Evaluation of in vitro incubation systems for the study of gonadotropin release.

A detailed study was undertaken in order to determine if a pituitary-half incubation system were a suitable model for the study of anterior pituitary response to estradiol and LHRH. Considerable variation in the gonadotropin content of randomized pituitary halves was observed. Much less variation was found in matched halves. During the initial thirty minutes incubation of pituitary halves, a large spontaneous release of gonadotropins was observed. Time course secretion studies indicated that by four hours incubation, in the presence of 50 ng/ml LHRH, cumulative secretion of LH and FSH had far exceeded that of controls. Elevations in both cumulative secretion and rate of secretion were evident within 15-30 minutes of incubation. Regardless of LHRH dose, only 2-4% of either gonadotropin was secreted. Estradiol in the range of 10, 100, 500, 1,000 and 50,000 pg/ml had no significant effect on pituitary response to LHRH or on basal release, tissue levels or total gonadotropin. Based on these results, it was concluded that while the pituitary-half incubation system may be suitable for studying LHRH induced gonadotropin secretion, it is apparently of insufficient sensitivity to allow the collection of meaningful data concerning the effects of estradiol alone on gonadotropin secretion or estradiol modulation of LHRH induced gonadotropin secretion.     

Triiodothyronine (T3) modulation of gonadotropin secretion in the female rat.

The effect of T3 upon gonadotropin secretion was examined in ovariectomized (Ovarx), Ovarx thyro-parathyroidectomized (Ovarx-TxPx), or proestrus rats. T3 (50 microgram/-100 gBW), administered late diestrus-2, abolished the LH surge during the critical period of proestrus in 7 out of 9 rats; the rise in sera FSH was not inhibited, although a distinct peak was absent. Administration of 5 or 50 microgram T3/100gBW 2.5h before the critical period resulted in either a suppression or an alteration of the timing of LH release. In the 5 microgram T3/100gBW treated animals the sera FSH peak was delayed in timing, whereas in the 50 microgram T3/100gBW treated rats sera FSH demonstrated two separate peaks during the critical period. Treatment with various dosages of T3 of Ovarx-TxPx rats resulted in significant suppressions (p less than 0.05) of sera LH and FSH. Despite depressed concentrations of sera LH and FSH in T3-treated rats pituitary sensitivity to a challenge of 3LHRH was enhanced. Hence, the pituitary was not the site of T3 inhibition of gonadotropin secretion. Additionally, T3 did not modify pituitary LH content or hypothalamic LH3 releasing activity (LHRH). Since T3 did not inhibit gonadotropin secretion at the pituitary level, a neural site of T3 action is suggested.     

The rapid effects of luteinizing hormone releasing hormone agonists and antagonists on the mouse pituitary in vitro

The effect of luteinizing hormone releasing hormone (LHRH) and its analogs on the release of FSH and LH by 20 day old whole mouse pituitary incubated in vitro for 3-4 hrs was investigated. Three agonistic analogs (AY 25650, 25205 and Buserelin) all of which are reported to be superactive in vivo showed approximately the same potency in this in vitro test system. Preincubation of the pituitaries for 1 h with the antagonistic analogs [Ac Dp Cl Phe1,2, D Trp3, D Phe6, D Ala10] LHRH and [Ac Dp Cl Phe1,2, D Trp3, D Arg6, D Ala10] LHRH inhibited the secretion of LH and FSH induced by 2.5 x 10(-9)M LHRH. The inhibitory response was dose dependent. The continued presence of the antagonists was not required for effective suppression of the LHRH effect. Experiments designed to find out the minimum time required for eliciting suppression of LHRH revealed that preincubation of the pituitary with the second antagonist for 5 mins followed by removal was adequate to produce effective inhibition of gonadotropin release. At lower doses of the antagonist, LH release was more effectively inhibited than FSH release. The results suggest that antagonistic analogs can effectively bind to LHRH receptors in the whole pituitary incubation preventing the subsequent action of LHRH. With the present incubation system assessment of bioactive LH and FSH release is possible within 24 hrs.     

Effects of the gonadotropin-releasing hormone associated peptides (GAP) on the release of luteinizing hormone (LH), follicle stimulating hormone (FSH) and prolactin (PRL) in vivo

Six peptide sequences residing between basic amino acid residues in GAP were tested for effects on the release of FSH, LH and PRL in vivo in ovariectomized, estrogen-progesterone-primed (OEP) rats. Synthetic GAP peptides (1–13, 1–23, 15–23, 25–36, 38–53 and 41–53) were injected intravenously (IV) into conscious OEP rats and plasma levels of FSH, LH and PRL were measured by RIA. The activity of GAP peptides in the control of PRL was further examined in ether-stressed male rats which were injected IV with GAP peptides just prior to a 1-min etherization. GAP(1–13) significantly stimulated FSH release at doses of 1, 10 and 100 μg, whereas it stimulated LH release only at the highest dose of 100 μg. GAP(1–23) elevated plasma levels of FSH and LH only at a dose of 100 μg. The other 4 peptides had no effect on the release of gonadotropins. Of these 6 peptides, only GAP(1–13) partially lowered the plasma levels of PRL at the high dose of 100 μg in OEP rats, but it had no effect on the ether-induced PRL surge at doses of 10 and 100 μg. In conclusion, both GAP(1–13) and GAP(1–23) stimulate FSH and LH release in vivo; these 2 peptides are much less potent in stimulating gonadotropin release than is LHRH. GAP(1–13) exerts a preferential FSH-releasing activity, but its PRL-inhibiting activity is minimal.     

Effect of luteinizing hormone releasing hormone pulse characteristics on comparative luteinizing hormone and follicle stimulating hormone secretion from superfused rat anterior pituitary cell cultures

We have shown that 4 ng luteinizing hormone releasing hormone (LHRH) pulses induced significantly greater luteinizing hormone (LH) release from proestrous rat superfused anterior pituitary cells with no cycle related differences in follicle stimulating hormone (FSH). Current studies gave 8 ng LHRH in various pulse regimens to study amplitude, duration and frequency effects on LH and FSH secretion from estrous 0800, proestrous 1500 and proestrous 1900 cells. Regimen 1 gave 8 ng LHRH as a single bolus once/h; regimen 2 divided the 8 ng into 3 equal 'minipulses' given at 4 min intervals to extend duration; regimen 3 gave the 3 'minipulses' at 10 min intervals, thereby further extending duration: regimen 4 was the same as regimen 2, except that the 3 'minipulses' were given at a pulse frequency of 2 h rather than 1 h. In experiment 1, all four regimens were employed at proestrus 1900. FSH was significantly elevated by all 8 ng regimens as compared to 4 ng pulses; further, 8 ng divided into 3 equal 'minipulses' separated by 4 min at 1 and 3 h frequencies (regimens 2 and 4) resulted in FSH secretion that was significantly greater than with either a single 8 ng bolus (regimen 1) or when the 'minipulses' were separated by 10 min (regimen 3). In experiment 2, at proestrus 1500, FSH response to the second pulse of regimen 4 was significantly greater than in regimen 2; LH release was significantly suppressed at pulse 2 compared to regimen 2 accentuating divergent FSH secretion. At estrus 0800, FSH response to the second pulse of regimen 4 was significantly stimulated FSH at proestrus 1900, 1500 and estrus 0800, FSH divergence was most marked at proestrus 1500. These data indicate a potential role for hypothalamic LHRH secretory pattern in inducing divergent gonadotropin secretion in the rat.     

Inhibitory effects of a luteinizing-hormone-releasing hormone agonist implant on ovine fetal gonadotrophin secretion and pituitary sensitivity to luteinizing-hormone-releasing hormone.

Sheep fetuses at day 70 of gestation (term = 145 days) were implanted subcutaneously with a biodegradable implant containing a luteinizing-hormone-releasing hormone (LHRH) agonist (buserelin) to investigate whether treatment with LHRH agonist would induce a state of desensitization of the fetal gonadotrophs and thus influence fetal gonadal development. Treatment with the LHRH agonist for 35-40 days caused a significant reduction in mean fetal plasma concentrations of LH and follicle-stimulating hormone (FSH) compared with control fetuses. LH pulses were evident in control fetuses but were completely abolished by buserelin treatment. Furthermore, the pituitary content of LH and FSH was significantly depleted in fetuses implanted with LHRH agonist. A bolus intravenous injection of 500 ng LHRH given to control fetuses caused a rapid and significant increase in plasma LH and FSH concentrations which was sustained for at least 60 min after injection. Pretreatment with buserelin completely abolished the LH and FSH responses to a bolus injection of LHRH. There were no differences between the sexes in fetal gonadotrophin concentrations or pituitary sensitivity to LHRH in control or agonist-treated fetuses. Furthermore, buserelin treatment for 35-40 days had no effect on the morphological appearance of the fetal gonads when compared with control fetuses, at least to day 110 of pregnancy. These results provide evidence for the induction of a state of desensitization of the LHRH receptors of the fetal pituitary gonadotrophs following long-term treatment with an LHRH agonist, but provide no evidence for a role for gonadotrophin secretion in gonadal development at this stage in fetal life.     

The release of pituitary gonadotrophins by luteinizing hormone releasing hormone in the intact,castrated and aspermatogenic rat

The change in serum gonadotrophin concentration in response to synthetic Luteinizing Hormone Releasing Hormone (LHRH - 400 ng i.v.) was investigated under barbiturate anaesthesia in adult male rats either chronically castrated, rendered aspermatogenic by the administration of α-chlorohydrin 12–16 weeks previously (to remove inhibin), or treated with vehicle. A single injection of LHRH increased serum LH and FSH concentrations similarly in both intact and aspermatogenic rats. In castrated rats the amount of LH released was much greater and the FSH secretion sustained. A second injection produced a similar increase although a second peak of FSH could not be detected in castrated rats as the FSH level was still elevated. The increase in LH levels was two to three times larger in response to the second injection of LHRH than to the first in all groups. The results do not support the hypothesis that the enhanced gonadotropin response to castration in the aspermatogenic rat is due to increased pituitary sensitivity to LHRH.     

The prolonged action of the LHRH agonist buserelin (HOE 766) may be due to prolonged binding to the LHRH receptor

Hemi-pituitary glands of ovariectomized rats were superfused for 4 h with either LHRH or the analog buserelin (HOE 766) at several concentrations, and thereafter with medium only for another 1.5 h. In a further experiment glands were exposed for 2.5 h to LHRH or buserelin at a single concentration (5 ng/ml) and subsequently for another 2.5 h to either the same agonist (LHRH or buserelin) alone (5 ng/ml), the agonist plus an LHRH-antagonist (ORG 30093, 1000 ng/ml), the LHRH- antagonist alone, or medium alone. LHRH and buserelin stimulated gonadotropin release equally well. After cessation of this stimulation, the gonadotropin release by the buserelin-treated pituitary glands and the glands, treated with the highest dose of LHRH (1000 ng/ml), continued, while the release by the glands, treated with the lower doses of LHRH, declined. The LHRH-antagonist completely blocked the release of LH, stimulated by buserelin or LHRH, as well as the prolonged activation of the release, caused by buserelin pre-treatment. In a superfusion experiment with pituitary cell aggregates of 14-day-old intact female rats, buserelin stimulated the release of LH much more effectively than LHRH itself. Moreover, the release caused by buserelin declined more slowly after cessation of the stimulation. Finally, in a pituitary cell monolayer culture the Kd's of LHRH, buserelin and the antagonist were determined as 4.7 X 10(-9) M, 2.4 X 10(-10) M and 4.6 X 10(-9) respectively. It was concluded that the estimates of the potencies of LHRH and buserelin depend on the choice of the test-system. It is suggested that the long duration of action of buserelin is at least partly due to prolonged binding to the LHRH-receptor.     

Selective release of follicle-stimulating hormone by 5 alpha-dihydroprogesterone in immature ovariectomized estrogen-primed rats

The effect of 5 alpha-dihydroprogesterone (5 alpha-DHP) on gonadotropin release was examined in the immature acutely ovariectomized (OVX) rat primed with a low dose of estradiol (E2). Treatment with various doses of 5 alpha-DHP given in combination with E2 increased levels of follicle-stimulating hormone (FSH) but had no effect on serum luteinizing hormone (LH). A single injection of a maximally stimulating dose of 5 alpha-DHP (0.4 mg/kg) stimulated increases in serum FSH at 1200 h and, 6 h later, at 1800 h. Pituitary LH and FSH content was dramatically enhanced by 1600 h and levels remained elevated at 1800 h. The administration of pentobarbital at 1200 h, versus 1400 h or 1600 h, prevented the increase in basal serum FSH levels at 1800 h, implying that the release of hypothalamic LH releasing hormone (LHRH) is modulated by 5 alpha-DHP. In addition, changes in pituitary sensitivity to LHRH as a result of 5 alpha-DHP were measured and a significant increase in the magnitude of FSH release was observed at 1200 h and 1800 h. Although the LH response to LHRH in 5 alpha-DHP-treated rats was not different from controls, the duration of LH release was lengthened. These results suggest that 5 alpha-DHP may stimulate FSH release by a direct action at the pituitary level. Together, these observations support the theory that 5 alpha-DHP mediates the facilitative effect of progesterone on FSH secretion and further suggests an action of 5 alpha-DHP in this phenomenon at both pituitary and hypothalamic sites.     

Modulation of pituitary responsiveness to LHRH by androgens in ovariectomized female rats.

The effect of androgens on pituitary response to luteinizing-hormore-releasing hormone (LHRH) and their ability to modify effects of 17beta-estradiol (E2) on pituitary responsiveness to LHRH were tested in ovariectomized rats maintained on a daily dose of 0.25 microgram estradiol benzoate per rat for 6 d before androgen administration. Testosterone propionate (TP) (4, 40, 400, or 4000 microgram per rat), administered 24 h before LHRH (500 ng per rat), had no significant effect on luteinizing hormone (LH) or follicle-stimulating hormone (FSH) response. Similar doses of dihydrotestosterone (DHT) did not significantly alter the LH response but significantly suppressed the FSH response. Even the lowest dose completely blocked the FSH response to LHRH. TP in combination with 4 or 400 microgram of E2 suppressed the stimulatory effect of E2 on both LH and FSH response to LHRH in a dose-related manner. DHT and E2 in combination affected LH response inconsistently, whereas their ratio determined FSH response; there was pronounced inhibition of FSH response in rats given high doses of DHT combined with low doses of E2; DHT inhibition of FSH response in animals receiving 4 microgram of DHT with 400 microgram E2 was partially overcome by the stimulatory effect of E2. Our results indicate that TP and DHT affect LH and FSH response to LHRH differently. The ratio of androgen to estrogen is important in determining the response to LHRH.     

Melanin-concentrating hormone stimulates the release of luteinizing hormone-releasing hormone and gonadotropins in the female rat acting at both median eminence and pituitary levels

The purpose of this study was to investigate whether melanin-concentrating hormone (MCH) acts directly on the median eminence and on the anterior pituitary of female rats regulating LHRH and gonadotropin release. In addition, immunohistochemistry was used to examine the density and distribution of MCH-immunoreactive fibers in the median eminence of proestrous rats. MCH-immunoreactive fibers were found in both the internal and external layers of the median eminence and in close association with hypophysial portal vessels. In the first series of in vitro experiments, median eminences and anterior pituitaries were incubated in Krebs-Ringer bicarbonate buffer containing two MCH concentrations (10(-10) and 10(-8) M). The lowest MCH concentration (10(-10) M) increased (P < 0.01) LHRH release only from proestrous median eminences. Anterior pituitaries incubated with both MCH concentrations also showed that 10(-10) M MCH increased gonadotropin release only from proestrous pituitaries. In the second series of experiments, median eminences and pituitaries from proestrous rats were incubated with graded concentrations of MCH. MCH (10(-10) and 10(-9) M) increased (P < 0.01) LHRH release from the median eminence, and only 10(-10) M MCH increased (P < 0.01) LH and FSH release from the anterior pituitary. The effect of MCH on the stimulation of both gonadotropins from proestrous pituitaries was similar to the effect produced by LHRH. Simultaneous incubation of pituitaries with MCH and LHRH did not modify LH but increased the FSH release induced by LHRH. The present results suggest that MCH could be involved in the regulation of preovulatory gonadotropin secretion.     

Effects of intermittent pulsatile infusion of luteinizing hormone-releasing hormone on dihydrotestosterone-suppressed gonadotropin secretion in castrate rams

The feedback effects of dihydrotestosterone (DHT) on gonadotropin secretion in rams were investigated using DHT-implanted castrate rams (wethers) infused with intermittent pulsatile luteinizing hormone-releasing hormone (LHRH) for 14 days. Castration, as anticipated, reduced both serum testosterone and DHT but elevated serum LH and follicle-stimulating hormone (FSH). Dihydrotestosterone implants raised serum DHT in wethers to intact ram levels and blocked the LH and FSH response to castration. The secretory profile of these individuals failed to show an endogenous LH pulse during any of the scheduled blood sampling periods, but a small LH pulse was observed following a 5-ng/kg LHRH challenge injection. Dihydrotestosterone-implanted wethers given repeated LHRH injections beginning at the time of castration increased serum FSH and yielded LH pulses that were temporally coupled to exogenous LHRH administration. While the frequency of these secretory episodes was comparable to that observed for castrates, amplitudes of the induced LH pulses were blunted relative to those observed for similarly infused, testosterone-implanted castrates. Dihydrotestosterone was also shown to inhibit LH and FSH secretion and serum testosterone concentrations in intact rams. In summary, it appears that DHT may normally participate in feedback regulation of LH and FSH secretion in rams. These data suggest androgen feedback is regulated by deceleration of the hypothalamic LHRH pulse generator and direct actions at the level of the adenohypophysis.     

Human chorionic gonadotropin and luteinizing hormone-releasing hormone reverse the blockade of ovulation in pregnant mare's serum gonadotropin-primed immature rats by the anti-androgenic drug, hydroxyflutamide

The present study was designed to examine mechanism(s) of the anti-ovulatory action of the anti-androgen, hydroxyflutamide (OH-F). Prepubertal rats were treated with 4 IU pregnant mare's serum gonadotropin (PMSG) (day -2) to induce first estrus and ovulation. They received OH-F in sesame oil or oil alone at 08:00 and 20:00 h on day 0 (the day of proestrus) and ovulations were assessed on the morning of day 1. Eighty-three percent of control animals ovulated with a mean of 7.7 +/- 1.1 corpora lutea per rat. Hydroxyflutamide blocked ovulation in all but 2 of the 12 rats receiving this drug alone. All of OH-F treated rats that received 5 and 25 IU human chorionic gonadotropin (hCG) ovulated with means +/- SEM of 9.1 +/- 0.1 and 7.3 +/- 1.4 corpora lutea per rat, respectively. The dose of 0.2 IU hCG was essentially ineffective, while the effect of 1.0 IU hCG was intermediate. At the dose of 20 ng and above (100 and 500 ng) luteining hormone-releasing hormone (LHRH) completely overcame the ovulation blockade in the OH-F treated animals, while a 4-ng dose was ineffective. At 18:00 h on the day of proestrus, serum LH levels in control animals were 17.56 +/- 2.60 ng/mL, which were 920% above basal levels (1.90 +/- 0.13) indicating a spontaneous LH surge. This surge was suppressed in OH-F treated rats. Injection of LHRH, at the dose of 20 ng and above, reinstated the LH release in OH-F treated animals. Thus, the anti-androgen, OH-F, inhibits ovulation in PMSG-treated immature rats through its interference with the preovulatory LH surge; the inhibition can be reversed by hCG or LHRH. Hydroxyflutamide does not appear to interfere at the level of the pituitary, but may have direct action at the hypothalamic and (or) extrahypothalamic sites involved in the generation of positive feedback signals that control LH release.     

Possible negative ultra-short loop feedback of luteinizing hormone releasing hormone (LHRH) in the ovariectomized rat

To determine if LHRH might act within the brain to modify its own release, repeated blood samples were removed from conscious ovariectomized rats and minute doses of LHRH were injected into the third ventricle (3V). The effect of these injections on plasma LH and FSH was measured by radioimmunoassay (RIA). The higher dose of intraventricular LHRH (10 ng in 2 microliter) induced an increase in plasma LH within 10 min after its injection. Plasma LH decreased for the next 60 min. This was followed by restoration of LH pulses characteristic of the ovariectomized rat. This dose of LHRH slightly elevated plasma FSH concentrations. In stark contrast, a 10 fold lower dose of 1 ng of LHRH injected into the ventricle resulted in a highly significant decrease of plasma LH at 10 min following injection, followed by return of LH pulsations. There was no effect on the pulsatile release of FSH. The results are interpreted to mean that at the higher dose, sufficient LHRH reached the site of origin of the hypophyseal portal vessels in the median eminence so that it diffused into portal vessels and was delivered to the gonadotrophs to induce LH release. In contrast, the lower dose provided sufficient hypothalamic concentrations of the peptide to suppress the discharge of the LHRH neurons, thereby leading to a decline in plasma LH, indicative of an ultrashort-loop negative feedback of LHRH to suppress its own release.     

Regulation of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) gene expression by gonadotropin-releasing hormone (GnRH) and sexual steroids in the Mediterranean Sea bass

The secretion of gonadotropins, the key reproductive hormones in vertebrates, is controlled from the brain by the gonadotropin-releasing hormone (GnRH), but also by complex steroid feedback mechanisms. In this study, after the recent cloning of the three gonadotropin subunits of sea bass (Dicentrarchus labrax), we aimed at investigating the effects of GnRH and sexual steroids on pituitary gonadotropin mRNA levels, in this valuable aquaculture fish species. Implantation of sea bass, in the period of sexual resting, for 12 days with estradiol (E2), testosterone (T) or the non-aromatizable androgen dihydrotestosterone (DHT), almost suppressed basal expression of FSHbeta (four to 15-fold inhibition from control levels), while slightly increasing that of alpha (1.5-fold) and LHbeta (approx. twofold) subunits. Further injection with a GnRH analogue (15 microg/kg BW; [D-Ala6, Pro9-Net]-mGnRH), had no effect on FSHbeta mRNA levels, but stimulated (twofold) pituitary alpha and LHbeta mRNA levels in sham- and T-implanted fish, and slightly in E2- and DHT-implanted fish (approx. 1.5-fold). The GnRHa injection, as expected, elevated plasma LH levels with a parallel decrease on LH pituitary content, with no differences between implanted fish. In conclusion, high circulating steroid levels seems to exert different action on gonadotropin secretion, inhibiting FSH while stimulating LH synthesis. In these experimental conditions, the GnRHa stimulate LH synthesis and release, but have no effect on FSH synthesis.     

Luteinizing hormone releasing hormone mediates naloxone's effects on serum luteinizing hormone levels in normal and morphine-sensitized male rats

Naloxone produces large increases in serum luteinizing hormone (LH) levels in normal males and females, supporting a role for endogenous opioids (EOP) in the tonic inhibition of LH. Since the antagonist apparently exerts no important effects on the pituitary, the reasonable assumption has been made that it elevates gonadotropin levels by affecting the release of LH-releasing hormone (LHRH) from the hypothalamus. However, at present there is no direct in vivo evidence supporting this widely-held view. In an attempt to directly demonstrate that naloxone increases the secretion of LHRH, and thereby elevates serum LH levels, we examined whether a potent synthetic antagonist of LHRH ( [D-p Glu1, D-Phe2, D-Trp3,6]-LHRH, GPT-LHRH) blocked the effects of naloxone in male rats with a normal response to naloxone and in those with a markedly enhanced sensitivity to the drug induced by a brief period of morphine pellet implantation. Our results demonstrated that GT-LHRH antagonized equipotent doses of LHRH (100 ng/kg) and naloxone (0.5 mg/kg) over a similar time course with approximately the same AD50. Most importantly, however, we showed that the GPT-LHRH produced equivalent, parallel shifts to the right in the dose-response curves for LHRH and naloxone, indicative of competitive inhibition. We also found that GPT-LHRH completely abolished the enhanced response to naloxone's effects on LH which occurs in morphine-pretreated rats. Since we observed no competition between LHRH and naloxone for their binding sites in pituitary or brain, the only viable interpretation of our results is that naloxone increases LH by inducing the release of LHRH.     

Effects of complete hypothalamic deafferentation on the estrous phase of follicle-stimulating hormone release in the cyclic rat

We investigated whether neural afferents to the medial basal hypothalamus play an acute role in the estrous phase of FSH release in the 4-day cyclic rat. A cannula was inserted into the right atrium of the heart under brief ether anesthesia during the early afternoon of proestrus for subsequent blood collections and injection of LHRH. In some of the rats, the medial basal hypothalamus was surgically isolated from the rest of the brain with a small knife under brief ether anesthesia between 2000 h and 2130 h of proestrus. Control groups consisted of naive rats which were not treated during the night of proestrus and sham-operated animals in which the knife was lowered to the corpus callosum between 2000 h and 2130 h or proestrus. Rats were bled at 2200 h of proestrus and at 0200 h, 0600 h and 1000 h of estrus for radioimmunoassay of plasma FSH and LH. The plasma FSH levels in all 3 groups between 2200 h of proestrus and 1000 h of estrus were elevated above levels observed in other cannulated rats bled to the onset of the proestrous phase of FSH release at 1400 h of proestrus. There were no statistically significant differences in plasma FSH or LH concentrations at any of the time periods between the 3 groups of serially bled rats. The deafferentation procedure did not appear to impair the pituitary gland's ability to secret gonadotrophins as injection of 50 ng of LHRH after the bleeding at 1000 h of estrus caused substantial elevations in plasma FSH and LH concentrations which were not different between the 3 groups. The results suggest that neural afferents to the medial basal hypothalamus play no acute role in the estrous phase of FSH release in the cyclic rat.     

A possible role for cyclic GMP in mediating the effect of luteinizing hormone releasing hormone on gonadotropin release in dispersed pituitary cells of the female rat.

In continuing studies on cyclic nucleotide involvement in the regulation of gonadotropin release, we have measured the cyclic nucleotide content and rate of LH and FSH release during stimulation by LHRH of dispersed overnight cultured cells from the pituitaries of adult female rats. The minimal effective concentration of LHRH was 0.1 nM and half maximal stimulation of gonadotropin release was observed in the presence of 1.0 nM LHRH. Significant release of both LH and FSH was detectable after only 10 min in the presence of 5 nM LHRH. The presence of fetal calf serum (FCS) in the overnight culture medium increased basal cGMP levels significantly, whereas horse serum (HS) had no effect, therefore all experiments were conducted on cells cultured in the presence of HS. Treatment of the cultured cells with the phosphodiesterase inhibitors theophylline (TH) or isobutyl-methyl-xanthine (MIX) revealed a preferential stimulatory effect of TH on basal cAMP levels and of MIX on cGMP levels. Throughout these experiments, LHRH had no effect on cAMP levels. In the presence of MIX, concentrations of the releasing hormone as low as 1 nM induced a significant rise in the level of cGMP whereas in its absence, cGMP levels appeared to be unchanged by LHRH. The increase was detectable after 10 min of incubation. MIX alone slightly increased LH and FSH release and significantly potentiated the response of the cells to increasing doses of LHRH up to, but not beyond, 10 nM. The data support the possibility that cGMP may be involved in the mechanism of action of LHRH.     

Effect of dexamethsone on luteinizing hormone and follicle stimulating hormone responses to LHRH and to clomiphene in the follicular phase of women with normal menstrual cycles.

In an attempt to elucidate the mechanism of suppressive action of glucocorticoids on the hypothalamo-pituitary-ovarian axis, we studied the effects of short-term high dose dexamethasone administration of the LH and FSH responses to LHRH and to clomiphene in healthy women with normal menstrual cycles. Seven women, 21--35 years of age, received 100 micrograms of LHRH i.v. on day 6 of two consecutive menstrual cycles, once with and once without pre-treatment with dexamethasone 2 mg orally every 6 hrs. on days 2 through 5 of the menstrual cycle. Seven other women (ages 21--35 years) received clomiphene citrate 100 mg on days 2 through 5 of their menstrual cycle, once with and once without simultaneous administration of dexamethasone 2 mg orally every 6 h. The administration of dexamethasone suppressed baseline serum levels of LH and FSH and blunted LH and FSH response to both LHRH and clomiphene. The results indicate that short-term administration of pharmacological doses of glucocorticoids suppress the secretion of LH and FSH by a direct effect on the anterior pituitary and possibly by an effect at the hypothalamic level with inhibition of the release of LHRH.     

The induction of divergent gonadotropin secretion in vitro by variations in luteinizing hormone releasing hormone pulse regimen

The effects of luteinizing hormone releasing hormone (LHRH) pulse amplitude, duration, and frequency on divergent gonadotropin secretion were examined using superfused anterior pituitary cells from selected stages of the rat estrous cycle. Cells were stimulated with one of five LHRH regimens. With low-amplitude LHRH pulses (regimen 1) in the presence of potentially estrogenic phenol red, LH response in pituitary cells from proestrus 1900, estrus 0800, and diestrus 1,0800 were all significantly larger (P less than 0.05) than the other stages tested. In the absence of phenol red, responsiveness at proestrus 1900 was significantly larger than proestrus 0800, proestrus 1500, and estrus 0800 (P less than 0.01, 0.05, and 0.05, respectively); other cycle stages tested were smaller. No significant differences were observed between cycle stages for follicle-stimulating hormone (FSH) secretion in the presence or absence of phenol red. Because pituitary cells at proestrus 1900 were the most responsive to low-amplitude 4 ng LHRH pulses, they were also used to study the effects of LHRH pulses of increased amplitude or duration and decreased frequency. Increasing the amplitude (regimen 2) or the duration (regimens 3 to 5) increased FSH secretion; this effect was greatest with regimens 3 and 5. When regimens 3 and 5 were studied in pituitary cells obtained at proestrus 1500, FSH was significantly increased by both regimes, but most by regimen 5; furthermore, LH release was significantly reduced. When regimens 3 and 5 were studied in pituitary cells obtained at estrus 0800, FSH release was elevated most significantly by regimen 5. Thus, variations in LHRH pulse regimen were found to be capable of inducing significant divergence in FSH release from superfused anterior pituitary cells derived from specific stages of the estrous cycle.