Effects of glucocorticoids on responsiveness of luteinizing hormone and follicle-stimulating hormone to gonadotropin-releasing hormone by male rat pituitary cells in vitro

To determine if the inhibitory effects of glucocorticoids on GnRH-stimulated secretion of LH observed in male rats in vivo are exerted directly on the pituitary, dispersed pituitary cells from adult male rats were treated with 60 or 600 ng/ml cortisol (F) or corticosterone (B) during one or two 48-h incubations. Control cells received no glucocorticoids. During the second 48 h, some cells from each group were treated with GnRH (2.4 X 10(-11)-6.2 X 10(-8) M). Concentrations of LH and FSH in media and cells were measured by RIA. Treatment with steroids had no effect on basal secretion or maximal GnRH-stimulated secretion of LH, or on maximal secretion of FSH. Treatment with 600 ng/ml B for 96 h increased basal secretion of FSH relative to controls. All treatments with glucocorticoids increased the slopes of the GnRH dose-response curves for both LH and FSH, cell content of LH, total (cells + medium) LH, and total FSH. Incubation with 6 micrograms/ml F or B or 60 ng/ml dexamethasone gave similar results. Decreasing the time period of the second incubation to 6 h results in no significant differences between control cells and cells treated with B or F. These results show that glucocorticoids have different effects in vivo and in vitro, suggesting that inhibitory effects of glucocorticoids on secretion of LH in vivo may not be exerted directly on the pituitary but are exerted elsewhere, perhaps by altered hypothalamic secretion of GnRH. Also, these results show that male and female pituitaries in vitro respond differently to glucocorticoids.     

Effects of glucocorticoids on secretion of luteinizing hormone and follicle-stimulating hormone by female rat pituitary cells in vitro

To determine if the divergent effects of glucocorticoids on the circulating levels of LH and FSH in female rats are exerted directly on the pituitary, adult female pituitary cells were treated either with no glucocorticoids or with 60 or 600 ng/ml cortisol or corticosterone during one or two 48-h incubations. During the second 48 h, some cells from each group were treated with GnRH (1.7 X 10(-12) - 4.6 X 10(-9) M). Concentrations of LH and FSH in media and cells were measured by RIA. Basal secretion of LH was inhibited 38-43% by different glucocorticoid treatment during the first 48 h and 21% by 600 ng/ml corticosterone during the second 48 h. In contrast, basal secretion of FSH was enhanced 22-64% during the first 48 h and 25-124% during the second 48 h. Secretion of LH in response to maximal stimulation with GnRH was unaffected by glucocorticoids, but maximal secretion of FSH was increased 68%. The responsiveness of the cells to GnRH, as determined from the slope of the GnRH dose-response curve for LH, was increased 43-50% by cortisol. The slope of the dose-response curve for FSH was unaffected, but the mean concentration of FSH as a function of the log dose of GnRH was increased 45-79%. Glucocorticoids had no effect on cell content of LH or total LH per dish, either under basal or maximal GnRH-stimulated conditions. Glucocorticoids increased basal cell content of FSH 41-82%, basal total FSH 35-93%, and maximal GnRH-stimulated total FSH 40-84%. These results suggest that the only negative effect of glucocorticoids on reproduction exerted at the level of the pituitary is a slight suppression of basal LH secretion, that glucocorticoids affect the pituitary directly by increasing FSH synthesis, and that the divergent effects of glucocorticoids on LH and FSH provide a novel model for differential regulation of the gonadotropins.     

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.     

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.     

In vitro pituitary responsiveness to gonadotropin-releasing hormone (LH-RH) in intact and castrated male and female rats

The in vitro response of pituitaries isolated from both normal and 18–21-day post-castration male and female intact rats to incremental doses of synthetic gonadotropin-releasing hormone (LH-RH) has been investigated. Intact male pituitaries released luteinizing hormone (LH) maximally at the smallest dose of LH-RH (0.1 ng/ml) whereas intact female pituitaries released LH in a dose-response fashion. FSH release from intact male pituitaries was considerably greater than that from intact female pituitaries. As with LH, intact male pituitaries appeared maximally stimulated at 0.1 ng/ml of LH-RH. Intact female pituitaries did not release FSH until a 10 ng/ml dose of LH-RH was used.Male and female castrate pituitaries were more susceptible to LH-RH-induced LH and FSH release than were their intact counterparts, although this was more pronounced with regard to LH release. In addition castrate male pituitaries were more sensitive to lower doses of LH-RH than were castrate female pituitaries, this being most pronounced regarding LH release. Castrate female pituitaries released less FSH at the 100 ng/ml dose than at the 10 ng/ml dose, possibly indicating inhibition at these higher doses.In addition, pituitary extraction and serum from normal and castrate male and female rats were examined for LH and FSH content. LH content of castrated rat pituitaries of both sexes was considerably greater than that of their intact counterparts, as expected. However, castrate male pituitaries contained significantly less FSH than intact male pituitaries, whereas the opposite was true for the female groups. Serum LH and FSH levels were increased in the castrate groups with no difference between sexes. Serum from intact males contained considerably more FSH than did the serum from intact females.     

The In Vitro Inhibitory Action of Antiprogestin RU486 on LH and FSH Secretion in the Absence of Progesterone in Rats Is Estrogen-Dependent

Previous in vivo findings show that in the virtual absence of progesterone (P), the antiprogestin RU486 reduces LH and FSH secretion in proestrous rats, indicating that activation of P receptor (PR) can occur in the absence of the cognate ligand. The present study investigates, in vitro, whether or not the inhibitory effect of antiprogestin RU486 on gonadotropin secretion in the absence of P is estrous cycle dependent, and whether its specific expression in proestrus mirrors the high estrogen (E₂) background.In the first experiment we investigated the effect of RU486 (10 nM) and/or LHRH (10 nM) on LH and FSH secretion in incubated pituitaries collected on each day of the estrous cycle of the rat. In the second experiment, we determined the effect of RU486 and/or LHRH on preovulatory LH and FSH release by pituitaries from female rats that were ovariectomized (OVX), treated with the antiestrogen LY117018-HCL (Eli Lilly & Co.), or injected with 20 g of estradiol benzoate (EB). The third experiment investigated the effect of RU486 and/or LHRH on LH and FSH release by pituitaries collected from intact or EB-treated (0.1 mg/kg over three consecutive days) male rats.RU486 reduced both basal and LHRH-stimulated LH and FSH secretion in proestrous pituitaries from normal 4-day cyclic rats. By contrast, in diestrous pituitaries, RU486 increased both parameters of LH secretion but was without effect on FSH release. RU486 was also without effect in pituitaries collected from rats in estrus or metestrus, or from OVX or antiestrogen-treated rats. Moreover, EB injection or treatment induced the full inhibitory effect of RU486 in pituitaries from female and male rats, respectively.The above results suggested that P occupancy of the receptor is not required for the formation or function of the active receptor and hence for preovulatory LH and FSH secretion, and that this form of PR activation at pituitary level is E₂-dependent and not genetically determined.     

Inhibin suppresses luteinizing hormone (LH)-releasing hormone self-priming: direct action on follicle-stimulating hormone secretion and opposition of estradiol-enhanced LH secretion.

Previous studies have suggested that the ovary produces a factor that maintains the pituitary in a state of low LHRH responsiveness that must be overcome by the self-priming action of LHRH. To determine the role of inhibin in maintaining low LHRH responsiveness in pituitaries of diestrous female rats, endogenous inhibin was passively immunoneutralized in vivo, and the pituitaries were removed 18-20 h later and examined for LHRH responsiveness in vitro. Pituitaries from diestrous control rats produced the biphasic pattern of gonadotropin secretion that typifies LHRH self-priming: an initial low secretory response to LHRH (lag phase), followed by a protein synthesis-dependent transition to an enhanced rate of secretion with continued LHRH exposure (primed phase). Immunoneutralization of endogenous inhibin [antiserum (AS) treated] resulted in an increased rate of LH secretion during the lag phase, while no change was observed in the primed phase rate of LH secretion. FSH secretion from pituitaries of AS-treated rats was increased during the lag phase to a rate of secretion similar to that observed during the primed phase of FSH secretion from control pituitaries, and it was increased further during the primed phase of secretion. These results suggest that inhibin is at least partially responsible for the low secretion of LH observed during the lag phase response to LHRH exposure and is totally responsible for the lowered rate of FSH secretion during the lag phase. The observation that the enhanced rate of gonadotropin secretion observed with AS-treated pituitaries during the lag phase was resistant to inhibition of protein synthesis provides further evidence that a partial transition from the lag to the primed phase had already occurred. Pituitaries from ovariectomized rats were also examined in order to place the contribution of inhibin in perspective with the total ovarian influence on pituitary responsiveness to LHRH. Unexpectedly, LH secretion during the lag phase was similar to the low secretion rate of diestrous control pituitaries, and the higher primed rate of secretion failed to fully develop, suggesting that an additional ovarian factor was required to induce and maintain pituitary responsiveness to LHRH in terms of LH secretion. FSH secretion from the ovariectomized rats was similar to that observed from pituitaries of AS-treated rats, thus further supporting the concept that inhibin is fully responsible for the suppression of FSH secretion in response to LHRH. Plasma from the AS-treated rats revealed a 2-fold increase in estradiol levels compared with diestrous control rats.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparison of the effects of antiprogestins RU38486, ZK98299 and ORG31710 on periovulatory hypophysial, ovarian and adrenal hormone secretion in the rat

The antiprogestin (AP) RU38486 (RU) blocks progesterone (P) and glucocorticoid (G) actions. Administration of 4 mg RU on proestrous morning to cyclic rats dissociates LH and FSH secretion on proestrous afternoon, early estrus and on estrous afternoon. In order to ascertain which action blocked by RU is predominant in the control of periovulatory LH and FSH secretion, a study was made on the effects of: a) 1 or 4 mg of ZK98299 (ZK) (type I P antagonist; Schering), b) 2 or 8 mg of Org31710 (OR) (type II P antagonist lacking anti-G actions; Organon) or c) 1 or 4 mg of RU (type II P antagonist; Exelgyn) to 4-day cyclic rats on proestrous morning on serum concentrations of LH, FSH, inhibin-alpha (I), estradiol-17beta (E), progesterone (P) and corticosterone (B) at 18:30 h on proestrus and at 02:00 and 18:30 h on estrus. Controls, receiving 0.2 ml oil, had elevated serum concentrations of all six hormones on proestrous afternoon; at early estrus, only serum concentrations of FSH and P remained elevated, and, on estrous afternoon, all hormones but I and B, that peaked again, had reached their lowest serum levels. All AP treatments except 1 mg ZK had the same effects. On proestrous afternoon serum LH concentrations were reduced and serum FSH concentrations were suppressed whereas serum levels of I, E, P and B were unaffected. At early estrus, basal serum concentrations of LH and E increased while FSH secretion was abolished. Serum levels of I, P and B did not differ from controls. AP treatments increased basal LH concentration, hyperstimulated FSH secretion and reduced serum I concentration on the afternoon of estrus. E, P and B serum levels did not differ from controls at this stage. Treatment with 1 mg ZK was less effective in reducing serum FSH on proestrous afternoon and at early estrus, and had no effect on serum concentrations of any hormone on estrous afternoon. These results indicate that blockade of P receptor activation by P is, predominantly, the mechanism of AP action on periovulatory gonadotropin secretion in rats.     

Kinetic characteristics of LH and FSH responses to LHRH in incubated pituitaries from ovariectomized or ovariectomized, estrogen-implanted rats

Incubated pituitary halves from ovariectomized, estrogen-implanted female rats were shown to be much more sensitive to LHRH than pituitaries from castrated, nontreated animals. LHRH in a concentration of 1,885 pg/ml increased the release of LH and FSH from 7.3 +/- 0.9 and 0.91 +/- 0.13 ng/h/hemipituitary respectively to 21.4 +/- 1.9 and 1.97 +/- 0.18 ng/h in animals implanted with the steroid. In contrast, 5,000 pg/ml of LHRH increased LH secretion from 3.4 +/- 0.3 to 8.4 +/- 0.4 ng/h in ovariectomized, nontreated animals. In pituitaries from both steroid and nontreated animals a highly significant dose response for LH and FSH secretion to the actual concentration of LHRH measured in each incubation tube by radioimmunoassay was observed. When expressed as percent of the corresponding control release, maximal stimulation of LH and FSH was comparable. Pituitaries from implanted animals provided a very sensitive bioassay for LHRH, in which amounts of the peptide lower than 100 pg/ml were detected. The apparent responsiveness to LHRH of pituitaries from estradiol-treated rats was found to be over 20 times greater than that of pituitaries from nontreated castrates.     

Effect of pulsatile gonadotropin-releasing hormone on the release of luteinizing hormone and follicle-stimulating hormone in vitro by anterior pituitaries from lactating and cycling rats

The ability of pituitaries from lactating animals to secrete LH and FSH in response to gonadotropin-releasing hormone (GnRH) was studied in vitro using a pituitary incubation system. Hemipituitaries were exposed to GnRH for 6 min during each hour of incubation. LH release by anterior pituitaries (APs) from day 5 postpartum rats nursing eight pups, in response to pulsatile exposure to GnRH, was significantly less than that released by APs from diestrous cycling females. Even though the amount of LH released by APs increased as lactation progressed, LH release by APs from day 15 postpartum rats nursing eight pups was still less than LH release by APs from diestrous females. In contrast pituitaries from lactating females nursing two pups released amounts of LH similar to that released by pituitaries from diestrous females, whereas females deprived of their litters for 48 h showed a greater response than diestrous females. Generally, there was a good quantitative relationship between the amount of LH released in vitro and plasma LH concentrations for all the intact groups studied. The ability of lactation to suppress the postcastration rise in serum LH also was demonstrated in vitro as pituitaries from ovariectomized or intact females nursing eight pups released similar amounts of LH on days 5 and 10 postpartum. However, by day 15 postpartum, even though serum LH concentrations were still very low, pituitaries from ovariectomized lactating females released LH in vitro at a rate similar to pituitaries from nonlactating rats. Serum FSH concentrations were not suppressed but similar in intact and cycling females. Also, the total amount of FSH released in vitro in response to GnRH by pituitaries from lactating and cycling females did not differ significantly, even though LH release differed greatly among these groups of animals. However, the patterns of GnRH-stimulating FSH secretion differed among intact lactating, ovariectomized lactating, and nonlactating females. Pituitary LH concentrations were similar on day 5 postpartum and diestrus and on day 15 postpartum and proestrus. Pituitary FSH concentrations on day 5 postpartum were similar to those during diestrus and proestrus and had increased 2-3 times by day 15 postpartum. Generally, there was no correlation between the amount of LH or FSH released by pituitaries in response to GnRH and pituitary gonadotropin content. In summary, the inability of pituitaries from lactating rats to respond adequately to large doses of GnRH in vitro suggests that the suckling stimulus indirectly suppresses pituitary responsiveness to GnRH. This suppression differentially affects basal LH secretion, but not basal FSH secretion, and may be the direct result of inadequate GnRH stimulation in vivo.     

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

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

Pituitary gonadotropin-releasing hormone receptor regulation in the hypogonadotrophic hypogonadal (hpg) mouse

Recent evidence indicates that endogenous GnRH is required for maintenance of its own pituitary receptors (GnRH-R). We have measured GnRH-R in pituitaries of hypogonadotrophic hypogonadal (hpg) mice, in whom hypothalamic GnRH is deficient or absent. The GnRH-R concentration in hpg male mouse pituitaries was 10.6 +/- 1 fmol/pituitary vs. 30.9 +/- 1 fmol/pituitary in normal male littermate pituitaries. Similarly, GnRH-R in female hpg mice (15.2 +/- 1.7 fmol/pituitary) were 30% those of normal random cycling females (51.4 +/- 3.5 fmol/pituitary). There was no difference in receptor affinity (Ka = 1.5-3 C 10(9) M-1) of hpg mouse pituitaries. The pituitary LH content in hpg male and female mice was very similar (range 3.4-4.8 micrograms/pituitary) representing 5% and 19% of normal male (95 +/- 7.2 micrograms/pituitary) and female (18.1 +/- 1.5 micrograms/pituitary) values, respectively. The administration of 50 ng GnRH sc 10 times daily to male hpg mice, increased GnRH-R to 80% of normal values within 3 days. Serum FSH and pituitary FSH content rose to normal male values after 7 days of GnRH injections. However, serum LH remained undetectable and pituitary LH reached only 20% of normal male levels, even after 15 days of GnRH administration. Treatment of hpg male mice with 60 ng GnRH either once daily for 6 days, or 12 times daily for 5 days, increased GnRH-R to 50% of normal male values. Twelve daily injections of GnRH elevated serum FSH to above the normal male range, whereas daily GnRH only doubled untreated hpg levels. Pituitary FSH was stimulated to 50% of normal with 12 daily injections, whereas once daily administration elevated pituitary FSH to 30% of normal values. Both pulsatile regimes depleted pituitary LH. These data demonstrate that: 1) despite absence of bioactive GnRH, GnRH-R values are only reduced to 30% of normal in hpg mouse pituitaries, suggesting that little, if any, endogenous GnRH is required for expression of GnRH receptors. 2) Pituitary GnRH-R number rapidly increase when GnRH is administered to hpg male mice indicating that, as in the rat, GnRH positively regulates its own receptor concentration. 3) The pituitary FSH and LH responses to GnRH treatment in hpg mice depends to a different extent on the frequency and duration of GnRH administration. 4) The hpg mouse provides an ideal animal model for investigating the interaction of defined regiments of exogenous GnRH and gonadal steroids on pituitary GnRH receptor and gonadotroph function.     

Glucocorticoid repression of the reproductive axis: effects on GnRH and gonadotropin subunit mRNA levels

Activation of the stress axis by glucocorticoids suppresses reproductive function in many species. Here, we performed studies to determine whether these effects are mediated at the level of the hypothalamus or pituitary or both, and to dissect the underlying molecular mechanisms, using two established rodent models. Rats were treated either chronically or acutely with glucocorticoids, and circulating gonadotropins, GnRH mRNA levels, and gonadotropin subunit mRNAs levels were measured. In model I, chronic treatment for 6 days with corticosterone (CORT) was used in adult intact male rats. CORT caused a significant decrease in serum LH but not FSH secretion compared to vehicle. Whereas pituitary LHbeta and FSHbeta mRNA levels were not affected by CORT treatment, hypothalamic GnRH mRNA was significantly decreased by 35-40%. In model II, acute blockade of the estradiol (E(2))-induced gonadotropin surge by dexamethasone (DEX) was used in 28-day-old female rats. DEX treatment resulted in substantially lower serum LH and FSH concentrations compared to vehicle, although DEX had no effect on GnRH mRNA and LHbeta mRNA levels. By contrast, FSHbeta mRNA levels were about 14-fold lower in DEX-treated females. Taken together, these results indicate that suppression of gonadotropin levels by chronic elevations in glucocorticoids/stress may be accounted for in part by suppression of GnRH mRNA levels, whereas short-term glucocorticoid treatment to block the gonadotropin surge appears to involve other mechanisms including decreased FSHbeta mRNA levels.     

A detailed examination of the in vivo and in vitro effects of ACTH on gonadotropin secretion in the adult rat

These studies were designed to: (1) determine the effects of continuous infusion of synthetic ACTH(1-24) on postcastration changes in serum and pituitary LH, FSH and prolactin in the male rat; (2) assess the effects of adrenalectomy on the gonadotropin and prolactin response to ACTH, and (3) test the hypothesis that ACTH may directly (not via adrenal factors) alter gonadotropin secretion at the brain and/or pituitary level. Adult male rats were either orchidectomized (ORX) or orchidectomized-adrenalectomized (ORX-ADX), and were treated continuously for 6 days with ACTH(1-24) (10 micrograms/day) or saline using an osmotic minipump. Animals were killed on day 6 following castration. ACTH treatment reduced serum LH and prolactin levels in ORX rats to mean values +/- SE of 204 +/- 25 and 37 +/- 3 ng/ml respectively, compared to 366 +/- 72 and 62 +/- 7 ng/ml in saline-treated ORX animals. Serum FSH concentrations were not altered by ACTH administration. Pituitary concentrations of LH and FSH, but not prolactin were enhanced by ACTH treatment. Adrenalectomy had no effect on serum and pituitary gonadotropin and prolactin levels, but abolished the effects of ACTH on these parameters. Central (intracerebroventricular) infusion of ACTH(1-24) (6 micrograms/day X 4 days) failed to alter the rise in serum LH in male rats following orchidectomy. Acute treatment with large doses of ACTH of perifused anterior pituitary glands from male rats and chronic treatment with ACTH of enzymatically dispersed anterior pituitary cells from female rats did not influence basal or GnRH-stimulated LH secretion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autoregulation of follicle-stimulating hormone secretion in the hamster: evidence for control at the level of the anterior pituitary gland

Pituitary tissue obtained from proestrous, phenobarbital-treated hamsters was placed in organ culture, and the release rates of FSH and LH were monitored. Addition of LHRH to the culture medium increased gonadotropin release rates. Preincubation of pituitaries in medium that contained 1 microgram purified FSH ( NIADDK rat FSH-I-5) resulted in enhanced basal and LHRH-induced FSH release rates. Full expression of FSH hypersecretion by pituitary tissue occurred after 3-h exposure to purified FSH. This phenomenon appears to require adequate tissue calcium concentrations. Further, exposure of pituitaries to purified FSH slightly but significantly enhanced basal but not LHRH-stimulated LH release compared with that in untreated controls. Experiments that employed labeled FSH, reduced exposure time to purified FSH, or decreased calcium concentration in the medium proved that the increased FSH secretory rate was not due to contamination of the medium with the purified FSH used to stimulate the pituitary. These studies suggest that FSH has the ability to influence its own secretion by an action at the level of the anterior pituitary gland.     

Prolactin suppresses luteinizing hormone secretion and pituitary responsiveness to luteinizing hormone-releasing hormone by a direct action at the anterior pituitary

In pathological or experimental hyperprolactinemia, the elevated circulating levels of PRL are the usual cause of the impairment in gonadotropic function. The present study was undertaken to determine whether PRL could suppress basal LH secretion and LHRH-stimulated LH release by a direct action at the anterior pituitary. Anterior pituitaries from ovariectomized rats were incubated in medium 199 alone or in medium 199 containing ovine PRL, and basal and the LHRH-stimulated LH release were followed for 2 or 3 h in vitro. Ovine PRL at 40 and 80 micrograms/ml suppressed basal LH release by 41% and 72%, respectively, at 2 h of incubation. This suppressive effect of both concentrations of PRL continued to the third hour of incubation. LHRH at 5 ng/ml increased the release of LH from pituitaries incubated in medium alone by 57%, 61%, and 107% at 1, 2, and 3 h of incubation, respectively. However, in the pituitaries treated with 40 micrograms/ml ovine PRL, the stimulatory effects of LHRH were diminished at all time points measured. Pretreatment of anterior pituitaries with ovine PRL for 6 h significantly inhibited by 81% the LHRH (5 ng/ml) stimulation of LH release at 2 h of incubation. On the other hand, inhibition of endogenous PRL release by 10(-6) M bromocriptine enhanced the stimulatory effects of 5 ng/ml LHRH by 2.5-fold at 2 h of incubation. The inhibitory effects of PRL on basal and stimulated LH secretion appeared unique, since neither BSA nor vasopressin could elicit similar suppressive effects on LH. These results suggest that in anterior pituitaries exposed to elevated levels of PRL, LH secretion and pituitary responsiveness to LHRH could be impaired. This phenomenon may contribute in part to the antigonadotropic effects of PRL.     

Epidermal growth factor stimulates secretion of rat pituitary luteinizing hormone in vitro

The effects of epidermal growth factor (EGF) on pituitary luteinizing hormone (LH) release and on the releases induced by oestradiol (E2) and LH-releasing hormone (LRH) were examined in a sequential double chamber perifusion system. In this system the mediobasal hypothalami (MBH) and/or pituitaries excised from normally cycling female rats in dioestrus were perifused with test media. Perifusion with EGF at 1 ng/ml for 30 min induced significant release (80-100% increase, P less than 0.05) of LH from hypothalamo-pituitary pairs, but not from the pituitary alone. Perifusion of the pituitary alone with medium containing 1 ng/ml EGF, resulted in significant release of LH (70-140% increase, P less than 0.05) after administration of 10(-7) M E2, but did not significantly influence LH release in response to 20 ng/ml LRH. These findings suggest that EGF may be involved in the regulation of pituitary gonadotrophin secretion by a direct effect on the hypothalamus and indirectly by increasing the pituitary responsiveness to E2.     

Steroid priming of the luteinizing hormone response to luteinizing hormone releasing hormone.

Perifusion experiments were performed to study the stimulatory effects of luteinizing hormone releasing hormone (LH-RH) on the release of LH from anterior pituitary tissue. Exposure of pituitary tissue from normal male rats to LH-RH (5 ng/ml for 5 min) induced a small release of LH; in tissue from ovariectomized rats receiving no pretreatment, the release was more than three times greater and in tissue from gonadectomized male or female rats pretreated with oestradiol benzoate and progesterone, the release was six times greater than that observed in normal rats. Further exposure of pituitary tissue from gonadectomized steroid-pretreated male and female rats to LH-RH (5 ng/ml) induced an increase in the level of LH even greater than that seen after the initial exposure (priming action of LH-RH); in tissue from ovariectomized rats receiving no pretreatment, less LH was released than after the first exposure to LH-RH and in tissue from normal male rats the response was unchanged.     

Sex and age differences in the spontaneous release of gonadotropins from immature rat pituitary glands in vitro

The present study was carried out to characterize the spontaneous release of LH and FSH from the pituitary gland of infant rats in an in vitro system. In addition, the responsiveness of their pituitary glands to synthetic LHRH in vitro was examined. Wistar-Imamichi male and female rats, aged between 1 and 21 days and androgenized female rats at 7 and 21 days of age were used. One-day-old female rats were androgenized by a subcutaneous injection of 1 mg of testosterone propionate. Animals were killed by decapitation, trunk blood was collected, and the pituitary gland was dissected free and weighed. Pituitaries were placed in 9 ml-test tube with 2 ml Krebs-Henseleit solution and incubation was carried out in a shaking incubator for 6 hours at 37 degrees C under the gassing of 5% CO2 and 95% O2. After preincubation for 15 min, medium was replaced with 2 ml fresh medium and LH and FSH concentrations released during the first 3 hr-incubation period were assessed as for the spontaneous release and the second 3 hr-incubation period assessed for the response to LHRH (10(-6)M) stimulation. In an experiment, time course changes of the spontaneous release of LH and FSH were studied using 7-day-old rat pituitaries. An aliquot of 0.5 ml of medium was taken at 30, 60, 120 and 180 min during the incubation. Gonadotropin contents in the pituitaries were determined by adding the residue in the pituitary gland and the amounts released into medium. Spontaneous release of LH and FSH increased with age in both male and female rats, and the released amounts of LH as well as FSH in female rats tended to be higher than those in males at 1, 3, 7 and 21 days of age examined. But significant sex differences in the spontaneous release of LH and FSH were only seen at 21-day-old; Spontaneous release of LH in female rats was 7 times higher than that in age-matched males. Serum LH and FSH concentrations in female rats were significantly higher than those in males at all ages examined, except the LH level at 1-day-old. In contrast to LH, age and sex differences in the magnitude of the spontaneous release of FSH from the pituitary paralleled with the age and sex differences in serum FSH concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Luteinizing-hormone-mediated precocious puberty induced in female rats by a prepuberal pituitary graft

The present study analyzes the mechanism of precocious puberty induced in female rats after a 'young' pituitary graft (obtained from 21-day-old animals). For this purpose, the following experiments have been performed: (1) female rats were grafted or sham-operated on day 21 with a littermate's pituitary the follicle-stimulating hormone (FSH), luteinizing hormone (LH) and estradiol plasma levels as well as the ovarian, uterine and adrenal weights were determined at different times after the graft; (2) female rats grafted or sham-operated on day 21 were treated with 0.2 ml of LH antiserum (LHAS) or the same volume of a normal horse serum (NHS); (3) female rats were injected on day 1 of life with 0.1 mg of estradiol benzoate or olive oil. Groups of these animals were decapitated daily between days 6 and 21 in order to measure gonadotropins and prolactin (PRL) pituitary content. Since on day 21 estrogenized females showed decreased gonadotropin content and normal PRL content, females in experiment 4 were grafted on day 21 with pituitaries obtained from control or neonatally estrogenized female rats. The results obtained showed that FSH, LH and estradiol plasma levels as well as ovarian and uterine weights increased after pituitary grafts. LHAS blocked the precocious puberty induced by the pituitary graft, and pituitaries obtained from neonatally estrogenized female rats were unable to modify the occurrence of puberty when grafted. In conclusion, this work evidences that precocious puberty induced by 'young' pituitary grafts was mediated by the increase in LH secretion from the graft.