Increased gonadotrophin releasing hormone receptors on pituitary gonadotrophs: effect on subsequent LH secretion

GnRH, high potassium concentrations, and cAMP derivatives have been previously shown to increase GnRH receptor levels (GnRH-R) in cultured rat pituitary cells. However, the effect of these changes in receptor number on subsequent stimulated LH release has not been investigated. In this study pretreatment of pituitary cells with either 1 nM GnRH, 58 mM KCl, or 1 mM dibutyryl cAMP (dbcAMP) resulted in a 70-100% increase in GnRH-R 7-10 h later. Subsequent LH responses to GnRH in those cells pretreated with GnRH and KCl were markedly reduced and the dose-response characteristics altered such that the curves were non-sigmoidal. When corrected for depletion of cellular LH during the pretreatment period these GnRH response curves were similar to control, implying that hormone depletion was the explanation for apparent desensitisation. By contrast, dbcAMP and low-dose calcium ionophore (0.1 microM A23187) pretreatment, which did not deplete cellular LH, neither enhanced nor decreased subsequent sensitivity to GnRH. Thus, 4 agents which all, under these conditions, increased GnRH receptors did not sensitise gonadotrophs to GnRH. By contrast, pretreatment with 10(-9) and 10(-8) M GnRH for either 12 or 16 h rendered cells completely or partially refractory to further GnRH stimulation, despite an increase in GnRH receptors. This desensitisation could not be explained by cellular LH depletion, and was specific to the homologous ligand since dose-responses to the Ca2+ ionophore A23187 and KCl were normal when corrected for LH depletion. Non-receptor-mediated depletion of cellular LH during A23187 pretreatment (10 microM for 10 h) did not alter subsequent GnRH dose-responses, after correction for LH content. These data indicate that, under these in vitro conditions, the increased GnRH receptors are not functionally linked to the secretory apparatus of the gonadotroph. Furthermore, homologous ligand-induced desensitisation is both time- and concentration-dependent and is mediated largely by post-receptor cellular events independent of cellular LH content. Therefore, post-receptor cellular processes may be more important than changes in GnRH receptors in regulating gonadotrophin secretion. It is suggested that an increase in GnRH receptors may represent a cellular response to generalised gonadotroph activation by a variety of agents, and does not necessarily signify enhanced responsiveness to GnRH.     

Pituitary gonadotropin-releasing hormone (GnRH) receptor responses to GnRH in hypothalamus-lesioned rats: inhibition of responses by hyperprolactinemia and evidence that testosterone and estradiol modulate gonadotropin secretion at postreceptor sites

Increased hypothalamic GnRH secretion appears to influence positively the number of pituitary GnRH receptors (GnRH-R). GnRH-R increase after castration in male rats, and this rise can be prevented by testosterone (T), anti-GnRH sera, or hypothalamic lesions. GnRH also increases serum LH and GnRH-R in hypothalamus-lesioned rats, and these animals injected with exogenous GnRH are, therefore, a good model in which to study the site of steroid feedback at the pituitary level. Adult male and female rats were gonadectomized, and radiofrequency lesions were placed in the hypothalamus. Males received T implants, and females received estradiol implants at the time of surgery. Empty capsules were placed in the control animals. Beginning 3-5 days later, animals in each group were injected every 8 h with vehicle (BSA) or GnRH (0.002-200 micrograms/day) for 2 days. After these GnRH injections, all rats received 6.6 micrograms GnRH, sc, 1 h before decapitation to determine acute LH and FSH responses. GnRH-R were determined by saturation analysis using 125I-D-Ala6-GnRH ethylamide as ligand. In males, GnRH injections increased GnRH-R. T inhibited acute LH and FSH responses to GnRH in all groups, but had little effect on GnRH-R, indicating that T inhibits gonadotropin secretion at a post-GnRH receptor site. In females, the GnRH-R response to GnRH was less marked, and only the 200 micrograms/day dose of GnRH increased GnRH-R, indicating that the positive feedback effects of estradiol at the pituitary level are also exerted at a site distal to the GnRH receptor. There was no positive correlation between the number of GnRH-R and GnRH-stimulated gonadotropin release in males or females. Female rats with hypothalamic lesions had markedly elevated serum PRL levels (greater than 300 ng/ml). Suppression of PRL secretion by bromocryptine resulted in augmented GnRH-R responses to GnRH, and GnRH-R concentrations rose to the same values induced in males. This suggests that hyperprolactinemia inhibits GnRH-R responses to GnRH in females by a direct action on the pituitary gonadotroph.     

Dose response effects of pulsatile GnRH administration on restoration of pituitary GnRH receptors and pulsatile LH secretion during lactation

Ovariectomized (OVX) rats suckling 8 pups have a complete suppression of pulsatile LH secretion and a decrease in pituitary GnRH receptor (GnRH-R) content. Removing the suckling stimulus for 24 h results in a sharp increase in GnRH-R and a restoration of pulsatile LH secretion. These findings suggest that the suckling stimulus induces a suppression of GnRH secretion, and removal of the suckling stimulus permits the restoration of GnRH secretion. Indeed, if GnRH antiserum is injected at the time of pup removal, the restoration of pituitary GnRH-R and LH secretion is prevented. The present studies were designed to test our hypothesis that the deficits in pituitary gonadotroph function observed during lactation are due to suckling-induced suppression of GnRH. Exogenous GnRH was administered in a pulsatile regimen to OVX lactating rats on days 10 and 11 postpartum, and the effects on pituitary GnRH-R levels, pituitary sensitivity to GnRH, and pulsatile LH secretion were assessed. GnRH doses of 0, 0.5, 2.0 or 5.0 ng/pulse were administered every 50 min for 24 h beginning on day 10. Administration of 0.5 ng GnRH/pulse for 24 h increased GnRH-R from 35 +/- 3 to 63 +/- 8 fmol/pituitary. There was a clear GnRH dose-related upregulation of GnRH-R to approach nonsuckling levels (140-160 fmol/pituitary) with the 5 ng GnRH dose. At the beginning of GnRH administration, the pituitary was very unresponsive to GnRH. Consistent LH pulses were only observed with 5 ng GnRH/pulse.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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

Implication of dopaminergic systems on GnRH and GnRHR genes expression in the hypothalamus and GnRH-R gene expression in the anterior pituitary gland of anestrous ewes.

We examined by Real-time PCR how prolonged inhibition of dopaminergic D-2 receptors (DA-2) in the hypothalamus of anestrous ewes by infusion of sulpiride into the third cerebral ventricle affected GnRH and GnRH-R gene expression in discrete parts of this structure and GnRH-R gene expression in the anterior pituitary. Blockaded DA-2 receptors significantly decreased GnRH mRNA levels in the ventromedial hypothalamus but did not evidently affect GnRH mRNA in the preoptic/ anteriorhypothalamicarea. Blockaded DA-2 receptors led to different responses in GnRH-R mRNA in various parts of the hypothalamus; increased GnRH-R mRNA levels in the preoptic/anterior hypothalamic area, and decreased GnRH-R mRNA amounts in the ventromedial hypothalamus stalk/median eminence. An infusion of sulpiride into the III-rd ventricle increased GnRH mRNA levels in the anterior pituitary gland and LH secretion. It is suggested that the increase of GnRH gene expression in the anterior pituitary gland and LH secretion in sulpiride-treated ewes are related with an increase of biosynthesis GnRH with concomitant decreased biosynthesis of GnRH-R protein in the ventromedial hypothalamus/stalk median eminence allowing to an increase of GnRH release.     

Pituitary receptor sites for gonadotropin-releasing hormone: effect of castration and substitutive therapy with sex steroids in the male rat

To examine the role of pituitary gonadotropin-releasing hormone (GnRH) receptors (pit GnRH-R) in the regulation of gonadotropin secretion, male rats were orchidectomized and then selectively received substitutive therapy with sex steroids. Pituitary content of GnRH-R was determined by saturation analysis, using radioiodinated [D-Trp6,(N-Et)Pro5,des-Gly10]GnRH as tracer. Castration produced a rapid and sustained increase of the number of GnRH-R, which doubled after 2 days, and after 10 days the pituitary content of GnRH-R was 258 +/- 23 fmol/pituitary compared to 103 +/- 12 fmol/pituitary for sham-operated control animals. No change of the affinity constant (Ka) was observed (Ka = 1.13 +/- 0.08 X 10(10) M-1; n = 14). Plasma LH increased 5- to 10-fold and FSH-2- to 3-fold after castration, and hypothalamic GnRH content was depleted by 30-60%. Immediate substitution of castrated rats with testosterone propionate (250 micrograms daily) prevented the increases of both plasma gonadotropins and of GnRH-R. Treatment of acutely castrated rats for 7 days with testosterone propionate (50-200 micrograms), 5 alpha-dihydrotestosterone propionate (25-400 micrograms), or estradiol benzoate (2 micrograms) prevented the rise in pit GnRH-R in a dose-related manner and normalized the other parameters studied except that plasma FSH remained slightly elevated. In contrast, when substitutive therapy was started 8 days after castration or later, the 7-day treatment with sex steroids reduced plasma gonadotropins, but pit GnRH-R remained elevated, and hypothalamic GnRH content remained depleted. These results indicate that the marked increase of gonadotropin secretion after castration is mediated at least in part, by an increase in the number of pit GnRH-R. Sex steroids were able to reverse all castration-induced endocrine changes in acutely castrated rats, but in long term castrated animals their action at higher centers to normalize hypothalamic GnRH content, and indirectly, to reduce pit GnRH-R content, was either delayed or ineffective. Thus, the rapid feedback action of sex steroids in long term castrated rats may be predominantly exerted at the pituitary level.     

Structure, function and transforming potential of the epidermal growth factor receptor

We have examined the pharmacology of the voltage-sensitive Ca2+ channels (VSCCs) that mediate gonadotropin secretion from primary cultures of rat pituitary cells, stimulated by either cell depolarization or by binding of gonadotropin-releasing hormone (GnRH). We also measured single-cell [Ca2+]i transients using fura-2 in gonadotropes identified by a reverse hemolytic plaque assay employing an antiserum to luteinizing hormone (LH). Cell depolarization evoked by either 50 mM K+ or 30 microM veratridine induced 2- to 6-fold increases in gonadotropin secretion over basal levels. GnRH caused 6- to 20-fold increases in follicle-stimulating hormone (FSH) and LH secretion, respectively, with maximal stimulation at 100 nM GnRH. K(+)- or GnRH-induced FSH release was largely prevented by co-incubation with 1 mM CdCl. Tetrodotoxin (TTX, 5 microM) prevented the veratridine-, but not the K(+)- or GnRH-induced, stimulation of FSH secretion. Nitrendipine (Ntd, 1 microM) produced 35-50% inhibition (NS) of both FSH and LH release stimulated by either 50 mM K+ or 100 nM GnRH. Ntd also inhibited the K(+)-induced [Ca2+]i rise (greater than 90%), as well as the secondary, plateau phase of the GnRH-induced elevation of [Ca2+]i (100% inhibition). Omega-conotoxin (omega-CgTx, 100 nM) partially suppressed FSH and LH release (NS) due to both K+ (33% each) and GnRH (44% and 18%, respectively). omega-CgTx showed variable effects on [Ca2+]i transients evoked by K+ or GnRH ranging from clear inhibition to no effect. We conclude that influx of extracellular Ca2+ is one of several fundamental events underlying the depolarization- or receptor-activated release of LH and FSH, and that this influx can be inhibited by dihydropyridine-sensitive ('L') Ca2+ channels. Two classes of L-channels may exist in gonadotropes, that differ in their sensitivity to omega-CgTx.     

Pituitary gonadotropin-releasing hormone receptor regulation in the hypogonadotrophic hypogonadal (hpg) mouse

Recent evidence indicates that endogenous GnRH is required for maintenance of its own pituitary receptors (GnRH-R). We have measured GnRH-R in pituitaries of hypogonadotrophic hypogonadal (hpg) mice, in whom hypothalamic GnRH is deficient or absent. The GnRH-R concentration in hpg male mouse pituitaries was 10.6 +/- 1 fmol/pituitary vs. 30.9 +/- 1 fmol/pituitary in normal male littermate pituitaries. Similarly, GnRH-R in female hpg mice (15.2 +/- 1.7 fmol/pituitary) were 30% those of normal random cycling females (51.4 +/- 3.5 fmol/pituitary). There was no difference in receptor affinity (Ka = 1.5-3 C 10(9) M-1) of hpg mouse pituitaries. The pituitary LH content in hpg male and female mice was very similar (range 3.4-4.8 micrograms/pituitary) representing 5% and 19% of normal male (95 +/- 7.2 micrograms/pituitary) and female (18.1 +/- 1.5 micrograms/pituitary) values, respectively. The administration of 50 ng GnRH sc 10 times daily to male hpg mice, increased GnRH-R to 80% of normal values within 3 days. Serum FSH and pituitary FSH content rose to normal male values after 7 days of GnRH injections. However, serum LH remained undetectable and pituitary LH reached only 20% of normal male levels, even after 15 days of GnRH administration. Treatment of hpg male mice with 60 ng GnRH either once daily for 6 days, or 12 times daily for 5 days, increased GnRH-R to 50% of normal male values. Twelve daily injections of GnRH elevated serum FSH to above the normal male range, whereas daily GnRH only doubled untreated hpg levels. Pituitary FSH was stimulated to 50% of normal with 12 daily injections, whereas once daily administration elevated pituitary FSH to 30% of normal values. Both pulsatile regimes depleted pituitary LH. These data demonstrate that: 1) despite absence of bioactive GnRH, GnRH-R values are only reduced to 30% of normal in hpg mouse pituitaries, suggesting that little, if any, endogenous GnRH is required for expression of GnRH receptors. 2) Pituitary GnRH-R number rapidly increase when GnRH is administered to hpg male mice indicating that, as in the rat, GnRH positively regulates its own receptor concentration. 3) The pituitary FSH and LH responses to GnRH treatment in hpg mice depends to a different extent on the frequency and duration of GnRH administration. 4) The hpg mouse provides an ideal animal model for investigating the interaction of defined regiments of exogenous GnRH and gonadal steroids on pituitary GnRH receptor and gonadotroph function.     

The frequency of gonadotropin-releasing hormone stimulation determines the number of pituitary gonadotropin-releasing hormone receptors

Gonadotropin-releasing hormone (GnRH) induces both synthesis and release of pituitary gonadotropins, but rapid or slow frequencies of stimulation result in reduced LH and FSH secretion. We determined the effects of frequency of GnRH stimulation on pituitary GnRH receptors (GnRH-R). Castrate male rats received testosterone implants (cast + T) to inhibit endogenous GnRH secretion. GnRH pulses were injected by a pump into a carotid cannula and animals received GnRH (25 ng/pulse) at various frequencies for 48 h. In control animals (saline pulses) GnRH-R was 307 +/- 21 fmol/mg protein (+/- SE) in cast + T and 598 +/- 28 in castrates. Maximum GnRH-R was produced by 30-min pulses and was similar to that seen in castrate controls. Faster or slower frequencies resulted in a smaller GnRH-R response and GnRH given every 240 min did not increase GnRH-R over saline controls. Equalization of the total GnRH dose/48 h (6.6 ng/pulse every 7.5 min or 200 ng/pulse every 240 min) did not increase receptors to the maximum concentrations seen after 30-min (25 ng) pulses. Serum LH responses after 48 h of injections were only present after 30-min pulses, and peak FSH values were also seen after this frequency. Serum LH was undetectable in most rats after other GnRH frequencies, even though GnRH-R was increased. These data show that GnRH pulse frequency is an important factor in the regulation of GnRH-R. A reduction of GnRH-R is part of the mechanism of down-regulation of LH secretion by fast or slow GnRH frequencies, but altered frequency also exerts effects on secretory mechanisms at a site distal to the GnRH receptor.     

Pituitary gonadotrophin-releasing hormone receptor up-regulation in vitro: dependence on calcium and microtubule function

Exogenous cyclic adenosine nucleotides increase gonadotrophin-releasing hormone (GnRH) receptors in intact cultured rat pituitary cells in a similar manner to that observed with GnRH itself. In this study the calcium and microtubule dependency of GnRH receptor up-regulation was examined in vitro. Treatment of pituitary cells in Ca2+ and serum-containing media with either GnRH (1 nmol/l), K+ (58 mmol/l) or dibutyryl cyclic AMP (dbcAMP; 1 mmol/l) for 7-10 h routinely resulted in a 50-100% increase in GnRH receptors. Incubation of pituitary cells with the calcium channel blocker verapamil, for 7 h, or the calcium chelator EGTA, for 10 h, had no effect on basal receptor levels but prevented the increase in GnRH receptors stimulated by either GnRH, K+ or dbcAMP. Luteinizing hormone release measured with the same stimulators over a 3-h period was prevented by both verapamil and EGTA. Calcium ionophore (A23187) increased GnRH receptors by 40-60% at low concentrations (10 and 100 nmol/l) while higher concentrations (10 and 100 mumol/l) reduced receptor levels. Luteinizing hormone release was not increased by receptor-stimulating concentrations of A23187, but was by higher concentrations (10 mumol/l). None of these pretreatments, for up to 10 h, impaired the subsequent LH response of the cells to increasing doses of GnRH. Vinblastine (1 mumol/l did not affect basal receptor levels but markedly reduced the increase in GnRH receptors stimulated by GnRH, K+ and dbcAMP. This concentration of vinblastine had no effect on LH release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Homologous ligand induction of pituitary gonadotrophin-releasing hormone receptors in vivo is protein synthesis dependent

This study demonstrates that a single subcutaneous injection of gonadotrophin-releasing hormone (GnRH) (60 ng) to GnRH-deficient (hpg) male mice causes a doubling of pituitary GnRH receptors (GnRH-R). No change in GnRH-R occurs during the time of LH release (15-60 min) or up until 4 h post-GnRH. Between 4 and 12 h there is a progressive increase in GnRH-R, which is still apparent 24 h later. No induction of GnRH-R occurs after the same treatment of intact adult normal mice. The same degree of GnRH-R induction occurs 12 h after a single GnRH injection (60 ng) to orchidectomized hpg male mice, indicating that this effect is mediated by a direct action of GnRH on the pituitary gonadotroph, rather than being secondary to stimulation of some gonadal product. Homologous ligand GnRH-R induction in hpg mouse pituitaries in vivo is prevented by prior treatment with cycloheximide, a non-specific protein synthesis inhibitor. Cycloheximide alone had no effect on GnRH-R in normal male mice but when combined with GnRH caused a 40% depletion of receptors, implying ligand-induced receptor loss without subsequent replenishment. The similarity between the extent, time-course, and dependence on protein synthesis of GnRH induction of its own receptors in vivo and in cultured pituitary cells in vitro indicates that the hpg mouse pituitary behaves like an in vivo pituitary cell culture system in this respect. Similarity of data derived from this in vivo model provides direct support for the view that in vitro studies on the cellular mechanism of GnRH action can be physiologically relevant to the intact animal.     

Pituitary gonadotropin-releasing hormone receptor regulation in mice. I: Males

The regulation of pituitary GnRH receptors (GnRH-R) has been examined in male mice (C3H/HeH/101H F1 hybrid) after castration and testosterone replacement. GnRH-R were quantified in individual mouse pituitaries by equilibration with 125I(D-Ser(tBut)6) des Gly10 GnRH N ethylamide and compared with serum and pituitary LH and FSH concentrations. The equilibrium association constant was 2.7 X 10(9) M-1 for both intact and castrated male mouse pituitary GnRH-R. Six hours after orchidectomy there was a transient 50% reduction in GnRH-R; 13.6 +/- 3.8 fmol/pituitary (castrate) vs. 25.4 +/- 2.5 (intact). A subsequent partial return of binding sites began at 12 h, reaching a peak value of 18.2 +/- 1.5 fmol/pituitary (33% increase vs. 6 h) at 24-h post orchidectomy. This was followed by a gradual decrease in GnRH-R, reaching a plateau by 72 h. The decrease in GnRH-R was associated with a rapid (6-12 h) increase in serum LH and serum FSH. The pituitary GnRH-R concentration remained 45% below intact control values for up to 3 months and was accompanied by a persistent 5-fold rise in serum LH values. Treatment of male mice with testosterone propionate (TP), 25 micrograms/day, completely prevented the GnRH-R fall and the serum and pituitary LH responses to castration, whereas 12.5 micrograms/day TP produced variable results and 5 micrograms/day TP were ineffective. In another strain of mouse (BALB/c white). GnRH-R values also fell by 66% at 7 days post orchidectomy, with no change in the receptor affinity. In mice with androgen resistance from birth due to absence of androgen receptors (Tfm mice), GnRH-R were 14.45 +/- 0.49 vs. 19.8 +/- 1.67 fmol/pituitary in normal male littermates, and serum LH was 472 +/- 78 ng/ml compared with 52.5 +/- 11.7 ng/ml in normals. These findings are qualitatively similar to those in orchidectomized normal adult mice. Thus, in contrast to reports in rats, pituitary GnRH-R content falls after orchidectomy in mice. Possible explanations for this consistent finding include: persistent receptor occupancy by increased endogenous GnRH secretion, endogenous GnRH-induced receptor loss (down-regulation), or a species difference in the pituitary GnRH-R response to removal of negative steroid feedback, unrelated to changes in endogenous GnRH secretion.     

Synthetic atrial natriuretic factors (ANFs) stimulate guanine 3',5'-monophosphate production but not hormone release in rat pituitary cells: peptide contamination with a gonadotropin-releasing hormone agonist explains luteinizing hormone-releasing activity of certain ANFs

The effects of atrial natriuretic factors (ANFs) on anterior pituitary hormone secretion and cyclic nucleotide production were investigated in cultured rat pituitary cells. ANF had no effect on ACTH, GH, PRL, and TSH release or on cAMP production either on basal hormone levels or during stimulation of their secretion by the appropriate releasing factor. However, ANF markedly stimulated cGMP production in both mixed anterior pituitary cells and enriched anterior pituitary cell populations fractionated by centrifugal elutriation. Unexpectedly, certain ANF preparations, Bachem rat ANF-(5-28) and rat ANF-(5-25), markedly stimulated LH release from cultured anterior pituitary cells and gonadotroph-enriched elutriated pituitary cells. The same ANFs also displaced [125I-D-Lys6]GnRH ethylamide from binding to anterior pituitary membranes with potencies similar to their LH-releasing activities. Immunoprecipitation of ANF with a specific antiserum abolished the effect of ANF on cGMP production, but did not change the effect of ANF on LH release. In conclusion, ANF did not affect anterior pituitary hormone secretion or cAMP production, but stimulated cGMP formation. The effect of certain ANF preparations on LH release appears to be attributable to peptide contamination with a potent GnRH agonist.     

Calcium-dependent actions of gonadotropin-releasing hormone on pituitary guanosine 3',5'-monophosphate production and gonadotropin release

The effects of gonadotropin-releasing hormone (GnRH) on cGMP production and LH release in cultured rat pituitary cells are markedly dependent upon the extracellular calcium concentration. The absence of calcium from incubation media caused almost complete loss of the GnRH effects on cGMP production and LH release but did not change the stimulation of cAMP accumulation by GnRH in the pituitary of the adult male rat. In female rat pituitary cells, reduction of the extracellular calcium concentration increased the concentration of GnRH required to produce half-maximal LH release and decreased the maximal gonadotropin output but had no significant effect on basal LH release. The divalent cation ionophore A23187 stimulated LH release, and this action was dependent on extracellular calcium. Both GnRH and A23187 were found to have maximal effects when the calcium concentration was 0.6 mM, and their actions were not additive. The calcium antagonists, verapamil and lanthanum, caused concentration-dependent inhibition of the actions of GnRH, with half-maximal blockade values of 10(-5) and 3 X 10(-6) M, respectively, and had no effect on basal LH release. The binding of a radioiodinated GnRH analog, [D-Ser(t-Bu)6]des-Gly10-GnRH-N-ethylamide, to pituitary GnRH receptors was unchanged in the absence of extracellular calcium. These observations demonstrate that stimulation of pituitary cGMP production and LH release by GnRH is dependent on extracellular calcium. The site at which calcium is required during GnRH action is at a postreceptor locus before cGMP formation.     

Expression of alpha-subunit and luteinizing hormone (LH) beta messenger ribonucleic acid in the rat during lactation and after pup removal: relationship to pituitary gonadotropin-releasing hormone receptors and pulsatile LH secretion

Pituitary GnRH receptor (GnRH-R) levels and LH secretion are suppressed in the lactating rat. To determine if LH synthesis is also inhibited, we have measured LH subunit mRNA levels in the pituitary of lactating rats. We have also examined the temporal relationship among restoration of GnRH-R, LH secretion, and LH synthesis after withdrawing the sensory stimulus of suckling. Pituitary alpha-subunit and LH beta mRNA levels were sharply reduced on day 10 of lactation in both intact and ovariectomized (OVX) animals compared with those in cycling diestrous rats or OVX controls. Removal of the suckling stimulus from OVX animals led to significant increases in alpha-subunit and LH beta mRNA levels by 24 h. Upon removal of the suckling stimulus from intact rats, alpha-subunit mRNA levels were restored by 48 h, but LH beta mRNA levels did not return to diestrous levels until 72 h. Pituitary GnRH-R levels were clearly up-regulated within 1 day after pup removal. Some LH pulses were observed by 48 h, but consistent plasma LH pulses were not detected until 72 h. When pulsatile GnRH was administered during the 24 h after pup removal from intact rats, the regimen of pulsatile GnRH was successful in inducing LH secretion; however, the restoration of pulsatile LH was not accompanied by increases in alpha-subunit and LH beta mRNA levels. The present studies provide further evidence to support the hypothesis that during lactation, the suppression of pituitary gonadotroph function is mainly due to the loss of hypothalamic GnRH secretion. Our data also show that 1) the restoration of GnRH-R alone is not sufficient to activate LH subunit mRNA and LH secretion; 2) the normal restoration of pulsatile LH secretion and increases in LH subunit mRNA are temporally correlated, as increases in LH secretion appear to precede increases in LH subunit mRNA; and 3) the restoration of pituitary LH subunit mRNA levels and pulsatile LH secretion took longer in the intact rat than in the OVX rat, suggesting that ovarian steroids may play a role in the inhibitory effect of lactation.     

LH down-regulates gonadotropin-releasing hormone (GnRH) receptor, but not GnRH, mRNA levels in the rat testis.

The demonstration of an inhibitory effect of gonadotropin-releasing hormone (GnRH) agonists upon steroidogenesis in hypophysectomized rats and the presence of mRNA coding for GnRH and GnRH receptors (GnRH-R) in rat gonads suggests that GnRH can act locally in the gonads. To assess this hypothesis, we investigated the effects of GnRH analogs, gonadotropins and testosterone on the levels of both GnRH and GnRH-R mRNA in the rat testis. Using dot blot hybridization, we measured the mRNA levels 2 to 120 h after the administration of the GnRH agonist, triptorelin. We observed an acute reduction of both GnRH and GnRH-R mRNAs 24 h after the injection (about 38% of control). However, the kinetics for testis GnRH-R mRNA were different from those previously found for pituitary GnRH-R mRNA under the same conditions. Initially, the concentrations of serum LH and FSH peaked, then declined, probably due to the desensitization of the gonadotrope cells. In contrast, the GnRH antagonist, antarelix, after 8 h induced a 2.5-fold increase in GnRH-R mRNA, but not in GnRH mRNA, while gonadotropins levels were reduced. Human recombinant FSH had no significant effect on either GnRH or GnRH-R mRNA levels. Inversely, GnRH-R mRNA levels markedly decreased by 21% of that of control 24 h after hCG injection. Finally, 24 h after testosterone injection, a significant increase in GnRH-R mRNA levels (2.3 fold vs control) was found, but a reduction in the concentration of serum LH, probably by negative feedback on the pituitary, was observed. In contrast, GnRH mRNA levels were not significantly altered following testosterone treatment. Since LH receptors, GnRH-R and testosterone synthesis are colocalized in Leydig cells, our data suggest that LH could inhibit the GnRH-R gene expression or decrease the GnRH-R mRNA stability in the testis. However, this does not exclude the possibility that GnRH analogs could also affect the GnRH-R mRNA levels via direct binding to testicular GnRH-R. In contrast, the regulation of GnRH mRNA levels appeared to be independent of gonadotropins. Taken together, our results suggest a regulation of GnRH and GnRH-R mRNA specific for the testis.     

Gonadotropin-releasing hormone action in cultured pituicytes: independence of luteinizing hormone release and adenosine 3',5'-monophosphate production.

The secretory response of pituitary gonadotropes to stimulation by gonadotropin-releasing hormone (GnRH) has been extensively studied, but the mechanism by which GnRH evokes gonadotropin synthesis and release has not been clarified. In particular, there has been conflicting evidence about the role of cAMP in GnRH-induced release of LH. To examine this question in more detail, the actions of GnRH on LH release and cAMP production were analyzed in primary cultures of collagenase-dispersed rat pituitary cells. In this system, addition of 10(-10)--10(-6) M GnRh to cultured pituicytes caused rapid release of LH into the incubation medium. In contrast, GnRH caused no significant change in intracellular or extracellular cAMP or in occupancy by cAMP of the regulatory subunit of protein kinse. Neither dibutyryl cAMP nor methyl isobutylxanthine (MIC) stimulated LH production to the same level as GnRH, and neither agent potentiated the effect of the releasing hormone. Cholera toxin and prostaglandin E1 (PGE1), both of which stimulated cAMP production in cultured pituicytes, did not raise LH levels as markedly as GnRH. These results demonstrate the independence of LH release from cAMP accumulation in cultured pituicytes, suggesting that cAMP is not required for stimulation of LH release from these cells and that GnRH acts on LH secretion by a different mechanism.     

Regulation of pituitary gonadotropin-releasing hormone (GnRH) receptors by pulsatile GnRH in female rats: effects of estradiol and prolactin

GnRH has been shown to modulate the concentration of its own pituitary receptors (GnRH-R), and changes in GnRH-R during the rat estrous cycle may reflect changes in GnRH secretion. To examine the relationship between GnRH and GnRH-R in female rats, we measured GnRH-R and serum gonadotropin responses to pulsatile GnRH in restrained ovariectomized (OVX) and ovariectomized estradiol-implanted (OVX-E2) rats. In addition, we examined the effects of suppression of serum PRL. Pulsatile injections of GnRH (10-250 ng/pulse) given every 30 min for 24 or 48 h did not increase GnRH-R in OVX or OVX-E2 rats compared to that in saline controls (246 +/- 27 fmol/mg). Bromocriptine treatment (2 mg/day) had no effect on GnRH-R in OVX animals. In contrast, OVX-E2 rats treated with bromocriptine showed significantly increased GnRH-R (500 +/- 43 fmol/mg) in response to GnRH injections. When ovine PRL was administered to bromocriptine-treated OVX-E2 rats, the GnRH induced rise in GnRH-R was abolished. Gonadotropin responses to GnRH were not correlated with changes in GnRH-R. In OVX animals, LH was only elevated in response to 250-ng pulses of GnRH. In OVX-E2 animals, basal LH was increased by all doses of GnRH, and acute responses to 50- and 250-ng pulses were observed. Bromocriptine treatment resulted in increased LH sensitivity to GnRH in OVX rats, but did not further enhance the responses in OVX-E2 animals. We conclude that in female rats, the presence of both E2 and a low serum PRL level is necessary for GnRH to increase GnRH-R, and the interaction of these factors may be involved in the regulation of GnRH-R during the estrous cycle.     

Modulation of the estradiol-induced luteinizing hormone surge by progesterone or antiestrogens: effects on pituitary gonadotropin-releasing hormone receptors

The present study examined the question of whether modulation of estradiol-induced LH surges by progesterone or antiestrogens in the immature rat might be related to changes in the concentration of pituitary GnRH receptors (GnRH-R). Rats (28 days old) that received estradiol implants at 0900 h had LH surges approximately 32 h later. Administration of progesterone or nafoxidine (U-11,100 A; 1-(2-[P-(3,4-dihydro-6-methoxy-2-phenyl-1-naphthyl)phenoxy]pyrrolidine hydrochloride) concomitantly with estradiol led to blockade of these LH surges (progesterone or nafoxidine inhibition), while progesterone treatment 24 h after estradiol brought about premature and enhanced LH release (progesterone facilitation). GnRH-R-binding capacity was determined by saturation analysis in homogenates of single pituitaries from immature rats treated with estradiol and progesterone or nafoxidine and controls treated only with estradiol using [125I]iodo-(D-Ala6,Des-Gly10)GnRh ethylamide. The affinity of GnRH-R for this analog ranged from 8.2-15.1 X 10(9) M-1 and was not affected by in vivo steroid or antiestrogen treatment. The number of GnRH-R in gonadotrophs from untreated 28-day-old rats (57.2 +/- 2.6 fmol/pituitary or 177 +/- 11 fmol/mg protein) was comparable to values previously reported for 30 day-old females. GnRH-R levels were first measured 1, 8, 24, 32, and 48 h after estradiol treatment. The pituitary content of GnRH-R paralleled changes in total pituitary protein (nadir at 24 h, rebound at 32 h, continued increase at 48 h), while their concentration (femtomoles per mg protein) was highest at 8 h. Next, GnRH-R levels were examined at 1200 h and at hourly intervals (1400-1800 h) on the afternoon of the LH surge. While GnRH-R concentrations were significantly lower at 1400 and 1700 h than at 1200 or 1800 h in animals treated with estradiol in the progesterone facilitation model, they did not change over time in the other two paradigms. There was no significant difference in pituitary content or concentration of GnRH-R at any time between immature rats treated with estradiol and progesterone or nafoxidine and their respective estradiol-treated controls. These results suggest that changes in GnRH-R levels in pituitary gonadotrophs do not play a major role in enhancement of LH surges by progesterone or in their suppression by progesterone or nafoxidine in the immature rat; therefore, these compounds may affect the pituitary at a site distal to the GnRH receptor.