Ovulation inhibition by administration of weekly gonadotropin-releasing hormone antagonist

To test the feasibility of administering the GnRH antagonist [(Ac-pClPhe1,pClPhe2,DTrp3,DArg6,DAla10) GnRH] intermittently to inhibit ovulation, this agent was given to normal ovulatory cynomolgus monkeys once weekly for 4 weeks. Ovulation was blocked in all females (eight of eight) throughout the 32 study weeks and resumed within 14.3 +/- 3.8 (+/- SEM) days in six of eight primates. Interestingly, mean tonic serum estradiol levels were not significantly reduced during treatment. Conversely, although midcycle levels of estradiol were not found, moderate estradiol levels occurred but they did not elicit preovulatory LH surges during the week after GnRH antagonist injection. In a second study directed at clarifying the mechanism(s) by which estrogen-induced LH surges were blocked, monkeys received GnRH antagonist in the early through the midfollicular phase of the menstrual cycle during which an estrogen (n = 3) or a GnRH (n = 4) challenge test was given on cycle day 6. Among monkeys receiving estradiol benzoate or a bolus dose of GnRH during the GnRH antagonist regimen, only those given GnRH (four of four monkeys) had increased LH secretion. These responses were similar to those of control monkeys (n = 3). Indeed, the pituitary was refractory (three of three monkeys) to estrogen-positive feedback for the LH surge. These findings indicate the potential utility of intermittent GnRH antagonist treatment to achieve contraception by ovulation inhibition, without creating a severely hypoestrogenic milieu attendant with the risks of negative sequelae effecting bone calcium loss, hot flushes, and atrophy of estrogen-dependent genital tissues.     

Suppression of pituitary-testis function in rats treated neonatally with a gonadotrophin-releasing hormone agonist and antagonist: acute and long-term effects

The acute and long-term effects of pituitary-testis suppression with a gonadotrophin-releasing hormone (GnRH) agonist, D-Ser(Bu(t))6des-Gly10-GnRH N-ethylamide (buserelin; 0.02, 0.1, 1.0 or 10 mg/kg body weight per day s.c.) or antagonist, N-Ac-D-Nal(2)1,D-p-Cl-Phe2,D-Trp3,D-hArg(Et2)6,D-Ala10 -GnRH (RS 68439; 2 mg/kg body weight per day s.c.) were studied in male rats treated on days 1-15 of life. The animals were killed on day 16 (acute effects) or as adults (130-160 days; long-term effects). Acutely, the lowest dose of the agonist decreased pituitary FSH content and testicular LH receptors, but with increasing doses pituitary and serum LH concentrations, intratesticular testosterone content and weights of testes were also suppressed (P less than 0.05-0.01). No decrease was found in serum FSH or in weights of accessory sex organs even with the highest dose of the agonist, the latter finding indicating continuing secretion of androgens. The GnRH antagonist treatment suppressed pituitary LH and FSH contents and serum LH (P less than 0.05-0.01) but, as with the agonist, serum FSH remained unaltered. Testicular testosterone and testis weights were decreased (P less than 0.01) but testicular LH receptors remained unchanged. Moreover, the seminal vesicle and ventral prostate weights were reduced, in contrast to the effects of the agonists. Pituitary LH and FSH contents had recovered in all adult rats treated neonatally with agonist and there was no effect on serum LH and testosterone concentrations or on fertility.(ABSTRACT TRUNCATED AT 250 WORDS)     

Suppression and recovery of pituitary gonadotrophin secretion in intact and orchidectomized rats treated neonatally with a gonadotrophin-releasing hormone antagonist

Suppression of neonatal rat pituitary-testis function by gonadotrophin-releasing hormone (GnRH) antagonists results in delayed sexual maturation and infertility. Since the mechanism is not understood, the acute effects of a GnRH antagonist on gonadotrophin secretion in neonatal male rats has been studied in more detail. Treatment with a GnRH antagonist analogue, N-Ac-D-Nal(2)1,D-p-Cl-Phe2,D-Trp3,D-hArg(Et2)6,D-Ala10 -GnRH (2 mg/kg per day) on days 1-10 of life had prolonged effects on gonadotrophin secretion; serum LH and FSH recovered in 1 week, but the pituitary content took 2 weeks to recover. Likewise, LH and FSH responses to acute in-vivo stimulation with a GnRH agonist were still suppressed 1 week after the treatment. Interestingly, a rebound (86% increase) in basal serum FSH was found 16 days after treatment with the antagonist. Whether testis factors influence gonadotrophin secretion during treatment with the GnRH antagonist and/or in the subsequent recovery period was also assessed. Neonatal rats were castrated on days 1, 5 or 10 of the 10-day period of antagonist treatment. Orchidectomy on days 1 and 5 only marginally affected gonadotrophin secretion. When orchidectomy was performed at the beginning of the recovery period, no effects on pituitary recovery were seen within 1 week of castration. After 16 days, serum LH and FSH in the antagonist-treated and control castrated rats were equally increased but the pituitary contents of the antagonist-treated rats were still suppressed. Finally, the effect of testosterone treatment on the recovery of gonadotrophin secretion after antagonist suppression was studied in intact and orchidectomized animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Pituitary responsiveness after administration of a GnRH agonist depot formulation: Decapeptyl CR

OBJECTIVE This study was focused on the pattern of LH release from the pituitary during the initial response to high dose GnRH agonist administration. Secondly, the pattern of LH release and the pituitary responsiveness to physiological and pharmacological stimulation during long-term pituitary suppression by a high dose GnRH agonist was studied. In addition, the relation between serum agonist levels and pituitary function and responsiveness was investigated. DESIGN oTrp'GnRH in microcapsules (Decapeptyl CR) was administered i.m. to 12 women on the third day of the cycle. High-rate blood sampling was carried out during the first 48 hours after the injection. Secondly, high-rate blood sampling for 6 hours and a GnRH challenge were performed before and weekly after administration, from week 4 till week 9. All samples were assayed for LH and FSH. LH patterns were analysed by applying a computerized pulse detection program. In the second or third week an oestradiol benzoate test was performed. Finally, trip-torelin levels were measured before and weekly after administration. PATIENTS Twelve patients, suffering from tubal infertility and recruited from the waiting list for in-vitro fertilization/ embryo transfer (IVF/ET) participated in the study. RESULTS During the first 48-hour period, LH and FSH levels demonstrated a rapid rise to peak values after 4 hours, subsequently declining to nearly normal levels. E2 rose to peak values at 12 hours and returned to the follicular range thereafter. LH pulse patterns showed a rapid increase in pulse intervals leading to a near absence of LH pulses at the end of the 48-hour period. From the fourth till the seventh week after agonist administration, LH pulse patterns showed a markedly increased pulse interval, decreased pulse amplitude, and a severely decreased mean LH level. In the same period, LH responses to GnRH were severely blunted or absent. Restoration of the pre-injection LH pulse pattern and the LH response to GnRH was observed during the eighth and ninth week. Oestradiol benzoate challenges showed an E2 rise to preovulatory levels in response to the injections. However, no changes were observed in LH and FSH concentrations. Triptorelin levels showed a peak within 48 hours and gradual decline towards pretreatment values in week eight. conclusions It is concluded from the study, that after administration of triptorelin depot in the early follicular phase, desensitization of the pituitary starts to develop within 24 hours. Pituitary responsiveness is completely absent in the second week and continues to exist until the eighth week after injection, when the agonist has disappeared from the circulation. These findings suggest profound alterations in GnRH receptor availability and post-receptor pathways, that prevent the pituitary from responding to physiological stimuli.     

Attenuation of preovulatory gonadotrophin surges by epostane: a new inhibitor of 3 beta-hydroxysteroid dehydrogenase

Epostane, an inhibitor of 3 beta-hydroxysteroid dehydrogenase, was administered orally to pro-oestrous rats to evaluate further a possible role for preovulatory progesterone secretion in eliciting surges of LH and FSH. Whereas a dose of 10 mg epostane/kg had essentially no effects on preovulatory gonadotrophin surges and ovulation, 200 mg epostane/kg markedly attenuated LH and FSH surges and blocked ovulation. A dose of 50 mg epostane/kg exerted effects on LH and FSH surges and ovulation intermediate between those of doses of 10 and 200 mg/kg. Plasma concentrations of progesterone were significantly lower in all anovulatory epostane-treated rats at 18.00 and 22.00 h on proestrus than those measured in vehicle-treated rats. Concurrent injection of 2 mg progesterone in rats given 200 mg epostane/kg restored gonadotrophin surges to normal, but consistently failed to reverse the inhibitory effects of epostane on ovulation. Peak plasma progesterone levels produced by the progesterone injections were eight- to tenfold higher than the highest levels measured in vehicle-treated rats during the afternoon of pro-oestrus. Insertion of progesterone capsules was less effective than injections of progesterone in restoring gonadotrophin surges to normal, even though peak plasma progesterone concentrations achieved after insertion of two 20 mm long progesterone capsules were double the peak progesterone concentrations measured in control rats. Nevertheless, taken together with recent reports showing attenuation of preovulatory gonadotrophin surges by the progesterone antagonist RU 486 (17 beta-hydroxy-11 beta-[4-dimethyl-aminophenyl]-17 alpha-[prop-1-ynl] estra-4,9-diene-3-one), the present results provide support for a role of preovulatory progesterone secretion in enhancing oestrogen-dependent LH/FSH surges on pro-oestrus.     

Influence of sodium valproate on late follicular phase pulsatile LH secretion in normal women

OBJECTIVE: The present study was designed to further assess the mechanism of action of GnRH and GnRH analogues. DESIGN AND PATIENTS: Both the Nal-Glu GnRH antagonist and the D-Trp6 GnRH agonist were administered sequentially to nine normal, post-menopausal women. MEASUREMENTS: A baseline study of pulsatile LH, FSH and free alpha-subunit secretion was performed, with sampling every 10 min for 8 h, and then repeated 8 h after a single subcutaneous injection of Nal-Glu GnRH antagonist (5 mg). Sampling was repeated 21 days after the intramuscular injection of a depot preparation of D-Trp6 GnRH (3.75 mg) in the same women. RESULTS: The baseline sampling period showed synchronous pulses of LH and free alpha-subunit. The antagonist Nal-Glu decreased plasma LH (71%) and free alpha-subunit (43%). However, with the single dose of 5 mg, pulsatile LH and free alpha-subunit release were not completely suppressed and remained temporally correlated. The GnRH agonist had a potent inhibitory action on plasma immunoreactive LH (IRMA) (93%). In contrast, it increased the mean plasma levels of free alpha-subunit from 1.66 +/- 0.01 to 5.06 +/- 0.02 micrograms/l (205%). The pulsatile secretory patterns of both LH and free alpha-subunit were abolished by the agonist. Immunoreactive FSH levels were decreased by the antagonist (24%) and suppressed by the agonist (93%). CONCLUSIONS: The pulsatile study confirms the different mechanism of action of GnRH analogues. Following antagonist administration, low amplitude free alpha-subunit pulses persist and are synchronous with residual LH pulses. In contrast, LH and free alpha-subunit are not maintained under agonist treatment. These data provide evidence for the differential regulation of LH and free alpha-subunit by GnRH.     

Gonadotropin-releasing hormone regulates follicle-stimulating hormone beta-subunit gene expression in the male rat

Since the role of GnRH in the control of FSH release and synthesis is controversial, we have examined the effect of elimination of GnRH action on gonadotropes on FSH beta gene expression, FSH release, and synthesis. GnRH stimulation of the pituitary was abolished by continuous infusion of either a GnRH antagonist or a GnRH antiserum. We also examined the effects of gonadotrope desensitization, using a continuous infusion of GnRH or GnRH agonist analog. FSH beta mRNA levels were determined by dot blot hybridization using rat FSH beta cDNA, and changes were related to pituitary and serum FSH concentrations. FSH beta mRNA levels increased after orchidectomy and correlated well with serum FSH concentrations. Overall FSH synthesis was increased after castration, as judged by elevated serum FSH and unchanged pituitary FSH content. In orchidectomized rats, continuous GnRH antagonist infusion prevented the postcastration rise in FSH beta mRNA levels and serum FSH. Pituitary FSH content was reduced at 7 days, but not at 14 days. In intact rats, GnRH antagonist infusion for 7 days had no effect on FSH beta mRNA levels, but after 14 days, there was a 33% reduction, and serum FSH was suppressed. Pituitary FSH content was decreased after GnRH antagonist treatment for 7 or 14 days. Daily injection of GnRH antiserum for 6 days abolished the increases in FSH beta mRNA levels and serum FSH in orchidectomized rats, but pituitary FSH content was unaffected. In intact rats, GnRH antiserum treatment reduced FSH beta mRNA levels by 38%, suppressed serum FSH, and decreased pituitary FSH content. When gonadotropes were desensitized by a continuous infusion of GnRH for 14 days or GnRH agonist analog for 28 days, FSH beta mRNA levels and pituitary FSH content were markedly reduced, and serum FSH was suppressed to undetectable levels. We concluded that 1) endogenous GnRH is required for the maintenance of FSH beta mRNA levels in both intact and orchidectomized rats; 2) FSH beta mRNA levels are coupled to the level of FSH biosynthesis, indicating the physiological importance of this pretranslational regulation; 3) desensitization is more effective at inhibiting FSH beta gene expression and FSH synthesis than preventing gonadotrope stimulation using the GnRH antagonist or antiserum; and 4) the actions of GnRH on FSH beta mRNA levels are paralleled by its effects on LH beta mRNA levels, suggesting that GnRH provides a common primary stimulus for the induction of both beta-subunit genes in vivo. These data provide further evidence for the crucial stimulatory role of GnRH in the control of FSH synthesis.     

Endocrine profiles after triggering of final oocyte maturation with GnRH agonist after cotreatment with the GnRH antagonist ganirelix during ovarian hyperstimulation for in vitro fertilization

In a randomized multicenter study, the efficacies of two different GnRH agonists were compared with that of hCG for triggering final stages of oocyte maturation after ovarian hyperstimulation for in vitro fertilization. Ovarian stimulation was conducted by recombinant FSH (Puregon), and the GnRH antagonist ganirelix (Orgalutran) was coadministered for the prevention of a premature LH rise. Luteal support was provided by daily progestin administration. Frequent blood sampling was performed at midcycle in the first 47 eligible subjects included in the current study, who were randomized for a single dose of 0.2 mg triptorelin (n = 17), 0.5 mg leuprorelin (n = 15), or 10,000 IU hCG (n = 15). Serum concentrations of LH, FSH, E2, and progesterone (P) were assessed at variable intervals. LH peaked at 4 h after both triptorelin and leuprorelin administration, with median LH levels of 130 and 107 IU/liter (P < 0.001), respectively. LH levels returned to baseline after 24 h. Subjects receiving hCG showed peak levels of 240 IU/liter hCG 24 h after administration. A rise in FSH to 19 IU/liter (P < 0.001) was noted in both GnRH agonist groups 8 h after injection. Within 24 h the areas under the curve for LH and FSH were significantly higher (P < 0.001) in both GnRH agonist groups compared with that for hCG. E2 and P levels were similar for all groups up to the day of oocyte retrieval. Luteal phase areas under the curve for P and E2 were significantly elevated (P < 0.001) in the hCG group. The mean (+/-SD) numbers of oocytes retrieved were 9.8 +/- 5.4, 8.7 +/- 4.5, and 8.3 +/- 3.3; the percentages of metaphase II oocytes were 72%, 85%, and 86%; and fertilization rates were 61%, 62%, and 56% in the triptorelin, leuprorelin, and hCG group, respectively (P = NS for all three comparisons). These findings support the effective induction of final oocyte maturation in both GnRH agonist groups. In summary, after treatment with the GnRH antagonist ganirelix for the prevention of premature LH surges, triggering of final stages of oocyte maturation can be induced effectively by a single bolus injection of GnRH agonist, as demonstrated by the induced endogenous LH and FSH surge and the quality and fertilization rate of recovered oocytes. Moreover, corpus luteum formation is induced by GnRH agonists with luteal phase steroid levels closer to the physiological range compared with hCG. This more physiological approach for inducing oocyte maturation may represent a successful and safer alternative for in vitro fertilization patients undergoing ovarian hyperstimulation.     

Endogenous excitatory amino acid involvement in the preovulatory and steroid-induced surge of gonadotropins in the female rat

The physiological role of N-methyl-D-aspartate (NMDA) receptors in the regulation of preovulatory and steroid-induced surges of gonadotropins in the female rat was examined. The specific and potent noncompetitive NMDA receptor antagonist MK801 was used for blockade of NMDA neurotransmission. MK801 treatment completely inhibited the ability of progesterone to induce LH and FSH surges in the estrogen-primed ovariectomized rat. Administration of MK801 on proestrus in the immature female rat primed with PMSG resulted in a significant attenuation of the proestrous LH, FSH, and PRL surge and a corresponding attenuation of ovulation. Similarly, in the adult cycling female rat, MK801 administration on proestrus led to a significant attenuation of the proestrous LH and PRL surges. Mean FSH levels were lower in MK801-treated adult rats than in vehicle-treated rats, but this effect was not significant. In the estrogen-primed ovariectomized immature rat, the agonist NMDA caused a rapid (less than 10 min) elevation of LH and FSH in vivo. The gonadotropin-releasing effect of NMDA may be mediated at the level of the hypothalamus, since the medial basal hypothalamus/preoptic area of NMDA-treated rats killed 3 and 5 min post-NMDA had a significantly greater release of GnRH in vitro than that of vehicle-treated rats. In conclusion, these findings demonstrate that the preovulatory gonadotropin surge in the female rat is dependent on NMDA neurotransmission for its expression and add further evidence for a critically important role for NMDA receptors in the physiological regulation of gonadotropin secretion in the female rat.     

Gonadotropin-releasing hormone analogs stimulate and testosterone inhibits the recovery of spermatogenesis in irradiated rats

We investigated the effects of GnRH analogs, different doses of testosterone (T), an androgen receptor antagonist (flutamide), and combinations of these on the recovery of spermatogenesis after irradiation. Treatment with a GnRH agonist (Lupron) for 10 weeks after irradiation reduced the intratesticular T concentration (ITT) to 4% of that in irradiated rats and serum FSH to undetectable levels without altering serum LH levels. Injection of a GnRH antagonist (Cetrorelix) at 3 weeks after irradiation suppressed LH, FSH, and ITT to <7%, 32%, and 10%, respectively, of levels in irradiated-only rats within 2 weeks; suppression was maintained for approximately 3 to 4 weeks. The percentage of tubules with differentiated germ cells (repopulation index, RI) was <0.6% at weeks 10 to 20 after irradiation. Spermatogenic recovery was induced by both the GnRH agonist (RI = 58% at week 10; 91% at week 20) and antagonist (RI = 70% at week 13). There was a dose-dependent suppression of testicular germ cell repopulation when T was combined with GnRH analogs. The ability of T to abolish the spermatogenic stimulatory effect of the GnRH antagonist was evident by the similar RI obtained for irradiated rats given antagonist + T or T alone. This suppression of GnRH-induced recovery of spermatogenesis by T could be reversed by flutamide. The RI best correlated with the degree of ITT suppression. In ITT-suppressed rats, the RI also showed an inverse correlation with serum T levels. Thus, T and/or its androgenic metabolites either directly or indirectly inhibit spermatogenic recovery after irradiation through an androgen receptor-mediated process. In addition, there was a close negative correlation between RI and FSH levels, and hence, a spermatogenic inhibitory role for FSH in the irradiated rats cannot be ruled out.     

Testosterone and dihydrotestosterone, but not estradiol, selectively maintain pituitary and serum follicle-stimulating hormone in gonadotropin-releasing hormone antagonist treated male rats

Recently it has been found that testosterone can maintain and restimulate serum and pituitary follicle-stimulating hormone (FSH) in the gonadotropin-releasing hormone (GnRH) antagonist treated adult male rat. The present investigation was undertaken to determine (1) which metabolite of testosterone, dihydrotestosterone (DHT), or estradiol accounts for the effects of testosterone in GnRH antagonist suppressed rats and (2) whether these effects of testosterone are influenced by other testicular factors. Eight groups of 6-8 adult male Sprague-Dawley rats were subjected to the following treatments: vehicle, GnRH antagonist (75 micrograms/day s.c.), testosterone-filled Silastic implants (3 x 5 cm, s.c.), DHT-filled Silastic implants (3 x 5 cm, s.c.), estradiol benzoate (15 micrograms/day s.c.), and combined administration of GnRH antagonist with either steroid. In addition, the GnRH antagonist/testosterone treatment regimen was applied to rats orchidectomized 72 h prior to initiation of treatments. After 3 weeks of treatment, serum was analyzed for concentrations of luteinizing-hormone (LH), FSH, testosterone, DHT, and estradiol. Pituitary extracts were analyzed for LH and FSH content. Except for the vehicle-treated groups, serum and pituitary LH concentrations were markedly suppressed by all treatments. In intact rats treated with GnRH antagonist alone and/or estradiol, the pituitary FSH level was reduced by more than 70% relative to controls, while both testosterone and DHT maintained pituitary FSH. Similarly, testosterone and DHT, but not estradiol, delayed the decline of serum FSH induced with GnRH antagonist alone. In orchidectomized animals, testosterone was also capable of preventing a reduction of pituitary FSH despite concomitant GnRH antagonist administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gonadotropin-releasing hormone agonists are unsuccessful in reducing tumoral gonadotropin secretion in two patients with gonadotropin-secreting pituitary adenomas

Whether GnRH agonist treatment leads to reduced gonadotropin secretion and tumor volume in patients with gonadotropin-secreting pituitary adenomas is controversial. We studied the effect of GnRH analog treatment in two such patients, one with a recurrent FSH- and LH-secreting pituitary adenoma (patient 1) and one with a recurrent FSH- and alpha-subunit-secreting pituitary adenoma (patient 2). Patient 1 was treated with 200 micrograms Buserelin daily for 65 days, and patient 2 received three injections of 3 mg [D-Trp6]-LHRH formulated in microcapsules at 21-day intervals. In both patients, plasma FSH, LH (RIA), and alpha-subunit concentrations increased initially and remained above the pretreatment values throughout the treatment period. Plasma LH, measured by immunoradiometric assay, remained well above the detection limit. Plasma bioactive LH and testosterone became undetectable in patient 2, but did not change in patient 1. In neither patient did pituitary tumor size (determined by computed tomographic scan) change during treatment. We conclude that 1) the overall effect of GnRH analogs in patients with gonadotroph cell adenomas is stimulation of gonadotropin release by the tumor, although LH release varies according to how plasma LH is measured, possibly related to the origin of the hormone (normal or tumor gonadotroph cells), and 2) GnRH analog treatment does not reduce tumor size.     

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.     

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.     

Dopamine agonist- and antagonist-induced modulation of pituitary gonadotrophin releasing hormone receptors are independent of changes in serum prolactin

The effect of drug-induced hypo- and hyperprolactinaemia on pituitary gonadotrophin releasing hormone receptors (GnRH-R), serum and pituitary gonadotrophins (LH and FSH) and prolactin was investigated in intact adult male and female rats. Hypoprolactinaemia (serum prolactin less than 20% of control values) resulting from dopamine agonist (bromocriptine) infusion (4 mg/kg per day for 7 days) was accompanied by a 40-50% increase in GnRH-R in both male and female animals, though this was not accompanied by any major change in serum or pituitary LH and FSH. Hyperprolactinaemia (serum prolactin greater than ten times control values) induced by the dopamine receptor antagonist metoclopramide (65 mg/kg per day for 7 days) increased GnRH-R between 35 and 45% in both male and female rats without altering serum gonadotrophins. Domperidone (1 mg twice daily for 14 days) also increased GnRH-R by 50% but only in female rats. Both dopamine antagonists significantly increased pituitary prolactin content. Pituitary FSH increased in female rats treated with both metoclopramide and domperidone. The stimulatory effects of bromocriptine and metoclopramide on GnRH-R in male rats were prevented by concurrent treatment with a GnRH antiserum, suggesting that the drug effects were mediated through alteration in endogenous GnRH secretion. Induction of massive (serum prolactin greater than 2000 micrograms/l) hyperprolactinaemia in male and female rats with a transplantable prolactin-secreting pituitary tumour did not reduce GnRH-R concentration, although serum gonadotrophins were suppressed and pituitary gonadotrophin content was increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of corticosterone and testosterone on pituitary gonadotropin content, secretion, bioactivity and messenger RNA levels in the presence or absence of GnRH in male rats

The effects of corticosterone (B) and testosterone (T) on pituitary and serum bioactive and immunoreactive gonadotropins and on gonadotropin hormone subunit messenger RNA levels were compared in the absence of GnRH. Male rats were implanted with pellets of either cholesterol, B or T. At implantation, 2 and 4 days later half of each group received GnRH antagonist and animals were killed 5 days after implantation. As expected, GnRH antagonist lowered bioactive and immunoreactive serum FSH and LH, pituitary FSH, LHβ and FSHβ mRNA. B treatment alone lowered bioactive and immunoreactive serum FSH and immunoreactive serum LH. B reversed the antagonist effect on bioactive and immunoreactive pituitary FSH and FSHβ mRNA. T alone lowered bioactive and immunoreactive serum FSH and LH levels. T reversed the antagonist effect on bioactive and immunoreactive pituitary FSH. T lowered bioactive and immunoreactive pituitary LH and LHβ mRNA and partially reversed the antagonist effect on FSHβ mRNA. The data suggest that either B or T enhance FSH synthesis by acting directly at the gonadotrope, but that B does not affect LH variables to the same extent as T. The results suggest that in stressed animals, when T levels are reduced, B can substitute for T in sustaining FSH synthesis.     

Gonadotrophin-releasing hormone modulation of gonadotrophins in the ewe: evidence for differential effects on gene expression and hormone secretion.

While the regulation of gonadotrophin secretion by gonadotrophin-releasing hormone (GnRH) has been well documented in both rats and sheep, its role in the synthesis of gonadotrophin subunits remains unclear. We have investigated the effects of the specific inhibition of GnRH by a GnRH agonist on the expression of gonadotrophin subunit genes and the subsequent storage and release of both intact hormones and free alpha subunit. Treatment with GnRH agonist for 6 weeks abolished pulsatile LH secretion, reduced plasma concentrations of FSH and prevented GnRH-induced release of LH and FSH. This was associated with a reduction of pituitary LH-beta mRNA and FSH-beta mRNA levels (to 5 and 30% of luteal control values respectively), but not alpha mRNA which was significantly increased (75% above controls). While there was a small decrease in the pituitary content of FSH (30% of controls), there was a drastic reduction in LH pituitary content (3% of controls). In contrast to the observed rise in alpha mRNA, there was a decrease in free alpha subunit in both the pituitary and plasma (to 30 and 80% of control levels). These results suggest that, while GnRH positively regulates the expression of both gonadotrophin beta-subunit genes, it can, under certain circumstances, negatively regulate alpha-subunit gene expression. Despite the complete absence of LH and FSH in response to GnRH, there remained a basal level of beta-subunit gene expression and only a modest reduction (50%) in the plasma levels of both FSH and LH, suggesting that there is a basal secretory pathway.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cortisol regulates secretion and pituitary content of the two gonadotropins differentially in female rats: effects of gonadotropin-releasing hormone antagonist.

To determine if LH and FSH respond to cortisol exposure the same way in females as they do in males, metestrous females were implanted with cholesterol or cortisol (F) subcutaneously, and either ovariectomized or left intact 4 days later. Tail vein injections of 1000 ng of GnRH in saline, or saline alone, were given 4.5, 23.5, or 47.5 h after the time of ovariectomy. Animals were killed 30 min after the injections at 5, 24, and 48 h after surgery. F attenuated the postovariectomy increase in serum LH at 48 h. F also suppressed GnRH-stimulated LH release 24 and 48 h after surgery in ovariectomized animals and in intact animals at 48 h. Pituitary content of LH was increased moderately by F at 5 h. These effects of F are similar to those seen in males. In contrast to LH, F increased serum FSH in intact females and suppressed levels in ovariectomized animals at 24 and 48 h, while inducing a remarkable increase in pituitary FSH content at all three times. These divergent effects of F on serum FSH (suppression in gonadectomized and stimulation in intact groups) were not seen in males, and the increase in pituitary FSH as a result of exposure to F was much more profound and reliable in females than in males. To determine if the F-induced increase in pituitary FSH was dependent on endogenous secretion of GnRH, intact metestrous females were implanted with either cholesterol or F pellets. Each implant group received sc injections of 100 micrograms GnRH antagonist or control injections every 48 h beginning at the time of steroid implantation. Animals were killed 5 days after implantation. The antagonist suppressed both serum and pituitary LH. F also suppressed serum LH levels, but had no effect on pituitary content of LH. Neither the antagonist nor F affected serum FSH. F greatly increased pituitary content of FSH in the presence or absence of GnRH antagonist. These data suggest that 1) LH responds to F treatment in a similar way in females and males; 2) pituitary FSH content is more sensitive to the enhancing effect of F in females than in males; 3) the ability of F to increase pituitary FSH in females is not dependent on GnRH.     

The antigonadotropic activity of a 19-nor-progesterone derivative is exerted both at the hypothalamic and pituitary levels in women

We have previously shown in postmenopausal women that a 19-nor-progesterone derivative, nomegestrol acetate (NOMA) had a strong antigonadotropic activity and that this effect was not mediated via the androgen receptor. The aim of the present study was to further assess the action of this progestin on gonadotropin secretion in women. To demonstrate at which level of the hypothalamo-pituitary-ovarian axis the gonadotropin inhibition was exerted, 10 normally cycling (NC) women, 3 women with a gonadotropin-independent ovarian function [McCune-Albright (MCA) syndrome], and 5 women with functional hypothalamic amenorrhea (FHA) participated in the study. NC women were treated orally with 5 mg NOMA for 21 days, after one control cycle. Plasma estradiol (E2) and progesterone, LH, and FSH levels were measured during each cycle. A frequent sampling study (every 10 min for 4 h), followed by a classic GnRH test (100 microg, i.v.), was performed on day 11. Women with MCA were studied before, during NOMA, and after long-acting GnRH agonist administration. In women with FHA, pulsatile GnRH (20 microg s.c., every 90 min) was given for two cycles with or without NOMA (5 mg for 21 days). In all NC women, ovulation was suppressed by NOMA. Mean plasma LH levels, LH pulse frequency, and the LH response to exogenous GnRH were significantly decreased. In MCA, neither NOMA nor GnRH agonist modified multiple ovarian cysts on ultrasound or plasma E2, levels which remained elevated, ruling out a direct ovarian effect. In FHA, pulsatile GnRH administration recreated a normal ovulatory menstrual cycle. Addition of NOMA prevented the increase of plasma E2, decreased the amplitude of LH pulses, and prevented ovulation. In view of this unexpected action of NOMA at the pituitary level, seven samples of normal human female pituitaries were tested for the presence of progesterone receptor (PR) using a double labeling immunocytochemical technique. The presence of PR was detected in the seven human pituitary tissues. In addition, PR was found to be expressed only in gonadotroph cells. In conclusion, NOMA, a 19-nor-P derivative, has a potent antigonadotropic activity exerted at the hypothalamic level, inhibiting ovulation in NC women. In women with FHA, NOMA decreased the gonadotropin stimulation induced by pulsatile GnRH administration. According to the presence of PR in gonadotroph cells of normal human pituitaries, 19-nor-progesterone derivatives may also act on the gonadotropin secretion at the pituitary level.