Pituitary gonadotropin-releasing hormone receptors during gonadotropin surges in ovariectomized-estradiol-treated rats

In intact cycling rats, the number of pituitary GnRH receptors varies markedly during the estrous cycle. Concentrations are maximal on diestrus and early proestrus, before falling rapidly for a brief period immediately before the preovulatory gonadotropin surge. In this study we investigated whether dynamic changes in ovarian steroids, pituitary hormones, and GnRH itself, all of which are changing at the time of the surge, play a role in the acute transient down-regulation of the pituitary GnRH receptors. We used the ovariectomized-estradiol-treated female rat as a model, as these animals exhibit daily gonadotropin surges at a predictable time of the day and also allow studies in a situation where concentrations of ovarian steroids are stable. The pituitary GnRH binding capacity (GnRH-BC) was measured using the analog D-Ala6des Gly10-GnRH ethylamide as ligand. GnRH-BC was stable between 0900-1530 h [range, 288 +/- 29 to 262 +/- 33 fmol protein (mean +/- SE)] and fell abruptly to 123 +/- 17 fmol/mg at 1630 h, before returning to the initial level by 1730 h. This abrupt fall in GnRH-BC preceded the afternoon gonadotropin surge and was similar in timing, magnitude, and duration to that observed in intact cycling rats. Serum PRL decreased from peak levels at 1630 h, coincident with the fall in GnRH-BC, before rebounding at 1730 h. Pentobarbital given at 1400 h abolished both the gonadotropin surge and the acute fall in GnRH-BC, but did not change serum PRL levels, suggesting that PRL is not causally related to the fall in GnRH-BC. The stable morning levels of GnRH-BC were not reduced after iv injections of LH, FSH, or both hormones despite elevations in serum gonadotropins to concentrations greater than those seen during the afternoon surge. Additionally, multiple iv injections of GnRH at 30- or 10-min intervals did not decrease the stable morning levels of GnRH-BC, although serum LH and FSH were markedly elevated. The data suggest that dynamic fluctuations in ovarian steroids, gonadotropins, PRL, and GnRH are not causally related to the acute transient reduction of pituitary GnRH receptors before the afternoon gonadotropin surge. These results also suggest that another hypothalamic or pituitary factor(s) is involved in the acute regulation of GnRH receptors, and the ovariectomized-estradiol-treated rat appears to be a good model for the elucidation of the factor(s) involved.     

Pituitary regulation of human growth hormone binding sites in rat liver membranes.

We have studied the binding of 125I-human growth hormone (hGH) to crude 100,000 X g membrane preparations from rat liver, and have studied factors which might regulate the capacity and affinity of hGH binding sites. Membrane preparations have livers of pregnant rats bound between 8% and 18% of the 125I-hGH initially added, and 70%-80% of that bound was displaced by 1 mug of unlabeled hGH. Humans prolactin (hPrl) displaced 125 I-hGH in a manner parallel to hGH itself but with about one-third the potency. Ovine, porcine, and rat Prl, and rat and bovine GH were much less effective. Scatchard analysis of specific hGH binding by a variety of different rat liver membrane preparations revealed a single order of binding site in each case with a binding affinity of 0.93-1.62 X 10(-9) M-1. Membranes from pregnant rats had twice the binding capacity of membranes from nonpregnant female rats, and about six times the capacity of sites present in preparations from normal adult male rats and hypophysectomized (Hx) male or female rats. Female or male rats with extremely high circulating GH an Prl levels, due to the presence of transplantable GH/Prl secreting pituitary tumors showed a significantly greater binding capacity than did the pregnant rats. Estradiol (E2) treatment (25 mug/day for 10-12 days) of normal male rats led to an increase in specific hGH binding. Treatment of hypophysectomized male rats with bovine GH (100 or 500 mug/day) +/- E2 (25 mug/day) for 5-10 days stimulated both body weight gain and the incorporation of sulfate by cartilage from the treated rats, but no significant increase was observed in the characteristics of 125I-hGH binding. These results indicate that high levels of E2, GH, and/or Prl play an important role in the regulation of hGH binding sites in rat liver membranes. The restoration of binding sites in liver from hypophysectomized rats, however, apparently requires additional factors which are as yet unidentified. The role of the hGH binding sites in the physiologic actions of GH also remains to be determined.     

Pituitary binding and internalization of radioiodinated gonadotropin-releasing hormone agonist and antagonist ligands in vitro and in vivo

In rat pituitary gonadotrophs, the rates of binding and endocytosis of two GnRH superagonist analogs, [D-Ala6,Pro9-NEt]GnRH and [D-Lys6,Pro9-NEt]GnRH, were compared with those of the potent antagonist analog [N-acetyl-D-pCl-Phe1,2,D-Trp3,D-Lys6,D-Ala10]GnRH by quantitative electron microscopic autoradiography. In dispersed pituitary cells, the two agonist analogs showed similar binding kinetics and comparable degrees of sequestration, as measured by their resistance to dissociation by low pH buffer. However, quantification of silver grain localization suggested that cellular internalization of the [D-Ala6]GnRH agonist increased more rapidly than that of the [D-Lys6]GnRH analog. These discrepancies, and the finding that a larger amount of the specifically bound 125I-[D-Ala6]GnRH agonist was removed during glutaraldehyde fixation, indicated that the proportional internalization of this analog was over estimated by quantitative autoradiography owing to loss of cell surface-bound radioligand. We, therefore, employed radioiodinated D-Lys6-substituted analogs to analyze the receptor binding and cellular uptake of GnRH agonist and antagonist derivatives in vivo. After iv injection, a high proportion of the 125I-[D-Lys6]GnRH agonist was translocated into pituitary gonadotrophs within 60 min, whereas the D-Lys6 antagonist was predominantly associated with the plasma membrane during that time. Four hours after injection of the antagonist, an appreciable proportion of silver grains was associated with intracellular organelles, and this trend increased progressively at later time points. The relatively prolonged cellular processing of the GnRH antagonist is consistent with in vivo binding kinetics, and its slower internalization may reflect the basal rate of GnRH receptor turnover in the cell membrane. In addition, the marked difference between the rates of internalization of the bound [D-Lys6]GnRH agonist and antagonist ligands supports the proposal that receptor activation is responsible for the rapid endocytosis of agonist ligands by the GnRH-stimulated pituitary gonadotroph.     

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.     

Pituitary gonadotropin-releasing hormone receptor regulation in mice. II: Females

The regulation of pituitary GnRH receptors (GnRH-R) by gonadal steroids was examined in female mice housed in a constant environment (six to 8 per cage in same room as males). A 60% decrease in GnRH-R occurred 7 days after ovariectomy (OVX) (9.2 +/- 0.9 fmol/pituitary OVX vs. 25 +/- 2 for intact random estrous cycle controls). The receptor affinity (Ka 1.86 X 10(9) M-1) remained constant in intact and OVX female mouse pituitary particles. The pattern of GnRH-R fall after OVX was similar to that found in male mice, except that the GnRH-R decrease began some 6 h later than in males and serum LH also rose more slowly. Serum FSH was significantly elevated 6 h post OVX. In contrast to males, pituitary LH, in spite of a rapid fall (60%) at 12 h, regained the random, estrous cycle control value by 4 days post OVX and then increased to above this level. Pituitary FSH, unlike in males, remained at the intact value (3.1 +/- 0.24 micrograms/pituitary) up to 24 h post OVX and then gradually rose to 7.9 +/- 0.37 micrograms/pituitary on day 4 and 15.5 +/- 0.32 micrograms/pituitary on day 7. Treatment of OVX female mice with estradiol-17 beta (300 ng/day) attenuated the postcastration GnRH-R fall, and was more effective when combined with progesterone (375 micrograms/day). Progesterone alone was ineffective. The GnRH-R fall post OVX persisted for up to 2 months, despite elevated serum and pituitary LH and FSH levels. GnRH-R fell by 40% in lactating mice (20.6 +/- 0.95-lactating vs. 32.4 +/- 1.25 fmol/pituitary-random, estrous cycling females). Serum LH was reduced by 70% in lactating mice. These findings are qualitatively and quantitatively similar to those in lactating rats suggesting that, in this physiological situation, a similar mechanism may account for the receptor fall in both species. In sex reversed (Sxr) mice (genotypic female-phenotypic male) GnRH-R values were about 50% higher than those of intact normal male and normal, random estrous cycling, female values. This was the only situation in mice in which pituitary GnRH-R increases were observed to date. Serum and pituitary LH and FSH values in Sxr mice were elevated, especially when compared with normal, random estrous cycling female controls. The results indicate that pituitary GnRH-R of female mice fall in response to removal of gonadal steroid feedback, in the same way as males.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pituitary luteinizing hormone responses to intravenous and subcutaneous administration of gonadotropin-releasing hormone in men

Although differences in plasma GnRH concentrations have been identified after iv and sc injection of this peptide, differences in pituitary LH responses to iv and sc GnRH have not been evaluated in detail. We studied the magnitude and contour of plasma GnRH and LH responses after low doses of iv and sc GnRH administered to men with idiopathic hypogonadotropic hypogonadism and compared them to LH pulses in normal men after endogenous GnRH secretion. Mean areas under the LH response curves differed significantly (P less than 0.01) after 25 ng/kg, but not 250 ng/kg, iv and sc GnRH doses. The mean time from basal to peak plasma LH concentrations was significantly longer with sc than iv GnRH (P less than 0.02). In addition, individual LH responses were more variable with sc GnRH. Intravenous administration produced greater GnRH amplitude (P less than 0.001) and area under the curve (P less than 0.005) and shorter time to peak (P less than 0.01) GnRH concentrations. When plasma LH responses of similar area and amplitude were compared, the contour of LH responses after iv GnRH more closely simulated the LH pulses in normal men. These data demonstrate that 1) significant differences exist in the amplitude, contour, and variability of plasma LH and GnRH pulses after iv and sc GnRH; and 2) iv GnRH elicits LH secretory episodes which closely resemble endogenous pulsations of normal men. These results suggest that iv GnRH administration may be preferred in physiological studies and, if the data can be extrapolated to women, may account for the greater success of ovulation induction reported with iv GnRH.     

Characterization of gonadotropin-releasing hormone binding sites in the pituitary of the three-spined stickleback, Gasterosteus aculeatus

Binding sites for gonadotropin-releasing hormone (GnRH) in stickleback pituitary homogenates were characterized using an iodinated, superactive analog of salmon GnRH (sGnRH), D-Arg6-Pro9-sGnRH-NEt (sGnRHa). Binding of 125I-sGnRHa reached equilibrium after 60 min incubation at 4 degrees and was a function of tissue concentration. The specificity of 125I-sGnRHa binding was demonstrated by displacement with sGnRHa, sGnRH, and Buserelin [D-Ser(t-Bu)6-Pro9-GnRH-NEt]. Both Scatchard analyses of saturation data and displacement curves revealed a single class of high-affinity binding sites (Ka = 0.71 +/- 0.03 X 10(9) M-1, Bmax = 1087 +/- 165 fmol/mg protein).     

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.     

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.     

Growth hormone advances spermatogenesis in premature rats treated with gonadotropin-releasing hormone agonist

To clarify the effect of GH on the development of seminiferous tubules in premature male rats, we investigated whether GH accelerates spermatogenesis under the condition of gonadotropin deprivation. Male Wistar rats aged three weeks were divided into three groups and subjected to administration of either long-acting GnRH agonist (GnRHa) or a combination of GnRHa and rat GH, with normal saline solution as control. After the 4-week treatment, sperm density and motility in the right epididymis were measured and seminiferous tubules of right testes were histologically examined. Sperm density and motility were significantly higher in GnRHa+GH-treated rats than in GnRHa-treated rats. In histological examination, the numbers of germ cells in various stages were increased in GnRHa+GH-treated rats compared with GnRHa-treated rats, with the number of mature spermatid being noticeably higher in GnRHa+GH-treated rats. These results suggest that administration of GH decreases loss of germ cells at various stages of spermatogenesis under the condition of gonadotropin withdrawal.     

Changes in cellular levels of messenger ribonucleic acid encoding gonadotropin-releasing hormone in the anterior hypothalamus of female rats during the estrous cycle

Cellular levels of messenger RNA encoding GnRH were measured using quantitative in situ hybridization in coronal sections through the area of the organum vasculosum of the lamina terminalis of female rats examined at various times of the 4-day estrous cycle. GnRH mRNA levels were high on the morning of diestrus day 1, but declined throughout the day of diestrus day 2 to a nadir on the morning of proestrus. Although GnRH message levels were lowest on the morning of proestrus, they rose nearly two-fold by 1900h that evening and remained high during the day of estrus. These data support the hypothesis that GnRH synthesis is coupled to GnRH release, and indicate that GnRH biosynthesis is not stimulated on the morning of proestrus in preparation for the ovulatory surge release of GnRH and LH in the afternoon.     

Growth-associated protein-43 messenger ribonucleic acid expression in gonadotropin-releasing hormone neurons during the rat estrous cycle

We have shown previously at the ultrastructural level that morphological changes occur in the external zone of the median eminence allowing certain GnRH nerve terminals to contact the pericapillary space on the day of proestrus. The present study was designed to determine whether the intrinsic determinant of neuronal outgrowth, growth-associated protein-43 (GAP-43), was expressed in GnRH neurons of adult female rats, and whether its expression varied throughout the estrous cycle. To accomplish this, we perfusion-fixed groups of adult female rats at 0800 and 1600 h on diestrous day 2 (diestrous II), at 0800 h and 1600 h on proestrus, and at 0800 and 1600 h on estrus (n = 4 rats/group) and used double labeling in situ hybridization and quantification to compare the levels of GAP-43 messenger RNA (mRNA) in cells coexpressing GnRH mRNA. GnRH mRNA was detected with an antisense complementary RNA (cRNA) probe labeled with the hapten digoxigenin, whereas the GAP-43 cRNA probe was labeled with 35S and detected by autoradiography. In addition, GAP-43 protein was identified with immunohistochemistry in the median eminence. The results show that many GnRH neurons expressed GAP-43 mRNA and that GAP-43 protein was present in many GnRH axon terminals in the outer layer of the median eminence. The number of GnRH neurons expressing GAP-43 mRNA was significantly higher on proestrus (64 +/- 5%) than on diestrous II (40 +/- 2%; P < 0.001) or on estrus (45 +/- 8%; P < 0.05), and the GAP-43 mRNA levels in GnRH neurons also varied as a function of time of death during the estrous cycle. The GAP-43 mRNA levels in GnRH neurons were higher on proestrus and estrus than on diestrous II (P < 0.05). These data show that 1) GAP-43 is expressed in adult GnRH neurons; 2) GAP-43 mRNA expression in GnRH neurons fluctuates during the estrous cycle; and 3) GAP-43 mRNA content in GnRH neurons is highest on the day of proestrus, before and during the onset of the LH surge. These observations suggest that the increased GAP-43 mRNA expression in GnRH neurons on the day of proestrus could promote the outgrowth of GnRH axon terminals to establish direct neurovascular contacts in the external zone of the median eminence and thus facilitate GnRH release into the pituitary portal blood.     

Gap junctions between neuronal inputs but not gonadotropin-releasing hormone neurons control estrous cycles in the mouse

The role of gap junctions in the neural control of fertility remains poorly understood. Using acute brain slices from adult GnRH-green fluorescent protein transgenic mice, individual GnRH neurons were filled with a mixture of fluorescent dextran and neurobiotin. No dye transfer was found between any GnRH neurons, although approximately 30% of GnRH neurons exchanged neurobiotin with closely apposed cells. Dual electrophysiological recordings from pairs of GnRH neurons revealed an absence of electrical coupling. Using adult connexin 36 (Cx36)-cyan fluorescent protein transgenic mice, Cx36 was identified in cells within several hypothalamic brain regions, including 64% of preoptic area kisspeptin neurons but not in GnRH neurons. To assess the potential role of Cx36 in non-GnRH neurons within the GnRH neuronal network (i.e. neurons providing afferent inputs to GnRH neurons), a calmodulin kinase IIα-Cre (CKC)-LoxP strategy was used to generate mice with a neuron-specific deletion of Cx36 beginning in the first postnatal week. Mutant female mice exhibited normal puberty onset but disordered estrous cyclicity, although their fecundity was normal as was their estrogen-negative and -positive feedback mechanisms. The effects of adult deletion of Cx36 from neurons were assessed using a tamoxifen-dependent inducible CKC-Cx36 transgenic strategy. Mutant mice exhibited the same reproductive phenotype as the CKC-Cx36 animals. Together these observations demonstrate that there is no gap junctional coupling between GnRH neurons. However, it is apparent that other neurons within the GnRH neuronal network, potentially the preoptic kisspeptin neurons, are dependent on Cx36 gap junctions and that this is critical for normal estrous cyclicity.     

Age-related differences in the release of luteinizing hormone and gonadotropin-releasing hormone in ovariectomized rats

The effect of aging on the release of gonadotropin-releasing hormone (GnRH) in vitro and of luteinizing hormone (LH) both in vivo and in vitro in ovariectomized (Ovx) rats was studied. Old (21-24 months) and young (3-4 months) rats were Ovx before use. They were injected subcutaneously with estradiol benzoate (25 micrograms/kg) or sesame oil for 3 days and then challenged with GnRH (0.5, 2 or 10 micrograms/kg) via a jugular catheter. Blood samples were collected immediately before and at 5, 10, 20, 40 and 60 min following GnRH injection. For in vitro study, Ovx rats were decapitated. The anterior pituitary glands (APs) were incubated with GnRH (0.1 or 10 nM) and estradiol (0, 0.1, 1 or 10 nM) at 37 degrees C for 30 min. The mediobasal hypothalamus was superfused with Locke's solution at 37 degrees C for 210 min, and stimulated with 60 mM KCl at 90 and 150 min. The medium samples were collected at 10-min intervals. Concentrations of GnRH and LH in plasma and medium samples were measured by radioimmunoassay. In all rats, the basal and GnRH-stimulated levels of plasma LH were lower in old than in young rats. The spontaneous release of LH in vitro from APs of Ovx rats was increased by aging, whereas GnRH-stimulated release of LH in vitro was lower in old than in young animals. The potassium-stimulated, but not spontaneous, release of GnRH was lower in old than in young Ovx rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.