Effect of gonadotropin-releasing hormone pulse frequency on gonadotropin secretion and subunit messenger ribonucleic acids in perifused pituitary cells.

Slow frequency GnRH pulses have been proposed to preferentially increase circulating FSH levels by increasing FSH synthesis and pulsatile release. Examination of this proposal using various in vivo models, however, has produced conflicting results. To examine directly the effects of GnRH pulse frequency on the pituitary, we compared the effects of 2.5-nM GnRH pulses administered every 1 h or every 4 h vs. no GnRH, using perifused rat pituitary cells. FSH secretion (total area under the response curve) was 2-fold greater (P less than 0.01) with every hour than with every 4 h GnRH pulses. This difference resulted from the increased number of GnRH pulses and increased (P less than 0.05) interpulse FSH secretion, whereas FSH pulse amplitude was unchanged. FSH beta mRNA levels at the completion of the 11-h perifusion were increased 4.5-fold by GnRH every h (P less than 0.01) and 3.3-fold by GnRH every 4 h (P less than 0.05) above levels in untreated cells. FSH beta mRNA levels were greater (P less than 0.05) at the faster GnRH pulse frequency. Because more frequent stimulation delivered more GnRH during the study, cells were next stimulated with 2.5 nM GnRH every 1 h for nine pulses, 7.5 nM GnRH every 4 h for three pulses to equalize the GnRH dose, or 2.5 nM GnRH every 4 h for three pulses. Interpulse FSH secretion and FSH beta mRNA levels were again greater (P less than 0.05) with every hour than every 4 h GnRH pulses. Interpulse LH secretion, FSH and LH pulse amplitude, and LH beta and alpha-subunit mRNA levels were not different between the groups. GnRH doses of 0.1-10 nM every hour increased FSH and LH pulsatile secretion dose-dependently, but FSH beta, LH beta, and alpha-subunit mRNA levels were similar. In conclusion, our data reveal that reducing the frequency of GnRH pulses from every hour to every 4 h reduces both FSH beta mRNA levels and FSH interpulse secretion, but does not change GnRH-stimulated FSH pulsatile release. We suggest that the finding by others that slow frequency GnRH pulses increase circulating FSH levels under certain experimental conditions in vivo may instead be explained by complex hormonal interactions or changes in FSH clearance.     

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.     

Secretion rates and short-term patterns of gonadotrophin-releasing hormone, FSH and LH in the normal stallion in the breeding season

Pituitary venous blood was collected by a painless nonsurgical cannulation method from five ambulatory stallions at 5-min intervals for 5-6 h during the breeding season. In four adult stallions, statistical analysis showed that pulses of gonadotrophin-releasing hormone (GnRH) and LH were coincident (P less than 0.01), as were pulses of FSH and LH (P less than 0.05). Furthermore, the patterns of changes in concentration of FSH and LH were highly correlated in each of the four stallions. However, seemingly ineffective pulses of GnRH were also observed, with 28% of GnRH pulses failing to induce a significant gonadotrophin pulse. In the four adult stallions the amplitude of pituitary venous gonadotrophin pulses varied markedly but no correlation with GnRH pulse amplitude was observed. Peak secretion of FSH, but not LH, during pulses was correlated with the length of the interpulse interval. Consequently, the ratio of FSH to LH during peaks was least (P less than 0.02) when the interpulse interval was 30 min or less. Thus, differential FSH and LH secretion was achieved within a constant steroid milieu. Two stallions had regular contact with oestrous mares, and in these horses the secretion of GnRH and gonadotrophins occurred almost continuously with rapid, rhythmic pulses superimposed upon a tonic background. Mean (+/- S.D.) interval between GnRH pulses was 31.4 +/- 9.8 min and 27.7 +/- 10.1 min. This secretory pattern was not observed in the two stallions which had infrequent contact with oestrous mares, although the small numbers precluded statistical testing of this apparent difference. No GnRH pulses were observed in one of these stallions, while in the other mean (+/- S.D.) GnRH pulse interval was 45.0 +/- 48.7 min, the large variance being partly due to rapid pulses during a period in which the stallion teased mares. The fifth stallion was pubertal, and GnRH and LH secretion occurred in 15 and 0% of samples respectively, while low levels of FSH secretion were observed in 37% of samples and jugular testosterone levels were immeasurably low. We conclude that there is a statistically significant synchrony between pulses of GnRH, LH and FSH in the pituitary venous blood of stallions. Furthermore, decreasing intervals between gonadotrophin pulses result in a significant reduction in secretion of FSH but not LH.     

Effects of inhibin from primate Sertoli cells on follicle-stimulating hormone and luteinizing hormone release by perifused rat pituitary cells

We used a pituitary cell perifusion system to investigate the time course and selectivity of the inhibin effect on pulsatile GnRH-stimulated LH and FSH release. Dispersed pituitary cells from 7- to 8-week-old male rats were perifused on a Cytodex bead matrix and stimulated with 10 nM GnRH for 2 min every hour for 8-11 h. The addition of a preparation of inhibin partially purified from primate Sertoli cells reduced pulsatile FSH release within 2 h. After removal of inhibin from the perifusion medium, the effect was reversed within 3 h. GnRH-stimulated LH release was also influenced by inhibin, although the decline in LH was less than that in FSH (80 +/- 3% vs. 68 +/- 4% of control; P less than 0.025). Smaller doses of inhibin suppressed GnRH-induced FSH secretion, but had no effect on LH release. Further, prolonged incubation of pituitary cells with inhibin at the higher dose reduced its FSH inhibitory effect and eliminated the effect on LH. These results indicate that inhibin can reduce both LH and FSH secretion in vitro, although the specificity and magnitude of the effect are a function of both the dose and duration of inhibin treatment. Further, the actions of inhibin and GnRH on the pituitary may be interrelated.     

Regulation of lutenizing hormone secretion and subunit messenger ribonucleic acid expression by gonadal steroids in perifused pituitary cells from male monkeys and rats.

The mechanisms by which gonadal steroids regulate gonadotropin secretion remain incompletely understood. As previous studies suggest that the pituitary actions of testosterone (T) and estradiol (E) differ in male primates and rodents, we compared the effects of 10 nM T, 0.1 nM E, and 10 nM dihydrotestosterone (DHT) on the LH response to hourly pulses of GnRH as well as the GnRH receptor (GnRH-R) and LH subunit messenger RNA (mRNA) levels in dispersed pituitary cells from intact male monkeys and rats. T suppressed (P < 0.01) and E increased (P < 0.05) GnRH-stimulated LH secretion by rat pituitary cells. With monkey pituitary cells, on the other hand, there was no significant effect of either T or DHT on GnRH-stimulated LH secretion. In E-treated monkey cells, a period of initial enhancement (P < 0.05) was followed by significant suppression (P < 0.05) of LH secretion. GnRH-R mRNA was unchanged by T or E in either rat or monkey cells. T suppressed LHbeta (P < 0.01) and alpha-subunit (P < 0.01) mRNAs, whereas E increased alpha-subunit (P < 0.01), but did not alter LHbeta mRNA levels in rat cells. In monkey cells, however, neither T nor E affected LHbeta or alpha-subunit mRNA levels significantly. Our results identify different regulatory mechanisms by which testicular steroid hormones control LH secretion by the pituitary in male primates and rodents. We propose that the primary site of androgen negative feedback in the male primate is to restrain GnRH pulsatile secretion, whereas in the male rat T also decreases gonadotropin synthesis and secretion by directly affecting the pituitary. E suppresses GnRH-stimulated LH secretion in the primate pituitary, but amplifies the action of GnRH in the rat. Our data also reveal that the action of T to suppress LH secretion and subunit mRNA in male rats is not through decreased GnRH-R gene expression.     

Action kinetics of inhibin in superfused pituitary cells depend on gonadotropin-releasing hormone treatment

We examined the effects of partly purified inhibin from porcine follicular fluid on FSH and LH release in superfused rat pituitary cell cultures exposed to different GnRH stimuli. Pituitary cells from immature male rats were cultured in chemically defined medium. After 4 days of static culture in the absence of inhibin preparation and GnRH, the cell monolayers were superfused for approximately 10 h at a constant speed (0.15 or 0.25 ml/min) with medium with or without inhibin preparation (1 micrograms/ml). During the superfusion, some cultures were stimulated with GnRH (10 nM) continuously or intermittently (1 min/0.5 h or 6 min/1 h). In the basal condition (no GnRH), inhibin suppressed FSH release after 5 h of exposure (P less than 0.01), whereas LH secretion was not affected. In cultures treated with GnRH pulses (of either frequency), the inhibitory effects on the GnRH-stimulated FSH and LH release were statistically significant (P less than 0.01) after 2 h of exposure, became more pronounced in the next several hours, then remained stable until the end of the experiment. In cultures exposed to GnRH continuously, the suppressing effects of inhibin preparation became significant (P less than 0.01) after 3 h of exposure and were maximal at 4 h (52% and 61% of control values for FSH and LH, respectively). Later, the suppressing effect became less pronounce due to the decreasing rate of gonadotropin secretion in control (no inhibin) cultures exposed continuously to GnRH. The magnitude of FSH and LH suppression after 9 h of exposure to the inhibin preparation was statistically different (P less than 0.05) for different GnRH treatments and was more pronounced with GnRH pulses (24-27% and 54-57% of control values for FSH and LH, respectively) than with cultures exposed to GnRH continuously (77% and 89% of control values for FSH and LH, respectively) or in the absence of GnRH (50% and 92% of control values for FSH and LH, respectively). We conclude that both the kinetics and magnitude of action of the inhibin preparation on FSH and LH release can differ significantly depending on the presence or absence of GnRH as well as on the mode of GnRH stimulation. Of particular importance is the observation that suppressive effects of inhibin preparation decline in cultures that have been desensitized to GnRH after prolonged continuous GnRH exposures. These differences stress the role of GnRH-inhibin interactions in the regulation of gonadotropin secretion and emphasize the importance of the mode of GnRH stimulation in studies concerning inhibin action on pituitary cells in vitro.     

Acute inhibitory effects of 17 beta-estradiol are observed on gonadotropin secretion from perifused pituitary fragments of metestrous, but not proestrous, rats

The in vivo suppression of LH by 17 beta-estradiol (E2) has been documented frequently. However, the demonstration of a direct inhibitory action of E2, in contrast to a stimulatory action, on the secretion of LH from the anterior pituitary has been inconsistent. The aim of this study was to determine if E2 can suppress either basal (unstimulated) or GnRH-stimulated gonadotropin secretion directly at the level of the anterior pituitary gland. Anterior pituitaries were obtained from metestrous and proestrous females rats at 0900 h, and trunk blood was collected for serum measurements of LH, FSH, E2, and progesterone (P). Each anterior pituitary was cut into eighths and placed into a microchamber for perifusion. Pituitary fragments were perifused at a rate of 10 ml/h using medium 199 (without phenol red) that contained E2 (1 nM) or ethanol as a control. Six pulses of GnRH (peak amplitude, 50 ng/ml; duration, 2 min) were administered one per h starting at 60 min. Fractions of perfusate were collected every 5 min for measurement of LH and FSH. The total amounts of LH and FSH secreted during the 1-h interval after each GnRH pulse or corresponding basal hour were calculated. Both basal and LH and FSH responses to GnRH were significantly greater from pituitaries of proestrous compared to metestrous rats. The selective suppression of LH secretion by in vitro treatment with E2 was demonstrated using pituitaries from metestrous rats receiving GnRH pulses, but not using pituitaries from proestrous rats. Thus, a negative feedback effect of E2 on LH secretion was observed only in pituitaries from donors with low serum levels of E2 and high P, but not from donors with high serum levels of E2 and low P. We believe that the in vivo steroid environment determined the subsequent responses to in vitro treatment with E2 on GnRH-stimulated gonadotropin secretion from the isolated pituitary gland.     

The negative feedback effects of testicular steroids are predominantly at the hypothalamus in the ram

This study aimed to delineate the hypothalamic and/or pituitary actions of testosterone and its primary metabolites 5 alpha-dihydrotestosterone and estradiol (E) in adult castrated rams (wethers) during the breeding season. In Exp 1, wethers were treated for a week with twice daily injections (im) of peanut oil, 8, 16 or 32 mg/day testosterone propionate (TP) or dihydrotestosterone benzoate (DHTB) or an sc silastic implant containing 1 or 3 cm E. TP decreased plasma LH concentrations, increased (P less than 0.05) LH interpulse interval, did not have consistent effects on LH pulse amplitude, and had minimal effects on plasma FSH concentrations. DHTB decreased LH and FSH concentrations and increased (P less than 0.05) LH interpulse interval. E reduced (P less than 0.05) plasma LH and FSH concentrations and increased LH interpulse interval but had no effects on LH pulse amplitude. In Exp 2, hypothalamo-pituitary disconnected wethers given 125 ng GnRH every 2 h, were treated with either peanut oil, 32 mg/day TP or DHTB or 3 cm E. None of the treatments affected plasma LH or FSH concentrations or LH pulse amplitude. Exp 3 investigated the effects on GnRH of treatment of wethers either with peanut oil or TP. TP reduced GnRH concentrations (P less than 0.05) and pulse amplitude (P less than 0.01) and increased interpulse interval (P less than 0.05). These data provide evidence that, during the breeding season, the principal site of negative feedback of testicular steroids in the ram is the hypothalamus, resulting in decreased GnRH secretion; feedback effects at the pituitary are minimal.     

Sex steroid control of gonadotropin secretion in the human male. II. Effects of estradiol administration in normal and gonadotropin-releasing hormone-deficient men

Although prior studies have suggested that estrogens exert their negative feedback effect at the pituitary level in men, these conclusions have been based on models that evaluate changes in LH pulse amplitude and frequency and, therefore, only provide indirect information concerning the site of action of estrogens. To assess whether estradiol (E2) inhibits gonadotropin secretion directly and solely at the pituitary level in men, we determined the pituitary responses to physiological doses of GnRH in six men with complete GnRH deficiency, whose pituitary-gonadal function had been normalized with long term pulsatile GnRH delivery, before and during a 4-day continuous E2 infusion (90 micrograms/day). To deduce whether E2 has an additional inhibitory effect on hypothalamic GnRH secretion, their responses were compared with the effects of identical E2 infusions on spontaneous gonadotropin secretion and the responses to a 100-micrograms GnRH bolus in six normal men. Both groups were monitored with 15 h of frequent blood sampling before and during the last day of the E2 infusion. In the GnRH-deficient men, the first three GnRH doses were identical and chosen to produce LH pulses with amplitudes in the midphysiological range of values in our normal men (i.e. a physiological dose), while the last four doses spanned 1.5 log orders (7.5, 25, 75, and 250 ng/kg). The 250-ng/kg dose was always administered last because it is known to be pharmacological. In the GnRH-deficient men, mean LH and FSH levels as well as LH pulse amplitude all decreased significantly (P less than 0.02) during E2 infusion, demonstrating a direct pituitary-suppressive effect of E2. Mean LH (P less than 0.01) and FSH (P less than 0.05) levels and LH pulse amplitude (P less than 0.01) also decreased significantly in the normal men. The degree of suppression of mean LH (52 +/- 3% vs. 42 +/- 12%) and FSH (49 +/- 10% vs. 37 +/- 10%) levels was similar in the two groups. These results provide direct evidence that E2 inhibits gonadotropin secretion at the pituitary level in men and suggest that the pituitary is the most important, and possibly the sole, site of negative feedback of estrogens in men.     

Testosterone administration inhibits gonadotropin secretion by an effect directly on the human pituitary

Testosterone (T) administration slows LH pulse frequency in man, presumably by an effect on the hypothalamic GnRH pulse generator, but it also may have a direct action on the pituitary. To determine if T does indeed affect gonadotropin secretion by acting directly on the pituitary, we studied the effect of T on GnRH-stimulated gonadotropin secretion. Six men with hypogonadotropic hypogonadism were treated with physiological doses of GnRH (5 micrograms every 2 h, sc by automatic infusion pump) for 6 weeks. Once their gonadotropin levels were normal, the men received a supraphysiological dosage of T enanthate (200 mg, im, weekly for 8 weeks) in addition to GnRH. They then received GnRH alone for a final 8-week period. Blood sampling was performed every 10 min for 8 h at the end of each of the three study periods. T administration suppressed the mean serum LH level to about 50% of the value during GnRH alone [18 +/- 2 (+/- SE) vs. 37 +/- 2 micrograms/L; P less than 0.05] and suppressed the mean serum FSH level to about 30% of the value during GnRH alone (39 +/- 6 vs. 128 +/- 28 micrograms/L; P less than 0.05). Eight weeks after stopping T, while continuing GnRH alone, serum LH and FSH levels were similar to those at the end of the first period of GnRH administration. The mean LH response to GnRH was reduced during T administration (17 +/- 3 micrograms/L) compared to that during the initial period of GnRH alone (31 +/- 4 micrograms/L; P less than 0.05). Serum T and estradiol levels were in the low normal range after GnRH alone before T administration (11 +/- 2 nmol/L and 105 +/- 17 pmol/L, respectively) and increased to just above the normal adult ranges after 8 weeks of T administration (36 +/- 5 nmol/L and 264 +/- 49 pmol/L, respectively). These results demonstrate that T and/or its metabolites inhibit LH and FSH secretion by a GnRH-independent mechanism, probably directly on the pituitary gland, in man.     

Effects of gonadectomy on the in vitro and in vivo gonadotropin responses to gonadotropin-releasing hormone in male and female rats

Pituitary gonadotropin responses to GnRH were measured using both in vitro and in vivo methods to investigate the contribution of increased pituitary responsiveness to GnRH in generating the rise in serum gonadotropin levels after gonadectomy. We compared in vitro GnRH-stimulated secretion rates of LH and FSH of perifused pituitaries obtained from intact female (metestrous) and male rats, and rats gonadectomized 2 or 6 days earlier. GnRH pulses (peak amplitude, 50, 500, or 5000 ng/ml; frequency, one per h) caused significant dose-dependent increases in gonadotropin secretion rates. However, gonadectomy resulted in decreased secretion rates of LH and FSH. Similar findings were observed for in vivo serum gonadotropin responses to a single iv injection of GnRH (males received 250 or 1000 ng; females received 1000 or 4000 ng). These results indicate that increases in serum LH and FSH levels 2 or 6 days after gonadectomy are not mediated by increased responses of the rat anterior pituitary to GnRH. We have also shown that perifused pituitaries from proestrous and diestrous rats exhibit significantly higher GnRH-stimulated gonadotropin secretion rates than pituitaries from metestrous and estrous rats. Therefore, we tested the effect of in vivo pretreatment with 17 beta-estradiol (E2) or testosterone (T) in both female and male rats on the in vitro secretion of LH and FSH. Rats were gonadectomized and received a sc Silastic implant containing E2, T, or no steroid as a control 6 days before perifusion. Perifused pituitaries received pulses of GnRH (peak amplitude, 50 ng/ml; frequency, one per h). In vivo pretreatment with E2, but not T, caused significant increases of in vitro LH and FSH secretion rates for pituitaries of both sexes. Overall, our data demonstrate that gonadectomy does not cause increases in LH and FSH secretory responses to GnRH, and that prior exposure to E2 in vivo has a major stimulatory influence on the in vitro secretion of both gonadotropins regardless of sex.     

Effects of recombinant human inhibin and testosterone on gonadotropin secretion and subunit mRNA in superfused male rat pituitary cell cultures stimulated with pulsatile gonadotropin-releasing hormone.

Effects of recombinant human inhibin (rh inhibin) and testosterone on follicle-stimulating hormone (FSH) and luteinizing hormone (LH) secretion and mRNA levels of gonadotropin subunits were investigated in superfused male rat pituitary cell cultures. During superfusion, the cells were stimulated with gonadotropin-releasing hormone (GnRH) pulses (10 nM, 6 min/h) and exposed to rh inhibin (2 ng/ml) and/or testosterone (10 nM) for up to 20 h. The concentrations of FSH and LH were measured in effluent media by radioimmunoassay (RIA), and subunit mRNAs were determined by Northern blot hybridizations using rat FSH beta, LH beta and alpha genomic and cDNA probes. Rh inhibin suppressed the secretion of FSH (30-40% of control) and the secretion of LH to 50-60% of control, but inhibited only FSH beta mRNA (to non-detectable levels). Testosterone alone suppressed the release of LH to 50% of control, whereas FSH release was increased to 130-160% (P less than 0.05) of control. This increase was due to higher interpulse values without significant changes in the pulse amplitude. Also FSH beta mRNA level was increased (1.5-fold, P less than 0.05) but only after 17-20 h of treatment. On the other hand, testosterone had no effect on LH beta and alpha subunit mRNA levels. Testosterone in combination with rh inhibin showed an inhibitory effect on LH beta mRNA; however, the pattern of LH release was not significantly different from that observed with rh inhibin or testosterone alone. Combined effects of testosterone and rh inhibin on FSH secretion and FSH beta mRNA were similar to those observed with rh inhibin alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

Gonadotropin-releasing hormone-induced stimulation and desensitization of free alpha-subunit secretion mirrors luteinizing hormone and follicle-stimulating hormone in perifused rat pituitary cells

A pulsatile pattern of hypothalamic GnRH stimulation is necessary for the maintenance of pituitary LH and FSH secretion, with continuous GnRH leading to a decrement in response. Although the physiological pattern of free alpha-subunit secretion closely mimics that of LH, several reports have indicated that free alpha-subunit is not desensitized by continuous GnRH stimulation. To explore the basis of this phenomenon, we have evaluated the responses of all three gonadotrope secretory products to carefully coordinated administration of pulsatile and continuous GnRH in a dispersed rat pituitary perifusion system. Sensitivities (ED50) to GnRH fell within a narrow range for free alpha-subunit (11.5 nM), LH (12.9 nM), and FSH (17.3 nM), although a greater mass of LH than free alpha-subunit or FSH was released after each pulse of GnRH. The response to a standard GnRH pulse (10 nM) administered every 15, 30, or 120 min for 9 h was very stable, with no evidence of priming, summation, or loss of response. LH, FSH, and free alpha-subunit did, however, show significantly (P less than 0.05) higher pulse amplitude with longer interpulse intervals. In contrast to previous observations in vivo, the three gonadotrope secretory products showed parallel desensitization in response to continuous infusions of GnRH. This loss of response was significant (P less than 0.05) after exposure to as little as 0.1 (FSH) to 0.5 nM (LH and alpha-subunit) GnRH for 2 h or to higher concentrations of GnRH (10 nM) for as little as 15 min (LH, FSH, and alpha-subunit). These concentrations and durations of GnRH stimulation are within the range of values measured in vivo. We conclude that 1) free alpha-subunit, LH, and FSH have similar concentration and frequency responses to pulsatile GnRH, although the absolute amount of hormone released is different for each secretory product; 2) the frequency of pulsatile GnRH stimulation can function as an independent determinant of secretion for each of the three products; and 3) in contrast to observations in vivo, free alpha-subunit, LH, and FSH secretion desensitize similarly after exposure to concentrations or durations of GnRH that may occur in vivo. These observations raise the possibility that desensitization plays a role in the physiological regulation of gonadotrope secretion.     

Chronic inhibitory effect of follicle-stimulating hormone (FSH)-suppressing protein (FSP) or follistatin on activin- and gonadotropin-releasing hormone-stimulated FSH synthesis and secretion in cultured rat anterior pituitary cells

The effects of bovine FSH-suppressing protein (FSP) or follistatin on activin- and GnRH-stimulated FSH synthesis and secretion have been studied using cultured pituitary cells from adult male Sprague-Dawley rats. Exposure to FSP (0.001-10 nM) for 3 days dose-dependently suppressed basal FSH secretion (IC50 = 146 +/- 21 pM., mean +/- SE), cellular content (IC50 = 269 +/- 8 pM) and total FSH (IC50 = 181 +/- 25 pM), with no effect on LH. Activin (0.3 nM) increased FSH secretion 2.1-fold, cellular content 1.3-fold, and total FSH 1.9-fold during a 3-day incubation, but these increases were dose-dependently inhibited by concomitant treatment with 35-kDa bovine FSP (0.1-3 nM), with complete inhibition occurring at concentrations between 1 and 3 nM. The 31- and 39-kDa forms of bovine FSP also antagonized the actions of activin. GnRH (1 nM) increased FSH secretion 1.8-fold and total FSH 1.6-fold during a 3-day incubation, effects that were dose-dependently inhibited by concomitant treatment with 35-kDa bovine FSP. The highest tested concentration of FSP (3 nM) suppressed GnRH-stimulated FSH secretion and total FSH to 59 and 57%, respectively, of the levels found in untreated cultures. All three forms of bovine FSP produced a significant inhibition of FSH secretion and total FSH stimulated by GnRH. FSP also suppressed FSH secretion and total FSH in response to activators of protein kinase C including 100 nM phorbol 12-myristate 13-acetate (43 and 59%, respectively) and 100 nM mezerein (40 and 60%, respectively). Finally, treatment of cultured pituitary cells with 35-kDa FSP at 1 and 3 nM for 3 days resulted in 21 and 24% decreases in GnRH binding sites, respectively. It is concluded that (i) FSP inhibits not only the secretion but also the synthesis of FSH induced by activin and GnRH in long-term culture, and (ii) FSP may cause its inhibitory effects on GnRH by suppression of the protein kinase C system, and possibly by reduction of GnRH binding sites.     

Hypothalamic-pituitary-gonadal axis in two men with aromatase deficiency: evidence that circulating estrogens are required at the hypothalamic level for the integrity of gonadotropin negative feedback

BACKGROUND: In men, the feedback of gonadotropins is regulated by estrogens that come from the aromatization of testosterone, but the relative contribution to the inhibition of LH and FSH secretion by the amount of locally produced estrogens within the hypothalamus and/or the pituitary, and the amount of circulating estrogens still remains unknown. OBJECTIVE: In order to evaluate the effect of regulation induced by estradiol on the hypothalamic-pituitary-gonadal (HPG) axis, we studied the pulsatility of LH and FSH in two aromatase-deficient men (called subject 1 and subject 2), in which the production rate of estrogen (both local and circulating) is completely, or at least severely, impaired. DESIGN: FSH and LH were evaluated in terms of their pulsated secretion and as GnRH-stimulated secretion in two phases: phase 1, before estrogen treatment; and phase 2, during estrogen treatment with 25 microg transdermal estradiol twice weekly. METHODS: Blood samples were taken during phase 1 and phase 2 at 0800 h for basal measurements of LH, FSH, inhibin B, testosterone, and estradiol. The analysis of the pulsatility of LH and FSH was performed by sampling every 10 min for 8 h in the two phases. Gonadotropin response to GnRH-stimulation test was studied by serial standard sampling after 100 microg GnRH i.v. bolus in phases 1 and 2. RESULTS: Estrogen treatment led to a significant reduction in both LH-pulsated frequency (7.5 +/- 0.7 in phase 1, 4.5 +/- 0.7 in phase 2) and amplitudes (3.5 +/- 0.006 in phase 1, 1.9 +/- 0.4 in phase 2) of peaks, whereas FSH showed only a conspicuous reduction in serum levels and a trend towards the reduction of the amplitudes of its peaks without modification of the frequency of the pulses. Both testosterone and gonadotropins decreased during phase 2, whereas estradiol reached the normal range in both subjects. Transdermal estradiol treatment significantly lowered the peaks of both serum LH and FSH after GnRH as well as the incremental area under the curve after GnRH administration in both subjects. Basal serum inhibin B levels were slightly higher before transdermal estradiol treatment (phase 1) than during estrogen treatment (phase 2) in both subjects. CONCLUSIONS: The administration of estrogen to aromatase-deficient men discloses the effects of circulating estrogens on LH secretion, exerted both at pituitary level, as shown by the decrease of basal and GnRH-stimulated secretion of LH and the LH pulsed amplitude, and at hypothalamic level as shown by the reduction of the frequency of LH pulses. The present study, coupling the outcomes of basal, GnRH-stimulated and the pulsatile evaluation of LH and FSH secretion in two aromatase-deficient men, demonstrates that circulating estrogens play an inhibitory role in LH secretion by acting on the hypothalamus and the pituitary gland of men. The discrepancy among testosterone levels, the arrest of spermatogenesis and a slightly inappropriate respective increase of serum FSH (lower than expected) suggests a possible role of estrogens in the priming and the maturation of HPG axis in men, an event that has never occurred in these two subjects as a consequence of chronic estrogen deprivation.     

Effects of bovine follicular fluid on the secretion of LH and FSH in inhibin-immunized seasonally anoestrous ewes

It has been shown previously that treatment of seasonally anoestrous ewes with steroid-free bovine follicular fluid (FF), a crude inhibin-containing preparation, leads to a decrease in plasma FSH level which is accompanied by a marked increase in pulsatile LH secretion. Since FF contains several factors (e.g. activin, follistatin, unidentified components) other than inhibin, which might act to modify gonadotrophin secretion, it was of interest to establish whether these concurrent effects of FF on FSH and LH secretion persisted in ewes which had been actively immunized against a synthetic peptide replica of the alpha subunit of bovine inhibin. In June 1989 (anoestrous period) groups of inhibin-immune and control ewes (n = 5 per group) received 6-hourly s.c. injections of either bovine serum (2 ml) or one of two doses of FF (0.5 ml or 2 ml) for 3 days. Blood was withdrawn at 6-h intervals for 6 days beginning 24 h before the first injection. On the final day of treatment, additional blood samples were withdrawn at 15-min intervals for 8 h to monitor pulsatile LH secretion. Ewes were then challenged with exogenous gonadotrophin-releasing hormone (GnRH; 2 micrograms i.v. bolus) to assess pituitary responsiveness. In control ewes, FF promoted a dose-dependent suppression of basal (maximum suppression 65%; P less than 0.01) and post-GnRH (maximum suppression 72%; P less than 0.01) levels of FSH in plasma. This was accompanied by an increase (P less than 0.01) in LH pulse frequency from 1.40 +/- 0.24 (S.E.M.) to 3.20 +/- 0.37 pulses/8 h. In contrast, FF did not affect secretion of either FSH or LH in inhibin-immunized ewes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of progesterone on pulsatile luteinizing hormone (LH) secretion and LH subunit messenger ribonucleic acid during lactation in the rat

In ovarian-intact lactating rats, removal of the suckling stimulus leads to restoration of pituitary LH beta mRNA levels and pulsatile LH secretion after 72 h, which correlates with a sharp decrease in plasma progesterone concentrations to basal levels. In contrast, in ovariectomized lactating rats, the increase in pituitary LH function is observed by 24 h after pup removal. To determine if progesterone secretion from the ovary participates in the delayed recovery of LH secretion, we treated lactating rats with the progesterone antagonist RU 486 and determined the effects on the time course of recovery of pulsatile LH secretion and LH subunit mRNA after pup removal and on pituitary responsiveness to GnRH. In ovarian-intact lactating rats treated with RU 486, pulsatile LH secretion was observed in about 40% of the rats within 24 h after pup removal (LH interpulse interval, 43.7 +/- 8.3 min) and in about 90% of the rats within 48 h after pup removal (LH interpulse interval, 46.1 +/- 3.6 min). The mean plasma LH level in the RU 486-treated rats was 10.1 +/- 2.2 ng/ml 24 h after removal of pups (control, less than 5 ng/ml) and had increased to 35.1 +/- 6.4 ng/ml 48 h after pup removal (control, 9.1 +/- 2.5 ng/ml). However, RU 486 treatment had no significant effect on LH mRNA subunit levels. To determine whether progesterone acts at the pituitary to block GnRH stimulation of LH secretion, we tested the effects of RU 486 on LH secretion in response to 2- and 5-ng pulses of GnRH. Pituitary responsiveness was tested 24 h after pup removal. We found that both doses of GnRH were effective in stimulating pulsatile LH secretion, and treatment with RU 486 had no significant effect on this response. We conclude from these studies that progesterone secretion from the ovary contributes to the inhibition of LH secretion that occurs after pup removal, since antagonizing progesterone's action resulted in an earlier restoration of pulsatile LH secretion. The increase in LH secretion occurred in the absence of any significant changes in responsiveness of the pituitary to GnRH stimulation or in LH subunit mRNA levels. Therefore, the primary site of action of progesterone would appear to be at the hypothalamus to suppress pulsatile GnRH secretion.     

Acute inhibitory effect of follicle-stimulating hormone-suppressing protein (FSP) on gonadotropin-releasing hormone-stimulated gonadotropin secretion in cultured rat anterior pituitary cells

Follicle-stimulating hormone (FSH)-suppressing protein (FSP) or follistatin, a novel gonadal glycoprotein hormone, has been shown to have chronic inhibitory effects on the secretion of both FSH and luteinizing hormone (LH) in response to gonadotropin-releasing hormone (GnRH) in vitro. The present study was designed to investigate the acute effects of bovine FSP on GnRH-stimulated gonadotropin secretion and to examine the potential subcellular sites of this action of FSP using cultured pituitary cells. Anterior pituitaries from adult male Sprague-Dawley rats were enzymatically dispersed and cultured for 48 h, after which the cells were treated with bovine FSP for 6 h, followed by a 4 h stimulation with secretagogues in the continued presence of FSP. Results showed that the 35 kDa form of bovine FSP (0.1-3 nM) dose-dependently suppressed GnRH-stimulated FSH and LH secretion, with inhibition of 38 and 25%, respectively, at 3 nM. In addition, FSP suppressed gonadotropin secretion in response to activators of protein kinase C (phorbol 12-myristate 13-acetate (PMA) and mezerein) and a calcium ionophore (A23187). However, FSP had no effect on gonadotropin secretion evoked by melittin, an activator of phospholipase A2. Furthermore, 35 kDa bovine FSP did not compete with GnRH for GnRH binding sites in a direct competition study and treatment of cultured pituitary cells with FSP (0.1-3 nM) for 10 h did not alter the number of GnRH binding sites on the cell membranes. Finally, similar inhibitory effects on gonadotropin secretion in response to GnRH, PMA and mezerein were obtained with the 31 and 39 kDa forms of bovine FSP, each at a concentration of 1 nM. We conclude from the present study that FSP acutely inhibits GnRH-stimulated gonadotropin secretion in cultured pituitary cells, and that FSP exerts its action beyond the GnRH receptor, possibly by affecting the protein kinase C and/or the calcium-calmodulin systems.     

The rapid ovarian secretory response to pituitary stimulation by the gonadotropin-releasing hormone agonist nafarelin in sexual precocity

We evaluated a GnRH agonist (GnRHa) as a potential single stimulus to both pituitary and ovarian secretion in 13 girls with true precocious puberty. We compared the GnRH agonist [6-D-(2-naphthyl)alanine]GnRH acetate (nafarelin, Syntex) administered as a single sc injection of 0.2 microgram/kg to GnRH infused iv in a dose of 2 micrograms/kg X h for 3 h and assessed the response of plasma steroid intermediates in estradiol (E2) biosynthesis. Although serum LH and FSH levels increased to similar peaks 3 h after commencing GnRH and nafarelin testing, they rose faster (P less than 0.01 at 1 h) and remained elevated longer (P less than 0.05 at 24 h) after nafarelin administration. At the third hour of testing with either agent, LH and FSH rose 8.8- and 3.4-fold, respectively (P less than 0.001 vs. baseline), whereas the rise in E2 was inconsistent and averaged only one third (P less than 0.02). However, plasma E2 increased later after nafarelin, but not after GnRH, rising from a baseline level of 30 +/- 6 (+/- SEM) to 115 +/- 13 pg/ml at 24 h (P less than 0.001). The least E2 response to nafarelin at this time was 150%. This rise is probably an underestimate of the maximum E2 rise, since a 6-fold response to nafarelin was found at 12 h in patients sampled then. Measurement of steroid intermediates from progesterone and 17 alpha-hydroxypregnenolone to E2 indicated that the response to nafarelin was typical of normal ovarian follicular secretion. That is, plasma levels of the intermediates in E2 biosynthesis rose less than 2-fold, and only the elevations in androstenedione, from 58 +/- 10 to 78 +/- 16 ng/dl (P less than 0.05), and estrone, from 14 +/- 3 to 38 +/- 7 pg/ml (P less than 0.02), at 24 h were significant. The greater effectiveness of nafarelin than GnRH in stimulating E2 secretion appears to be related to the more prolonged gonadotropin response. The magnitude, consistency, specificity, and rapidity of the gonadotropin and E2 responses to nafarelin indicate that this is a promising agent for rapidly testing pituitary and ovarian function simultaneously.