Corticotropin-releasing hormone inhibition of gonadotropin release and the effect of opioid blockade

We studied the inhibitory effect of exogenous CRH on pulsatile gonadotropin secretion and the role of endogenous opioid peptides in this phenomenon in normal women. To do so, we infused human CRH (100 micrograms/h for 3 h) into 15 normal women during the midluteal phase of their menstrual cycle and studied its effect on both basal (10 women) and GnRH-stimulated (5 women) plasma gonadotropin levels. CRH infusion induced a significant decrease in plasma LH and FSH levels in all women. The decline in plasma LH (62%) was greater than that in FSH (36%). Plasma LH and FSH concentrations returned to basal levels within 30 min after the end of the CRH infusion. CRH infusion did not alter the gonadotropin response to GnRH. We also infused naloxone plus CRH in the 10 women who had received CRH alone during the midluteal phase of a different cycle. Addition of naloxone to CRH (5 women) reversed the LH and FSH inhibition when naloxone was started 1 h after the start of the CRH infusion. When naloxone was started 1 h before CRH infusion (5 women), plasma LH and FSH concentrations did not change. Plasma cortisol increased similarly during both the CRH and CRH plus naloxone infusions; the mean cortisol levels at the end of the CRH and CRH plus naloxone infusions were 497 +/- 40 (+/- SE) and 484 +/- 41 nmol/L, respectively, compared to 240 +/- 14 nmol/L after saline infusion (P less than 0.001). These results demonstrate that in normal women during the midluteal phase of the menstrual cycle, CRH inhibits the secretion of both LH and FSH. The CRH-induced inhibition of gonadotropin secretion is primarily mediated by endogenous opioid peptides, and this effect is not dependent on glucocorticoid levels. We suggest that the disruptive effect of stress on reproductive function in the women could be, at least in part, dependent on decreased gonadotropin secretion induced by elevated endogenous CRH levels.     

Frequency and amplitude of gonadotropin-releasing hormone stimulation and gonadotropin secretion in the rhesus monkey

In adult ovariectomized rhesus monkeys bearing hypothalamic lesions which reduced circulating LH and FSH to undetectable levels, sustained elevated gonadotropin concentrations were reestablished by the intermittent administration of gonadotropin-releasing hormone (GnRH) at the rate of 1 microgram/min for 6 min once every hour. The effects of varying either the frequency or the amplitude of these GnRH pulses on gonadotropin secretion were examined in such animals. Increasing the frequency of GnRH administration from the physiological one pulse per h to two, three, or five pulses h while maintaining a constant infusion rate and pulse duration resulted in gradual declines in plasma gonadotropin concentrations. These declines were most profound at the highest frequencies and the consequence of reduced pituitary responses to individual GnRH pulses. Decreasing the frequency of GnRH pulses from one per h to one every 3 h led to variable declines in plasma LH levels, but circulating FSH invariably rose. Reducing the GnRH infusion rate from 1 to 0.1 mg/min while maintaining constant frequency and pulse duration resulted in abrupt declines in plasma LH and FSH to immeasurable levels, although pulsatile increments in circulating GnRH concentrations without a concomitant reduction in plasma LH concentrations, which remained unchanged. An infusion rate of 0.5 microgram/min resulted in unstable plasma LH and FSH levels. These results demonstrate that changes in the frequency or amplitude of hypophysiotropic stimulation have profound effects on plasma gonadotropin levels as well as on FSH to LH ratios in the circulation. The physiological implications of these observations are discussed.     

Inhibition of pituitary gonadotropin secretion by the gonadotropin-releasing hormone antagonist antide. I. In vitro studies on mechanism of action

The GnRH antagonist antide is among the most promising "third generation" compounds available for clinical evaluation. In primates, antide manifests prolonged (several weeks) and reversible inhibition of pituitary gonadotropin secretion after a single high dose injection. In the present study, we have examined the effects of antide on pituitary gonadotropin secretion in vitro. Dispersed anterior pituitary cells from adult female rats were plated (48 h; 5 x 10(5) cells/well), washed, and exposed to increasing concentrations of antide for up to 48 h. Media were removed, and cells were washed twice and then incubated with GnRH (1 x 10(-8) M) plus antide for 4 h. Media and cell lysates were assayed for LH/FSH by RIA. Antide had no effect on basal LH/FSH secretion at any dose tested (10(-6)-10(-12) M). In contrast, GnRH-stimulated LH/FSH secretion was inhibited by this GnRH antagonist in a dose- and time-dependent manner. When incubated simultaneously, antide blocked GnRH-stimulated gonadotropin secretion, with a maximal effect at 10(-6) M (ED50, 10(-7) M). Preincubation of pituitary cells with antide for 6-48 h before GnRH exposure shifted the dose-response curve to the left; the maximally effective dose was 10(-8) M; the ED50 was 10(-10) M antide after 48-h preincubation. Intracellular LH/FSH levels increased concomitant with the decrease in secreted gonadotropins. Total LH/FSH levels (secreted plus cell content) remained unchanged. The inhibition of LH secretion by antide was specific for GnRH-stimulated gonadotropin secretion; antide had no effect on K(+)-stimulated LH secretion. Moreover, antide had little or no residual effect on LH secretion; full recovery of GnRH responsiveness in vitro occurred within 4 h after removal of antide. Lineweaver-Burke analysis of antide inhibition of GnRH-stimulated LH secretion indicated that antide is a direct competitor of GnRH at the level of the pituitary GnRH receptor. In summary, antide is a pure antagonist of GnRH stimulation of gonadotropin secretion; no agonistic actions of antide were manifest in vitro. Moreover, antide has no apparent noxious or toxic effect on pituitary cells in culture; the actions of antide are immediately reversible upon removal of antide from pituitary gonadotropes. We conclude that the long term inhibition of gonadotropin secretion by antide in vivo is not due to deleterious effects of this compound at the level of the pituitary gonadotrope.     

Pharmacodynamics of gonadotropin-releasing hormone (GnRH). II. Pattern of GnRH delivery alters pituitary luteinizing hormone secretion in women

The pulse frequency, amplitude, and mode of administration of GnRH all influence gonadotropin secretion and, ultimately, pituitary-gonadal function. We studied plasma LH responses to repetitive iv administration of GnRH given hourly for 5 h as a 2-microgram rapid (less than 15 s) bolus dose or a 2-microgram dose infused for 15 min of each hour in seven women deficient in endogenous GnRH and sex steroids. Plasma LH levels, measured at 10-min intervals throughout the 5-h period, rose more briskly (pattern X time course interactions: F = 3.33; P less than 0.0001) to higher levels overall (F = 11.7; P = 0.014) after rapid bolus GnRH administration than after GnRH infusion. Plasma FSH levels increased during both modes of delivery, with higher responses to rapid bolus GnRH administration (P = 0.005). Plasma estradiol levels did not change during either 5-h study. We conclude that the pattern of delivery of GnRH is a determinant of pituitary LH and FSH secretion in untreated hypogonadotropic women, and therefore, that alterations in the GnRH wave form and/or peak plasma GnRH concentrations consequent upon different rates of GnRH entry into the blood-stream may explain the different responses that occur when GnRH is given by different routes.     

Calcium antagonists and hormone release. II. Effects of verapamil on basal, gonadotropin-releasing hormone- and thyrotropin-releasing hormone-induced pituitary hormone release in normal subjects

Although it has been well established that Ca2+ plays an essential role in the release of several hormones, very little is known of the interactions between Ca2+ and secretagogues in the process of pituitary hormone release. One possible way of studying the mechanism of action of hypothalamic releasing hormones is to study how organic calcium antagonists affect their action. Consequently, we infused the commonly used calcium antagonist, verapamil, into 20 normal subjects (10 men and 10 women; aged 19-37 yr) and studied its effects on both basal pituitary hormone levels and augmented hormonal release induced by gonadotropin-releasing hormone (GnRH) and TRH. Verapamil, infused at a rate of 5 mg/h for 3 h, induced a significant and marked suppression of circulating LH and FSH levels in both men and women. By the end of the infusion, the suppression of release was greater for LH (60%) than for FSH (54%). After the termination of the infusion, plasma gonadotropin concentrations returned progressively to basal levels within 2 h. Verapamil was also capable of blunting the peak incremental gonadotropin response to GnRH. Although the basal TSH concentration was apparently unaffected by verapamil, the incremental TSH response to TRH was significantly inhibited in both men and women. Verapamil infusion did not affect either the basal PRL concentration or the PRL response to TRH. Our data provide evidence that verapamil exerts different effects on the release of pituitary hormones in normal subjects. It inhibits the centrally mediated as well as the peripherally mediated gonadotropin release and blunts the TSH response to TRH. On the contrary, verapamil does not seem to affect basal or TRH-mediated PRl release. The use of organic calcium antagonists in experimental models in vitro as well as in vivo appears to offer a promising tool for further studies on the mechanism of action of secretagogues in the process of hormone release.     

Reduction in pituitary desensitization and prolongation of gonadotropin release by estrogen during continuous administration of gonadotropin-releasing hormone in women: its antagonism by progesterone

The present study was designed to elucidate gonadal steroid influences on gonadotropin release and subsequent pituitary desensitization to GnRH. Sixteen women, 10 of whom were normal and 6 of whom had hypogonadism, were infused with GnRH at rates ranging from 0.313-10 micrograms/h via an indwelling iv catheter for 66 h. Blood samples obtained throughout the GnRH infusion were analyzed for LH, FSH, estradiol, and progesterone. A prompt and substantial release of gonadotropin occurred in women with ovarian failure or during the luteal phase in normal women compared with that during the follicular phase of the menstrual cycle. Thereafter, a gradual decrease in gonadotropin secretion occurred due to pituitary desensitization, which was slower in the follicular phase than in other groups. A dose-related increase in integrated LH release occurred during GnRH infusion, but this response tapered off with administration of large doses of GnRH to women with ovarian failure or during the luteal phase. In contrast, it increased linearly up to the maximum dose of GnRH in the follicular phase. These data suggest that 1) basal levels of estrogen suppress the early rapid release of gonadotropin in response to GnRH and reduce subsequent pituitary desensitization, resulting in the prolonged release of LH; 2) estrogen widens the range of dose-related increases in gonadotropin in response to GnRH; and 3) these effects of estrogen are antagonized by progesterone.     

Effects of testosterone on gonadotropin subunit messenger ribonucleic acids in the presence or absence of gonadotropin-releasing hormone.

There is accumulating evidence that the negative feedback actions of testosterone on the pituitary may contribute to the differential regulation of FSH and LH secretion in males. In the present study we measured steady state levels of the mRNAs encoding the gonadotropin subunits in pituitary cell cultures treated with 10 nM testosterone (T) as well as in T-treated pituitary cells perifused with pulses of GnRH to explore further the direct actions of T on the pituitary. T treatment of pituitary cells in monolayer culture for 72 h increased FSH beta mRNA 1.5-fold (P less than 0.05), decreased alpha-subunit mRNA to 45% of the control level (P less than 0.05), and decreased LH beta mRNA to 75% of the control level (P less than 0.05). FSH and uncombined alpha-subunit secretion were increased and decreased by T, respectively, whereas basal LH secretion was unchanged. Treatment with 0.1 nM estradiol, a physiological concentration for males, did not change gonadotropin secretion or subunit mRNA concentrations. Between days 2 and 5 in culture in the absence of steroid treatment, steady state levels of LH beta and alpha-subunit mRNA declined (P less than 0.01) 52% and 61%, respectively, but FSH beta mRNA levels were unchanged. Pulsatile stimulation with 2.5 nM GnRH every 1 h for 10 h increased FSH beta mRNA 2.8-fold (P less than 0.05) and increased (P less than 0.05) alpha-subunit mRNA to 117% of the control level. When cell cultures were pretreated with T for 48 h and then perifused with pulses of GnRH, FSH beta, LH beta, and alpha-subunit mRNA levels were 66%, 74%, and 70% of the value during GnRH alone (P less than 0.05). T treatment also reduced (P less than 0.01) the amplitudes of FSH, LH, and alpha-subunit secretory pulses by 18%, 26%, and 41%, respectively. These data indicate that a portion of the negative feedback action of T is at the pituitary to regulate gonadotropin subunit gene expression. Our data reveal two opposing effects of T on FSH beta mRNA: a stimulatory action, which is GnRH independent, and an inhibitory effect, which is related to the actions of GnRH. These divergent actions of T represent one mechanism through which FSH and LH are differentially regulated.     

Progesterone blocks the estradiol-induced gonadotropin discharge in the ewe by inhibiting the surge of gonadotropin-releasing hormone.

Previous studies indicate an elevation of circulating progesterone blocks the positive feedback effect of a rise in circulating estradiol. This explains the absence of gonadotropin surges in the luteal phase of the menstrual or estrous cycle despite occasional rises in circulating estradiol to a concentration sufficient for surge induction. Recent studies demonstrate estradiol initiates the LH surge in sheep by inducing a large surge of GnRH secretion, measurable in the hypophyseal portal vasculature. We tested the hypothesis that progesterone blocks the estradiol-induced surge of LH and FSH in sheep by preventing this GnRH surge. Adult Suffolk ewes were ovariectomized, treated with Silastic implants to produce and maintain midluteal phase concentrations of circulating estradiol and progesterone, and an apparatus was surgically installed for sampling of pituitary portal blood. One week later the ewes were allocated to two groups: a surge-induction group (n = 5) in which the progesterone implants were removed to simulate luteolysis, and a surge-block group (n = 5) subjected to a sham implant removal such that the elevation in progesterone was maintained. Sixteen hours after progesterone-implant removal (or sham removal), all animals were treated with additional estradiol implants to produce a rise in circulating estradiol as seen in the follicular phase of the estrous cycle. Hourly samples of pituitary portal and jugular blood were obtained for 24 h, spanning the time of the expected hormone surges, after which an iv bolus of GnRH was injected to test for pituitary responsiveness to the releasing hormone. All animals in the surge-induction group exhibited vigorous surges of GnRH, LH, and FSH, but failed to show a rise in gonadotropin secretion in response to the GnRH challenge given within hours of termination of the gonadotropin surges. The surges of GnRH, LH, and FSH were blocked in all animals in which elevated levels of progesterone were maintained. These animals in the surge-block group, however, did secrete LH in response to the GnRH challenge. We conclude progesterone blocks the estradiol-induced gonadotropin discharge in the ewe by acting centrally to inhibit the surge of GnRH secreted into the hypophyseal portal vasculature.     

The frequency of gonadotropin-releasing-hormone stimulation differentially regulates gonadotropin subunit messenger ribonucleic acid expression

The hypothalamic decapeptide GnRH is known to regulate the synthesis and secretion of LH and FSH by pituitary gonadotrope cells. The frequency of pulsatile GnRH secretion changes and LH and FSH are differentially secreted in various physiological situations. To investigate the potential role of altered frequency of GnRH stimulation in regulating differential secretion of LH and FSH, we examined the effects of GnRH frequency on expression of the alpha, LH beta, and FSH beta genes. GnRH pulses (25 ng/pulse) were administered to castrate testosterone-replaced rats at intervals of 8-480 min to cover the range of physiological pulsatile GnRH secretion. Fast frequency GnRH pulses (8-min pulse intervals) increased alpha-subunit mRNA concentrations 3-fold above those in saline-pulsed controls (controls, 1.01 fmol cDNA bound/100 micrograms pituitary DNA) and LH beta mRNA by 50% (controls, 0.18 fmol cDNA bound), but FSH beta mRNA was unchanged (controls, 0.38 fmol cDNA bound). GnRH pulses given every 30 min increased all three subunit mRNAs (alpha, 3-fold, LHbeta, 2-fold; FSH beta, 2-fold), and acute LH release and serum FSH concentrations were maximal after this frequency. Slower frequency GnRH stimuli (120- to 480-min pulse intervals) did not change alpha and LH beta mRNA levels, but increased FSH beta mRNA 2- to 2.5-fold, and FSH secretion was maintained. Equalization of the total dose of GnRH given at different intervals over 24 h confirmed the frequency dependence of subunit mRNA expression. Fast frequency GnRH stimuli (8 min) increased alpha mRNA 1.5- to 2.5-fold, while the same total GnRH doses were ineffective when given at slow frequency (480 min). Similarly, LH beta mRNA was only increased by GnRH pulses given at 8-min intervals. In contrast, FSH beta mRNA increased 2-fold after pulses given every 480 min, and the 8-min pulse interval was ineffective. The data show that the frequency of GnRH stimulation can differentially regulate gonadotropin subunit mRNA expression and may be a mechanism that enables a single GnRH peptide to selectively regulate gonadotropin subunit gene expression and hormone secretion.     

In vivo administration of a GnRH antagonist to male mice: effects on pituitary gonadotropin secretion in vitro

The potent luteinizing hormone-releasing hormone antagonist [N-Ac-D-p-Cl-Phe1,2,D-Trp3,D-Arg6,D-Ala10]GnRH (4 mg/kg) was administered sc once or daily for 21 days to immune-deficient (nude) and normal immune-competent (NIC) male mice derived from the same genetic background. Effects of in vivo pretreatment with the antagonist on gonadotropin secretion from hemipituitary glands from both types of mice were studied in vitro in the presence or absence of synthetic GnRH. Treatment with the GnRH antagonist caused differential effects on release of FSH and LH from and amounts of FSH and LH in hemipituitary glands. Pituitary FSH secretion was effectively inhibited, whereas effects on pituitary LH were less evident or nonsignificant under these experimental conditions. Long-term treatment with the antagonist caused larger effects on pituitary secretion and content of FSH, when compared with short-term treatment. No significant effects of duration of treatment on secretion or pituitary content of LH were detected. Addition of synthetic GnRH to the incubation medium caused stimulation of gonadotropin release. Therefore, it was concluded that the high doses of this GnRH antagonist were not able to block GnRH receptors effectively in the pituitary glands of nude and NIC male mice. The incomplete suppression of LH secretion by this high dose of the GnRH antagonist may partly explain the inability of the antagonist to suppress plasma testosterone levels and the growth of androgen-dependent tumours in male mice.     

Gonadotropin and alpha-subunit responses to chronic gonadotropin-releasing hormone analog administration in patients with glycoprotein hormone-secreting pituitary tumors

Pituitary tumors secreting intact glycoprotein hormones (LH, FSH, and TSH) and/or alpha-subunit are being increasingly recognized. Because chronic administration of GnRH analogs decreases gonadotropin secretion in normal subjects, we investigated gonadotropin and alpha-subunit responses to chronic GnRH analog administration in five men with glycoprotein hormone-secreting pituitary tumors. Two patients (patients A and B) received the GnRH agonist analog (D-Trp6-Pro9-NEt-LHRH) for 4 weeks as a daily sc dose (8 micrograms/kg.day). In both, secretion of LH and/or alpha-subunit increased markedly. Subsequently, three patients received a higher analog dose (32 micrograms/kg.day) for a longer duration (8 weeks). One patient with a LH- and FSH-secreting tumor (patient C) had a highly significant (P less than 0.001) fall in serum LH and FSH concentrations; however, alpha-subunit secretion increased. During a subsequent study, when this patient received a lower dose (8 micrograms/kg.day) for 8 weeks, gonadotropin suppression also occurred. In two additional patients who received this dose (32 micrograms/kg.day), it had a persistent agonist effect on FSH beta (patient D) and alpha-subunit secretion (patient E). A marked increase in alpha-subunit secretion occurred in all five patients, regardless of whether basal serum alpha-subunit concentrations were elevated. These patients received the GnRH analog at doses 2-8 times greater than those that suppress gonadotropin secretion in normal men. Serum LH and FSH concentrations decreased in only one patient with a gonadotropin-secreting adenoma. The serum LH and FSH responses to acute GnRH stimulation did not predict the gonadotropin responses to chronic GnRH analog administration. Thus, gonadotropin and alpha-subunit production by most pituitary adenomas is augmented during chronic GnRH analog administration, consistent with defective GnRH desensitization in the adenomatous tissue. Despite the heterogeneous gonadotropin responses to the GnRH analog in these patients, serum alpha-subunit levels increased in all patients, indicating dissociation in the secretion of intact gonadotropins and alpha-subunit.     

Immunoneutralization of circulating inhibin in the hypophysiotropically clamped male rhesus monkey (Macaca mulatta) results in a selective hypersecretion of follicle-stimulating hormone

The purpose of this experiment was to examine directly whether inhibin is involved in the testicular regulation of FSH secretion in the male rhesus monkey. To this end, the pituitary-testicular axis in eight juvenile monkeys was prematurely activated by a chronic iv infusion of GnRH (0.1 microgram/min for 3 min every 3 h). After a minimum of 5 weeks of pulsatile GnRH stimulation, four animals received a brief (30-min) iv infusion of an ovine antiserum to inhibin alpha-subunit (approximately 10 ml/kg BW), and four monkeys received a comparable volume of a control ovine immune serum. The pulsatile GnRH infusion continued without interruption throughout the entire experiment. The FSH response to passive immunization against inhibin was determined by measuring concentrations of this gonadotropin in sequential plasma samples collected immediately before a GnRH infusion and for 3 h thereafter (an inter-GnRH pulse interval) on days 0.5, 1, 2, 4, 8, and 16 after injection of the immune serum. Administration of the antiserum to inhibin alpha-subunit resulted, within 2 days, in a 2- to 3-fold increase in the mean concentration and pulse amplitude of plasma FSH. The hypersecretion of FSH induced by administration of the antiserum to inhibin alpha-subunit was maintained until day 4, and then mean concentrations and mean pulse amplitudes of plasma FSH declined, reaching preantibody control levels by day 16. The time course of the antiserum-induced hypersecretion of FSH was closely correlated to changes in circulating inhibin-binding activity. Most importantly, the hypersecretion of FSH observed during the first 2 days after immunoneutralization of circulating inhibin was indistinguishable from that elicited during the initial 2 days after subsequent bilateral orchidectomy and concomitant testosterone (T) replacement. Administration of a control immune serum did not influence circulating FSH concentrations, and neither the antiserum to inhibin alpha-subunit nor the control immune serum induced changes in pituitary LH secretion and testicular T release. Since the exogenous drive to the pituitary-testicular axis of the animals was clamped in a mode that produced a pattern of pulsatile LH and T secretion comparable to that observed in adult monkeys, the present findings provide evidence for the view that inhibin plays a major role in the testicular regulation of FSH secretion during adulthood by exerting a selective inhibition on the secretion of this gonadotropin directly at the level of the anterior pituitary gland.     

Sustained inhibitory actions of a potent antagonist of gonadotropin-releasing hormone in postmenopausal women

The inhibitory time course and dose-related characteristics of a new potent GnRH antagonist peptide, [N-acetyl-D-pCl-Phe1,2-D-Trp3-D-Lys6-D-Ala10]GnRH, on gonadotropin secretion were studied in nine postmenopausal women. Effective suppression of gonadotropin secretion was correlated with increased circulating concentrations of immunoassayable GnRH antagonist. Inhibition of gonadotropin secretion was achieved by a parenteral dose of 300 micrograms/kg GnRH antagonist. This dose reduced plasma bioactive LH concentrations by 49-59%, immunoactive LH by 41-46%, and immunoactive FSH by 25-40%. Blockade of gonadotropin secretion was sustained for 10-28 h after a single injection of the synthetic decapeptide. This prolonged action was associated with significant plasma protein binding of the GnRH antagonist and mean plasma half-times of disappearance of 1.5 and 21 h for the fast and slow components, respectively. In summary, we have described the biological actions of a potent GnRH antagonist that binds avidly to serum proteins, has a prolonged plasma residence time, and exerts sustained inhibitory effects on bio- and immunoactive LH release in man. The extended duration of action of this compound may reflect in part its significant binding to circulating plasma proteins.     

BROMOCRIPTINE AND OESTROGEN MODULATION OF GONADOTROPHIN RELEASE IN NORMO-AND HYPERPROLACTINAEMIC PATIENTS WITH AMENORRHOEA

Effects of oestradiol, bromocriptine, and bromocriptine plus oestradiol, on basal and GnRH stimulated gonadotrophin concentrations were studied in normo-(group 1, n= 7) and hyperprolactinaemic (group 2, n= 6) patients with secondary amenorrhoea. Before drug administration, hyper-responsiveness of LH, but normal FSH responses to GnRH were observed in most patients. Oral administration of 2 mg oestradiol for 4 days resulted in increased 17β-oestradiol levels in plasma in normal women (n= 6) in the early follicular phase of the cycle, and in groups 1 and 2. During oestradiol administration plasma LH concentration increased significantly and there was an increase of LH and FSH responses to GnRH in normal subjects, but not in amenorrhoeic women. In groups 1 and 2 basal FSH levels were suppressed but no change in GnRH stimulated gonadotrophin responses was seen. Bromocriptine (5 mg per day for 5 days) significantly decreased prolactin concentrations and increased 17β-oestradiol levels in plasma in group 2 but not in group 1. The mean plasma 17β-oestradiol concentration had increased to levels similar to those obtained during oestradiol administration alone. The mean LH response to GnRH was suppressed in group 2, but not in group 1. Basal and GnRH-stimulated plasma FSH concentrations were not changed by bromocriptine treatment. Compared with the GnRH induced LH response during bromocriptine alone, bromocriptine treatment plus oestradiol administration resulted in a significantly increased LH response in group 2. This was not found in group 1. The present results suggest that there is an increased dopamine activity and inhibition of GnRH at the hypothalamic level and a relative dopamine deficiency at the pituitary level in hyperprolactinaemic patients. Normoprolac-tinaemic patients with hypothalamic amenorrhoea have increased dopaminergic activity at the hypothalamic, as well as the, pituitary level, or alternatively that the LH release is not influenced by dopamine in these patients. Finally bromocriptine sensitizes LH-secreting cells to GnRH in hyperprolactinaemic, but not in normoprolactinaemic, patients.     

Dexamethasone reduces the postcastration gonadotropin rise in women

To investigate the influence of glucocorticoids on gonadotropin release in humans, we studied the effects of dexamethasone (DXM) administration on basal and GnRH-stimulated gonadotropin secretion in normal women after bilateral ovariectomy (OVR). From the 7th to the 14th day after OVR, 9 women received DXM (2.25 mg/day) and 13 women received placebo (control women). Plasma FSH and LH concentrations were measured before OVR and daily from the 7th to the 14th day after surgery. In addition, the FSH and LH responses to exogenous GnRH (10 micrograms, iv bolus dose) were determined in all DXM-treated women and in 5 control women on the 7th and 14th days after surgery. Plasma gonadotropin levels increased similarly in all women on the 7th day after OVR. DXM administration significantly limited (P less than 0.001) the progressive rise of basal LH and FSH levels from days 7 to 14. DXM treatment also blunted (P less than 0.005) the OVR-induced increase in the responsiveness of both LH and FSH to exogenous GnRH. These findings suggest that glucocorticoids inhibit the secretion of both gonadotropins at the pituitary level in ovariectomized women.     

Control of the preovulatory gonadotropin surge in the ewe

The aims of this study were to determine the effect of ovariectomy on the release of LH and FSH during the preovulatory gonadotropin surge and to ascertain, by the use of sodium pentobarbitone (NaPb), if the secretion of these pituitary hormones requires continuous stimulation from the hypothalamus. Sheep were treated with NaPb for 2 h beginning 1) immediately before the gonadotropin surge, 2) during the ascending limb of the gonadotropin surge, and 3) during the descending limb of the gonadotropin surge. Ewes were ovariectomized (ovx) at each of the time periods listed above, and intact ewes included were at times 2 and 3. A group of intact ewes was given 100 microgram gonadotropin-releasing hormone (GnRH) in addition to NaPb at time 2, NaPb given during the ascending limb of the gonadotropin surge caused a transient fall in peripheral LH and FSH; however, the release of gonadotropins was reinitiated and the surge continued when the ewes recovered from anesthesia. Treatment with NaPb after the apex of the gonadotropin surge did not affect circulating levels of LH and FSH. Ewes given NaPb and ovx before the initiation of the gonadotropin surge released significantly less LH and FSH during the surge than the other treatment groups. The total amounts of LH and FSH released in intact and ovx ewes treated with NaPb after the surge was initiated were not different than those levels in the saline-treated controls. Intact ewes treated with 100 micrograms GnRH also released an amount of LH similar to that in the control group. We conclude that gonadotropin release from the pituitary gland requires the continual presence of GnRH during the ascending limb of the preovulatory gonadotropin surge, and that once the surge has been triggered, the ovaries do not appear to be required for further hypothalamic stimulation.     

Photoperiodic differences in in vitro pituitary gonadotropin basal secretion and gonadotropin-releasing hormone responsiveness in the golden hamster

In order to examine pituitary gonadotropin secretion and responsiveness to GnRH after photic-induced changes in reproductive condition, an in vitro pituitary perifusion system was established for male golden hamster tissue. Anterior pituitaries from adult males which had been maintained on 14 h light:10 h dark (long days) or 6 h light:18 h dark (short days) for 10 weeks were perifused using an Acusyst perifusion system. Perfusates from unstimulated tissue (basal secretion) and from tissue stimulated with hourly pulses of GnRH (25, 50, or 100 ng/ml) were assayed for LH and FSH by RIA. Tissue from short-day animals had lower basal LH secretion than tissue from long day animals, but there were no significant photoperiodic differences for GnRH-stimulated LH secretion. In contrast, there were no photoperiodic differences in basal FSH secretion, but tissue from short-day animals secreted more FSH than tissue from long-day animals when stimulated with GnRH. Bioactivity of a small number of perfusate samples was assessed using in vitro rat granulosa cell and mouse Leydig cell assays for FSH and LH, respectively, and did not show any photoperiodic differences in LH or FSH bioactivity for GnRH-stimulated tissue. These studies indicate that the pituitaries of gonadally regressed hamsters are capable in vitro of responding to GnRH with similar or greater levels of gonadotropin release compared to pituitaries from animals with functional gonads. Therefore, it appears that the lowered serum gonadotropin levels seen in vivo in gonadally regressed animals are not due to a reduction in intrinsic pituitary sensitivity to GnRH.     

Effects of leptin on gonadotropin secretion in juvenile female rat pituitary cells

OBJECTIVE: Leptin is an adipocyte-derived hormone, which is the product of the obese gene and it is thought to play important roles in pubertal development and maintenance of reproductive function in the female. In a study using adult male or female rats, it was found that leptin stimulated the secretion of gonadotropin directly from the pituitary in a dose-related manner. However, there is no study in juvenile female rats before puberty. METHODS: In this study, we cultured pituitary cells from 4-, 6- and 8-week-old female Wistar rats with leptin (0-10(-7)mol/l) and gonadotropin-releasing hormone (GnRH) (0 or 10(-8) mol/l). Basal or GnRH-stimulated secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and their synthesis within cells were determined by radioimmunoassay (RIA). RESULTS: Leptin induced bell-shaped dose--response curves of basal LH and FSH secretion from cultured cells of every age-group of rats studied. The most effective concentration of leptin on the basal secretion of LH and FSH from 6- and 8-week-old cultured pituitary cells was 10(-10) mol/l. This leptin concentration was consistent with circulating physiological serum leptin levels at each age. As for juvenile 4-week-old pituitary cells, the most effective concentration was 10(-11) mol/l which was lower than that of 6- and 8-week-old rats. It was consistent with the circulating serum leptin levels of 4-week-old rats. Also, the synthesis and the GnRH-stimulated secretion of LH and FSH were effectively controlled by leptin at concentrations similar to the serum leptin levels of given ages. CONCLUSIONS: Leptin induced pituitary cells to synthesize and secrete both LH and FSH regardless of the presence or absence of GnRH. The concentration of leptin that induced the greatest synthesis and secretion of gonadotropins from pituitary cells changed around the pubertal period. The most effective leptin concentrations in each experiment were similar to the physiological serum leptin level at each animal age. These results indicate that leptin stimulates gonadotrophs not only in the pubertal and the mature period but also in the juvenile period before puberty. It is also conceivable that leptin may modulate the sensitivity of gonadotrophs until the appearance of GnRH stimulation, and may be the factor that brings about puberty onset.     

Abrogation by a potent gonadotropin-releasing hormone antagonist of the estrogen/progesterone-stimulated surge-like release of luteinizing hormone and follicle-stimulating hormone in postmenopausal women.

In both the rodent and primate, administration of progesterone elicits an acute surge-like release of LH in the setting of prior estrogen treatment. Whether these facilitative effects of estrogen and progesterone on gonadotropin secretion reside at pituitary or hypothalamic loci is not known. To further investigate the mechanisms by which estrogen combined with progesterone amplifies gonadotropin secretion, we studied the responses of seven estrogen-primed postmenopausal women to progesterone administration with or without cotreatment with a potent GnRH antagonist, [Ac-D2Nal1,D4ClPhe2,D3Pal3,Arg5,DGlu6(AA), DAla10]GnRH. Repetitive blood sampling for the later measurement of serum concentrations of LH, FSH, and PRL was begun 4 h before the administration of progesterone and continued for 36 h. We observed that progesterone administration after 72 h of priming with ethinyl estradiol resulted in a surge-like release of LH and FSH in all subjects. Concomitant administration of the GnRH antagonist abolished the surge-like release of both gonadotropins in all subjects. In contrast, administration of the antagonist had no effect on PRL release. These results indicate that endogenous GnRH action is an obligatory component of the progesterone-induced surge-like release of both gonadotropic hormones in the estrogen-primed human.     

Testosterone differentially modulates gonadotropin subunit messenger ribonucleic acid responses to gonadotropin-releasing hormone pulse amplitude.

Testosterone (T) inhibits GnRH secretion and can also modulate the effects of GnRH on gonadotropin synthesis and secretion. To assess the effect of T on GnRH stimulation of alpha, LH beta, and FSH beta mRNA expression, we replaced T at three levels to reproduce low (1.5 +/- 0.5 ng/ml), medium (3.5 +/- 0.3 ng/ml), and high (6.2 +/- 0.6 ng/ml) physiological plasma concentrations. Additionally, as peripheral conversion to dihydrotestosterone (DHT) or estradiol (E2) may mediate T action, the effects of GnRH pulses in the presence of DHT and E2 were also studied. Male rats were castrated, and steroids were replaced via implants containing either T (three doses) or DHT or E2 (two doses each). GnRH pulses (10-250 ng/pulse) were administered iv at 30-min intervals for 48 h. Pituitary subunit mRNA concentrations, gonadotropin content, and LH and FSH secretion were determined. The patterns of alpha, LH beta, and FSH beta mRNA responses to increasing GnRH pulse amplitude were similar at all concentrations of plasma T. Alpha mRNA concentrations were increased 2- to 4-fold by GnRH pulses. At the same plasma T concentration, all doses of GnRH produced similar increases in alpha mRNA, but the response tended to be lower at the higher (6.2 ng/ml) levels of T. LH beta mRNA showed a clear dependence on GnRH pulse amplitude, with the maximum responses (2- to 3-fold) occurring after 10- to 25-ng GnRH pulses. At the higher (3.5 and 6.2 ng/ml) T concentrations, the dose-response curve was shifted to the left. The lowest GnRH pulse dose (10 ng) produced maximum responses, and LH beta mRNA increments in response to the higher GnRH doses were suppressed. FSH beta mRNA concentrations were increased by T in saline-pulsed controls. FSH beta mRNA responses were similar (2- to 3-fold) after all GnRH doses and at all concentrations of T. Increasing GnRH pulse doses reduced the pituitary content of both LH and FSH at all levels of T. Acute LH secretion was maximal after 10- and 25-ng pulses of GnRH when plasma T was low, but increased progressively with GnRH dose at the highest plasma T concentrations. Plasma FSH did not show any differential responsiveness to GnRH pulse dose or to increasing plasma T. Thus, LH synthesis and secretion are affected more than those of FSH by changing plasma concentrations of T. T may modulate posttranslational events in LH secretion. The higher GnRH doses effected LH release without increasing LH beta mRNA in the presence of higher physiological concentrations of T.(ABSTRACT TRUNCATED AT 400 WORDS)