both cell preparations, Antide completely blocked LH and FSH release provoked by GnRH II as well as GnRH I. Furthermore, the combination of GnRH I and GnRH II was no more effective than either agonist alone. These results indicate that GnRH II stimulates FSH and LH secretion, but they also imply that this action occurs through the GnRH I receptor. The GnRH II receptors may have a unique function in the monkey brain and pituitary other than regulation of gonadotropin secretion.