Gonadotropin-releasing hormone receptor expression in Xenopus oocytes

The rodent GnRH receptor was characterized in Xenopus oocytes injected with RNA isolated from rat pituitary and from a gonadotrope cell line, alpha T3, derived from a transgenic mouse. Three to 4 days after 150-200 ng RNA injection, 93% of the oocytes, which were recorded by voltage clamp, responded to 10(-7) M GnRH. The mean inward currents obtained after RNA injection were 620 +/- 88 nA (n = 22) with pituitary RNA and 1415 +/- 598 (n = 4) with alpha T3 RNA. The threshold GnRH concentration able to evoke the dose dependent current after pituitary RNA injection was 3 x 10(-9) M GnRH. The GnRH receptor response of the oocyte was antagonized by [D-Phe2,6,Pro3] GnRH and [N-Ac-D-Na](2)1, D-alpha D-Me, pCl-Phe2, D-Arg6, D-Ala10-NH2]GnRH and could be elicited by D-Ser(But)6,Pro9-N-ethylamide GnRH (buserelin). The reversal potential of the GnRH generated current as determined by voltage-ramp was -22.5 +/- 1.0 mV (n = 7) and -25.6 +/- 3.3 mV (n = 3) in pituitary and cell line RNA-injected oocytes respectively, consistent with the chloride reversal potential. The GnRH receptor response was virtually eliminated by intracellular EGTA injection but was unaffected by ligand application in calcium-free perfusate. The GnRH-evoked response is mimicked by intracellular injection of inositol 1,4,5-trisphosphate. To determine the size of the GnRH receptor mRNA, alpha T3 RNA was size fractionated through a sucrose gradient. The maximal GnRH response was induced by a fraction larger than the 28S ribosomal peak. Thus we find that oocytes injected with RNA from an appropriate source develop an electrophysiological response to GnRH which is dependent on intracellular calcium mobilization, is independent of extracellular calcium, and may be mediated by inositol 1,4,5-trisphosphate.     

Modulation of gonadotropin-releasing hormone receptor concentration in cultured female rat pituitary cells by estradiol treatment

Short-term (0.5-4 h) treatment of rat pituitary cells in culture with estradiol (E2) results in a significant decrease of Gonadotropin-Releasing Hormone (GnRH) induced LH-release. We studied whether changes in the concentrations of GnRH-receptors (GnRH-R) might account for this phenomenon: pituitary cells from adult female rats were incubated for 4 or 24 h in the presence or absence of 10(-9) M E2. Then saturation curves of D-Ala6-des-Gly10-GnRH ethylamide binding were obtained. In addition, binding studies were carried out in cultures incubated for 0.5, 1, 2 or 4 h with or without 10(-9) M E2 using a near saturating concentration of GnRH-analog. No changes of GnRH-R affinity occurred (4 h experiments: Ka in vehicle treated cells: 0.94 +/- 0.2 x 10(9) M-1, Ka in E2 treated cells: 1.06 +/- 0.3 x 10(9) M-1; 24 h experiments: Ka vehicle: 0.95 +/- 0.2 x 10(9) M-1, Ka E2: 0.82 +/- 0.3 x 10(9) M-1). The GnRH-R concentrations, however, were significantly reduced (44 +/- 3%; P less than 0.001) by 4 h E2 treatment and increased (by 68 +/- 8%; P less than 0.01) by 24 h of E2 treatment. The GnRH induced LH-release in aliquots of the same cell preparations was significantly reduced after 4 h and markedly increased after 24 h of E2 treatment. The experiments on the time-course of the reduction of D-Ala6-GnRH-binding by E2 treatment showed that the number of GnRH-R was significantly decreased (24 +/- 1%; P less than 0.05) already after 0.5 h of exposure to the estrogen. This is also the time period after which the negative E2-effect on GnRH-induced LH-release becomes significant. These data provide first evidence that the short-term negative E2-effect on GnRH induced LH-release by rat pituitary cells in culture could be mediated via a reduction of available GnRH-R.     

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.     

Reversible,long-term inhibition of ovulation with a gonadotropin-releasing hormone antagonist in the marmoset monkey (Callithrix jacchus)

The effects of weekly injections of a gonadotropin-releasing hormone (GnRH) antagonist (GnRHa) ([N-acetyl-DβNal1-D-pCl-Phe2-D-Phe3-D-Arg6-Phe7-Arg8D-Ala10] NH2 GnRH) on pituitary and ovarian function were examined in the marmoset monkey, Callithrix jacchus. In experiment 1, five cyclic females were given weekly injections of vehicle (50% propylene glycol in saline) for 6 weeks followed by GnRHa for 20 weeks, animals receiving either 200 μg GnRHa/injection (n = 2) or 67 μg GnRHa/injection (n = 3) for 10 weeks, after which the treatment was reversed. Bioactive luteinizing hormone (LH) and progesterone (Po) were measured in blood samples (0.2–0.4 ml) collected twice weekly until at least 8 weeks after the last GnRHa injection. GnRHa treatment, timed to begin in the midluteal phase, caused a rapid decline in LH and Po and luteal regression after a single injection (both doses). Po levels were consistently low (<10 ng/ml), and ovulation was inhibited throughout 200 μg treatment in all animals. Short periods of elevated Po (>10 ng/ml) were, however, occasionally seen during 67 μg treatment, indicating incomplete ovarian suppression. Mean LH levels were significantly lower during GnRHa treatment compared with the period of vehicle injection (all animals 200 μg; three animals 67 μg), and there were significant differences in LH levels between GnRHa treatments (200 μg vs. 67 μg) in four animals. Four animals resumed normal ovarian cycles after the end of GnRHa treatment (15/16 days, three animals; 59 days, one animal); the fifth animal died of unknown causes 32 days after the last GnRHa injection. In a second experiment, pituitary responsiveness to exogenous GnRH was tested 1 day after a single injection of vehicle or antagonist (200 or 67 μg). Measurement of bioactive LH indicated that pituitary response to 200 ng native GnRH was significantly suppressed in animals receiving the antagonist, the degree of suppression being dose related. A third experiment examined the effect of four weekly injections of 200 μg GnRHa on follicular size and granulosa cell responsiveness to human follicle-stimulating hormone (hFSH) in vitro. Follicular development beyond 1 mm was inhibited by GnRHa treatment (preovulatory follicles normally 2-4 mm) although granulosa cell responsiveness to FSH during 48 hr of culture was not impaired. These results suggest that the GnRHa-induced suppression of follicular development and ovulation was mediated primarily by an inhibition of pituitary gonadotropin secretion and not by a direct action at the level of the ovary.     

Estrogen effects on osmotic regulation of AVP and fluid balance

To determine estrogen effects on osmotic regulation of arginine vasopressin (AVP) and body fluids, we suppressed endogenous estrogen and progesterone using the gonadotropin-releasing hormone (GnRH) analog leuprolide acetate (GnRHa). Subjects were assigned to one of two groups: 1) GnRHa alone, then GnRHa + estrogen (E, n = 9, 25 +/- 1 yr); 2) GnRHa alone, then GnRHa + estrogen with progesterone (E/P, n = 6, 26 +/- 3). During GnRHa alone and with hormone treatment, we compared AVP and body fluid regulatory responses to 3% NaCl infusion (HSI, 120 min, 0.1 ml. min(-1). kg body wt(-1)), drinking (30 min, 15 ml/kg body wt), and recovery (60 min of seated rest). Plasma [E(2)] increased from 23.9 to 275.3 pg/ml with hormone treatments. Plasma [P(4)] increased from 0.6 to 5.7 ng/ml during E/P and was unchanged (0.4 to 0.6 ng/ml) during E. Compared with GnRHa alone, E reduced osmotic AVP release threshold (275 +/- 4 to 271 +/- 4 mosmol/kg, P < 0.05), and E/P reduced the AVP increase in response during HSI (6.0 +/- 1.3 to 4.2 +/- 0.6 pg/ml at the end of HSI), but free water clearance was unaffected in either group. Relative to GnRHa, pre-HSI plasma renin activity (PRA) was greater during E (0.8 +/- 0.1 vs. 1.2 +/- 0.2 ng ANG I. ml(-1). h(-1)) but not after HSI or recovery. PRA was greater than GnRHa during E/P at baseline (1.1 +/- 0.2 vs. 2.5 +/- 0.6) and after HSI (0.6 +/- 0.1 vs. 1.1 +/- 1.1) and recovery (0.5 +/- 0.1 vs. 1.3 +/- 0.2 ng ANG I. ml(-1). h(-1)). Baseline fractional excretion of sodium was unaffected by E or E/P but was attenuated by the end of recovery for both E (3.3 +/- 0.6 vs. 2.4 +/- 0.4%) and E/P (2.8 +/- 0.4 vs 1.7 +/- 0.4%, GnRHa alone and with hormone treatment, respectively). Fluid retention increased with both hormone treatments. Renal sensitivity to AVP may be lower during E due to intrarenal effects on water and sodium excretion. E/P increased sodium retention and renin-angiotensin-aldosterone stimulation.     

Effects of the intensity of the suckling stimulus and ovarian steroids on pituitary gonadotropin-releasing hormone receptors during lactation

These studies examined whether the decrease in pituitary responsiveness to gonadotropin-releasing hormone (GnRH) observed during lactation in the rat results from a change in pituitary GnRH receptors. GnRH binding capacity was determined by saturation analysis using D-Ala6 as both ligand and tracer. During the estrous cycle, the number of GnRH binding sites increased from 199 +/- 38 fmol/mg protein on estrus to 527 +/- 31 fmol/mg protein on the morning of proestrus, whereas there was no change in receptor affinity (Ka, 6-10 X 10(9) M-1), During lactation, females nursing 8 pups on Days 5 or 10 postpartum had 50% fewer GnRH receptors (109-120 fmol/mg protein) than observed during estrus or diestrus 1 (199-242 fmol/mg protein) although receptor affinity was similar among all the groups. No deficits in pituitary GnRH receptors were observed in females nursing 2 pups on Day 10 postpartum. Removal of the 8-pup suckling stimulus for 24 or 48 h resulted in a dramatic increase in GnRH receptor capacity by 24 h from 120 +/- 16 to 355 +/- 39 fmol/mg protein. The rise in GnRH receptors after pup removal was accompanied by an increase in serum luteinizing hormone (LH) and estradiol concentrations. To assess the role of ovarian steroids in determining GnRH receptor capacity during lactation, females were ovariectomized (OVX) on Day 2 postpartum. Suckling of a large litter (8 pups) completely blocked the postcastration rise in serum LH and in pituitary GnRH receptors on Day 10 postpartum (OVX+ 8, 77 +/- 12 fmol/mg protein; OVX+ 0, 442 +/- 38 fmol/mg protein).(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional relationship between receptor binding and biological activity for analogs of mammalian and salmon gonadotropin-releasing hormones in the pituitary of goldfish (Carassius auratus)

The relationship between gonadotropin-releasing hormone (GnRH) receptor binding and biological activity in the goldfish pituitary for mammalian and salmon GnRH (sGnRH) analogs with structural modification at the C terminus involving replacement of glycine amide with an alkyl amine and replacement of the Gly6 residue with D amino acids was examined. The GnRH receptor binding data were analyzed with a computerized curve-fitting program (LIGAND) for a single as well as two classes of binding sites; analysis based on one site fit estimated binding affinity and capacity for one class of binding site, and analysis based on two-site fit estimated binding affinity and capacity for two classes of binding sites (high-affinity/low-capacity and low-affinity/high-capacity binding sites). The estimated receptor affinity values were then used to determine the correlation between binding affinity and gonadotropin (GTH)-release potency in vitro. The highest correlation between biological activity and receptor binding affinity was obtained for the high-affinity/low-capacity binding sites and GnRH analogs containing Trp7 and Leu8 residues (i.e., the salmon GnRH structural format) (R = 0.940 +/- 0.150). For the same group of GnRH analogs, there was no significant correlation between the relative GTH-release potency and binding affinity of the low-affinity/high-capacity sites (R = 0.159 +/- 0.434), or that obtained from a one-site fit (R = 0.198 +/- 0.431). Similarly, for mammalian GnRH analogs, significant correlation between binding affinity and biological activity (R = 0.406 +/- 0.049) was only obtained for the high-affinity sites, although the degree of correlation was significantly lower than that obtained for salmon GnRH analogs. The present findings provide strong support for the hypothesis that high-affinity GnRH receptors are involved in the control of GTH release in the goldfish pituitary. In addition, the results demonstrate clearly that the presence of Trp7, Leu8 residues in salmon GnRH molecule, a native peptide in goldfish, is important for recognition of the ligand by the GnRH receptors in the goldfish pituitary, and that structural modifications at positions 6 and 10 in this peptide can increase receptor binding affinity and biological activity at the pituitary level. The most active sGnRH analog identified to date is [D-Arg6, Pro9-NEt]-sGnRH.     

Binding of agonist but not antagonist leads to fluorescence resonance energy transfer between intrinsically fluorescent gonadotropin-releasing hormone receptors

We have used spot fluorescence photobleaching recovery methods to measure the lateral diffusion of GnRH receptor (GnRHR) fused at its C terminus to green fluorescent protein (GFP) after binding of either GnRH agonists or antagonist. Before ligand binding, GnRHR-GFP exhibited fast rates of lateral diffusion (D = 18 +/- 2.8 x 10(-10)cm2 x sec(-1)) and high values for fractional fluorescence recovery (%R) after photobleaching (73 +/- 1%). Increasing concentrations of agonists, GnRH or D-Ala6-GnRH, caused a dose-dependent slowing of receptor lateral diffusion as well as a decreased fraction of mobile receptors. Increasing concentrations of the GnRH antagonist Antide slowed the rate of receptor diffusion but had no effect on the fraction of mobile receptors, which remained high. To determine whether the decrease in %R caused by GnRH agonists was due, in part, to increased receptor self-association, we measured the fluorescence resonance energy transfer efficiency between GnRHR-GFP and yellow fluorescent protein-GNRHR: There was no energy transfer between GnRHR on untreated cells. Treatment of cells with GnRH agonists led to a concentration-dependent increase in the energy transfer between GnRH receptors to a maximum value of 16 +/- 1%. There was no significant energy transfer between GnRH receptors on cells treated with Antide, even at a concentration of 100 nM. These data provide direct evidence that, before binding of ligand, GnRHR exists as an isolated receptor and that binding of GnRH agonists, but not antagonist, leads to formation of large complexes that exhibit slow diffusion and contain receptors that are self-associated.     

Follicular growth, endocrine response and embryo yields in sheep superovulated with FSH after pretreatment with a single short-acting dose of GnRH antagonist

The objective of this study was to characterize follicular development, onset of oestrus and preovulatory LH surge, and in vivo embryo yields of sheep superovulated after treatment with a single dose of 1.5mg of GnRH antagonist (GnRHa). At first FSH dose, ewes treated with GnRH antagonist (n=12) showed a higher number of gonadotrophin-responsive follicles, 2-3mm, than control ewes (n=9, 13.5+/-3.8 versus 5.3+/-0.3, P<0.05). Administration of FSH increased the number of >or=4mm follicles at sponge removal in both groups (19.3+/-3.8, P<0.0005 for treated ewes and 12.7+/-5.4, P<0.01 for controls). Thereafter, a 25% of the GnRHa-treated sheep did not show oestrous behaviour whilst none control sheep failed (P=0.06). The preovulatory LH surge was detected in an 88.9% of control ewes and 66.7% of GnRHa-treated sheep. A 77.8% of control females showed ovulation with a mean of 9.6+/-0.9 CL and 3.3+/-0.7 viable embryos, while ewes treated with GnRHa and showing an LH surge exhibited a bimodal distribution of response; 50% showed no ovulatory response and 50% superovulated with a mean of 12.2+/-1.1 CL and 7.3+/-1.1 viable embryos. In conclusion, a single dose of GnRHa enhances the number of gonadotrophin-dependent follicles able to grow to preovulatory sizes in response to an FSH supply. However, LH secretion may be altered in some females, which can affect the preovulatory LH surge and/or can weak the terminal maturation of ovulatory follicles.     

Receptor-binding properties of gonadotropin-releasing hormone derivatives. Prolonged receptor occupancy and cell-surface localization of a potent antagonist analog

The receptor-binding properties and in vitro biological effects of a highly active gonadotropin-releasing hormone (GnRH) antagonist, [N-acetyl-D-p-chloro-Phe1,2D-Trp3,D-Lys6,D-Ala10]GnRH, were compared with those of the GnRH superagonist analog, [D-Ala6] des-Gly10-GnRH-N-ethylamide. In rat pituitary particles and isolated pituitary cells, the 125I-labeled GnRH antagonist showed saturable high-affinity binding (Ka v 8.4 +/- 1.4 X 10(9) M-1) to the same receptor sites which bound the GnRH agonist. The rate of dissociation of the receptor-bound antagonist from pituitary particles and cells was extremely slow in comparison with that of the agonist ligand. Also, dissociation of the antagonist analog was incomplete, with a residual fraction of tightly bound ligand that was proportional to the duration of preincubation. The [D-Lys6]GnRH antagonist prevented GnRH-induced luteinizing hormone release during static incubation and superfusion of cultured pituitary cells, but in contrast to the agonist did not cause desensitization of the gonadotroph. Although the antagonist caused a prolonged reduction in available GnRH receptor sites, this was attributable to persistent occupancy by the slowly dissociating ligand rather than to receptor loss. Autoradiographic analysis of [D-Lys6]GnRH-antagonist uptake by cultured pituitary cells revealed that the peptide remained bound at the cell membrane for up to 2 h, in contrast with the rapid endocytosis of GnRH agonists. The slow dissociation of receptor-bound antagonist was consistent with its ability to cause sustained blockade of GnRH actions, and its prolonged cell-surface location suggests that receptor activation is necessary to initiate the rapid internalization of hormone-receptor complexes that is a feature of the agonist-stimulated gonadotroph.     

A gonadotropin-releasing hormone (GnRH) antagonist distinguishes three populations of GnRH analog-responsive cells in human and rat pituitary in vitro and produces an acute increase in intracellular Ca2+ concentration without inducing gonadotropin secretion

We have examined the actions of the potent GnRH antagonist [N-acetyl-D beta Na11-D-pCl-Phe2-D-Phe3-D-Arg6,Phe7,Arg8-D-Ala10]NH2GnRH++ + (GnRHa) on basal and GnRH-stimulated LH secretion, inositol phospholipid turnover, and intracellular Ca2+ levels in dispersed rat anterior pituitary tissue. As expected, GnRHa was found to be a pure antagonist of secretion, but was paradoxically equipotent with GnRH in stimulating inositol phospholipid turnover. Examination of intracellular Ca2+ changes at the single cell level using digital video-enhanced fluorescence imaging demonstrated that dispersed rat pituitary cells appeared to contain three GnRH analog-responsive cell populations: those that increased intracellular Ca2+ in response to both GnRH and GnRHa, and those that responded to either GnRH or GnRHa only. These observations were extended to studies of the relatively homogeneous gonadotroph cell populations of endocrinologically inactive pituitary adenomas. Of five adenomas examined, one increased inositol phospholipid turnover in response to GnRHa plus GnRH, but not GnRH alone, three responded to GnRH only, and one responded to both GnRH and GnRHa. Our findings, therefore, suggest that three GnRH analog-responsive cell types are also present in human pituitary and that clonal expansion of any of these cell types may be responsible for tumor formation.     

Photoaffinity labelling of gonadotropin releasing hormone binding sites in human epithelial ovarian carcinomata

A photoaffinity labelled derivative of [D-Lys6]-GnRH was prepared with a bifunctional photolabile reagent (4-azidobenzoyl)-N-hydroxysuccinimide. In rat pituitary membranes, this analog retained high binding affinity (Ka = 0.12 x 10(9) M-1) consistent with a single class of receptors. The analog was iodinated and used for the identification of GnRH binding sites in human epithelial ovarian carcinomata. By sodium dodecyl sulfate electrophoresis in 10% polyacrylamide gel the presence of two labelled components could be demonstrated: a high molecular weight component of 63,200 and a smaller component of 46,000. Competition experiments with unlabelled ligand suggest that it is the high molecular weight component which specifically binds GnRH.     

Disruption of neuroendocrine control of luteinizing hormone secretion by aroclor 1254 involves inhibition of hypothalamic tryptophan hydroxylase activity

Mechanisms governing the effect of polychlorinated biphenyl (PCB) toxicity on hypothalamic serotonergic function and the neuroendocrine system controlling LH secretion were investigated in Atlantic croaker (Micropogonias unulatus) exposed to the PCB mixture Aroclor 1254 (1 microg x g body weight(-1) x day(-1)) in the diet for 30 days. PCB treatment caused a decrease in hypothalamic 5-hydroxytryptamine (5-HT) concentrations and significant inhibition of hypothalamic tryptophan hydroxylase (TPH), the rate-limiting enzyme in 5-HT synthesis, but did not alter the activity of monoamine oxidase, the catabolic enzyme. Further, PCB treatment caused significant decreases in GnRH content in the preoptic-anterior hypothalamic area. Significant decreases in pituitary GnRH receptor concentrations and the LH response to the GnRH analogue (GnRHa) were also observed in PCB-exposed fish, possibly as a consequence of a decline in GnRH release. The possible association between impaired serotonergic and neuroendocrine functions after PCB treatment was explored using serotonergic drugs. Treatment of croaker with p-chlorophenylalanine, an irreversible TPH inhibitor, mimicked the effects of PCB on the GnRH system and the LH response to GnRHa. Bypassing the TPH-dependent hydroxylation step with the administration of 5-hydroxytryptophan restored 5-HT to control levels and prevented the deleterious effects of PCB on the neuroendocrine parameters. Moreover, slow-release GnRH implants prevented the PCB-induced decline in GnRH receptors and restored the LH response to GnRHa, suggesting that GnRH therapy can reverse PCB-induced disruption of LH secretion. These results demonstrate that TPH is one of the targets of PCB neurotoxicity and indicate that a decrease in 5-HT availability in PCB-exposed croaker results in disruption of the stimulatory 5-HT/GnRH pathway controlling LH 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.     

Pituitary receptors for gonadotropin-releasing hormone in relation to changes in pituitary and plasma luteinizing hormone in ovariectomized-hypothalamo pituitary disconnected ewes. I. Effect of changing frequency of gonadotropin-releasing hormone pulses

Studies were undertaken to determine if changes in the amplitude of luteinizing hormone (LH) pulses that occur in response to changes in the frequency of gonadotropin-releasing hormone (GnRH) pulses are due to an alteration in the number of GnRH receptors. Ewes were ovariectomized (OVX) and the hypothalamus was disconnected from the pituitary (HPD). Ewes were then given pulses of GnRH at a frequency of 1/h or 1/3 h. Two control groups were included: OVX ewes not subjected to HPD, and HPD ewes that were not OVX. At the end of one week of treatment, blood samples were collected to determine the amplitude of LH pulses. The treated ewes were killed just before the next scheduled pulse of GnRH, and the content of LH and number of GnRH receptors were measured in each pituitary. The amplitude of LH pulses was highly correlated with the amount of LH in the pituitary gland (r = 0.71, p less than 0.01), and both LH content and pulse amplitude (mean + SEM) were higher in ewes receiving GnRH once per 3 h (189.7 +/- 39.3 microgram/pituitary, 10.3 +/- 1.1 ng/ml, respectively) than in ewes receiving GnRH once per h (77.8 +/- 11.4 microgram/pituitary, 5.2 +/- 1.3 ng/ml). The pituitary content of LH was highest in the OVX ewes (260.2 +/- 57.4 micrograms/pituitary) and lowest in the nonpulsed HPD ewes (61.7 +/- 51.2 micrograms/pituitary). The number of GnRH receptors was similar in all groups, and was not correlated with any other variable.(ABSTRACT TRUNCATED AT 250 WORDS)     

Seasonal variation in hypothalamic content of gonadotropin-releasing hormone (GnRH), pituitary receptors for GnRH, and pituitary content of luteinizing hormone and follicle-stimulating hormone in the mare

Seasonal changes in the hypothalamic-hypophyseal axis were investigated using tissue from 49 light-horse mares, of mixed breeding. Hypothalamic and pituitary tissues were collected at 5 intervals throughout the years 1981 and 1982, representing midbreeding season (July, n = 10), transition out of the breeding season (October, n = 11), midanestrus (December, n = 8), transition into the breeding season (March, n = 10), and again in the following midbreeding season (July, n = 10). The hypothalamic region was dissected into preoptic area, body and median eminence. Gonadotropin-releasing hormone (GnRH) was extracted from hypothalamic samples with methanol-formic acid and quantified by radioimmunoassay. The anterior pituitary was homogenized and receptors for GnRH were quantified in a crude membrane fraction. Concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured in the resulting supernatant. Content of GnRH in each of the 3 hypothalamic areas varied with season (P less than 0.01) and was lowest during midanestrus (P less than 0.05). There was no effect of season (P greater than 0.01) on either concentration or total number of receptors for GnRH, or concentration of FSH in the anterior pituitary. Concentrations of LH in the anterior pituitary varied with season (P less than 0.001). Means (+/- SEM) for the 5 collection times were 15.5 +/- 2.7, 9.7 +/- 2.4, 2.3 +/- 0.5, 2.7 +/- 0.4 and 11.7 +/- 1.5 microgram LH/mg anterior pituitary, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of gonadotropin-releasing hormone (GnRH) binding sites in the pituitary and testis of the frog, Rana esculenta

Frog, Rana esculenta, pituitary and testis gonadotropin-releasing hormone (GnRH) receptors were characterized by using 125I-chicken IIGnRH (cIIGnRH) as radiolabeled ligand. At 4 C equilibrium binding of 125I-cIIGnRH to pituitary homogenates was achieved after 90 min of incubation; binding of 125I-cIIGnRH to testis membrane fractions reached its maximum at 60 min of incubation. Binding of the radioligand was a function of tissue concentration, with a positive correlation over the range 0.5-2 tissue equivalents per tube. One pituitary and one testis per tube were used as standard experimental condition. Incubation of the pituitary homogenate with increasing concentrations of 125I-cIIGnRH indicated saturable binding at radioligand concentrations of 1 nM and above while for the testis membrane preparation saturation was achieved using 5 nM 125I-cIIGnRH. The binding of 125I-cIIGnRH was found to be reversible after addition of the cold analog and the displacement curves could be resolved into one linear component for both tissues. Scatchard analysis suggested the presence of one class of binding sites for both pituitary and testis (Pituitary: Kd = 1.25 +/- 0.14 nM and Bmax = 8.55 +/- 2.72 fmol/mg protein; testis: Kd = 2.23 +/- 0.89 nM and Bmax = 26.48 +/- 7.39 fmol/mg protein). Buserelin displaced the labeled 125I-cIIGnRH with a lower IC50 as compared with cIIGnRH cold standard, while Arg-vasopressin (AVP) was completely ineffective, confirming the specificity of binding.     

Gonadotropin-releasing hormone inhibits cyclic nucleotide accumulation in cultured rat granulosa cells

Ovarian granulosa cells obtained from hypophysectomized, diethylstilbestrol-treated rats were cultured in the presence of ovine follicle-stimulating hormone (FSH) and gonadotropin-releasing hormone (GnRH). FSH stimulated the production and accumulation of both cAMP and cGMP, as well as progesterone, during a 48-h incubation period. Addition of GnRH or an agonist analog, [D-Ala6]des-Gly10-GnRH N-ethylamide (GnRHa), did not influence the cyclic nucleotide response to FSH in the first 6 h of incubation, but caused dose-dependent inhibition of the FSH-induced rise in cyclic nucleotide production from 24 to 48 h of incubation. Cellular production of both cyclic nucleotides and progesterone was decreased by GnRHa concentrations as low as 10(-12) M, with maximum inhibition at 10(-9) M GnRHa. These results suggest that the in vitro antigonadal actions of GnRH and related peptides are expressed through inhibition of cyclic nucleotide production.     

Role of GnRH in the regulation of pituitary GnRH receptors in female mice

The fall in pituitary GnRH receptors in female mice after ovariectomy (Ovx) was further decreased (greater than 50%), rather than prevented, by treatment with a GnRH antiserum, despite suppression of the post-gonadectomy increase in serum gonadotrophins, suggesting that increased endogenous GnRH secretion is not the mediator of GnRH receptor fall after ovariectomy in mice. Furthermore, GnRH antiserum reduced GnRH receptors by 30-50% in intact normal females, without altering receptor affinity, and rendered serum LH and FSH undetectable but did not reduce receptors in GnRH-deficient, hpg mice. When GnRH was administered to ovariectomized mice this failed to restore receptor values (fmol/pituitary) (intact = 55.3 +/- 2.4; Ovx = 30.1 +/- 2; Ovx + GnRH = 31.6 +/- 2.8), but serum LH was reduced from high post-ovariectomy values (231 +/- 42 ng/ml) to values normal for intact females (24 +/- 2 ng/ml). In contrast, multiple GnRH injections to intact female mice increased GnRH receptor by 35%, while serum LH was reduced to just detectable levels. A marked dissociation between GnRH receptor and serum gonadotrophin concentrations was observed. Administration of oestrogen (E2) plus progesterone (P) to ovariectomized mice in which endogenous GnRH had been immunoneutralized reversed the inhibitory effect of GnRH antiserum on GnRH receptors and increased values above those of ovariectomized controls, although no increase in serum or pituitary gonadotrophin levels was seen in ovariectomized mice treated with E2 + P + GnRH antiserum. Treatment with E2 and P of intact females receiving GnRH antiserum did not prevent the inhibitory effect of antiserum on receptors, while E2 + P treatment alone of intact female mice reduced GnRH receptors by 30%. These data suggest that the gonadal steroids reduce GnRH receptors in intact female mice by inhibiting hypothalamic GnRH secretion, and that a certain degree of pituitary exposure to GnRH is required for maintenance of a normal receptor complement. These results suggest that (1) the fall in GnRH receptors after ovariectomy is primarily attributable to removal of gonadal factors. The fall is not a reflection of alteration in endogenous GnRH interaction with the gonadotroph; (2) homologous ligand 'up-regulation' of GnRH receptors in female mice depends upon the presence of the ovaries; (3) endogenous GnRH is also required for GnRH receptor maintenance in intact female mice; and (4) GnRH receptor and serum gonadotrophin responses to hormonal changes can be dissociated and their relationship is complex.