Effects of a novel gonadotropin-releasing hormone antagonist (ORG 30850) on gonadotropin and prolactin secretion by rat pituitary cells in culture

The effect of a new GnRH antagonist (ORG 30850 ANT) on FSH, LH, and PRL secretion was studied using male rat pituitary cells in monolayer cell culture. In the absence of GnRH, ORG 30850 ANT did not alter spontaneous FSH and LH secretion into culture medium or the cell content of these hormones. In the presence of GnRH (10(-8) mol/l), ORG 30850 ANT significantly and dose-dependently inhibited FSH and LH secretion into culture medium while increasing their cell content. Conversely, in the presence of a single dose of ORG 30850 ANT, FSH and LH secretion rose significantly when subjected to increasing amounts of GnRH, whereas the hormonal cell content diminished. Furthermore, inhibition of GnRH-induced FSH and LH release by ORG 30850 ANT was not changed by pre-incubation with the GnRH antagonist regardless of the pre-incubation time. The inhibitory effect of the GnRH antagonist was observed early, with its peak occurring within 6 h of culture. These short-term studies indicate that ORG 30850 ANT specifically inhibits GnRH-induced gonadotropin release into culture medium, exerts no effect on the rate of gonadotropin production in the presence or absence of GnRH, competitively and reversibly inhibits the binding of natural GnRH to its receptors, and does not lead to any modifications in PRL secretion.     

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

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

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

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

Adrenocorticotropin-induced changes in ovine pituitary gonadotropin secretion in vitro

In vitro pituitary perifusion experiments were conducted to examine the effect of ACTH and related peptides on basal and GnRH-stimulated gonadotropin release. Treatments of 5 X 10(-7) M ACTH-(1-39), ACTH-(1-24), or ACTH-(18-39) were examined for their ability to influence basal gonadotropin secretion and the subsequent response to a 10(-9)- or 10(-8) M GnRH challenge. Administration of the 1-39 or 18-39 peptide sequences of ACTH similarly stimulated the release of LH and FSH (P less than 0.01). ACTH-(1-24) had no effect on basal gonadotropin secretion. Pretreatment with ACTH-(1-39) inhibited the LH and FSH responses to 10(-9) and 10(-8) M GnRH (P less than 0.05). Suppression of the LH response to 10(-8) M GnRH (P less than 0.05) and the FSH response to 10(-9) M GnRH (P less than 0.05) was observed after ACTH-(1-24) treatment. The administration of ACTH-(18-39) had no significant effect on GnRH-induced gonadotropin release. PRL concentrations were not affected by any of the ACTH peptides. Exposure to 10(-10) M GnRH or 5 X 10(-7) M synthetic ACTH-(1-39) produced an equivalent stimulation of LH secretion. GnRH pretreatment enhanced (P less than 0.05), while ACTH-(1-39) diminished (P less than 0.05), the subsequent response to GnRH. The GnRH receptor antagonist [D-pGlu1, D-Phe2, D-Trp3,6]GnRH attenuated the LH and FSH responses to GnRH and ACTH-(1-39) (P less than 0.05). The results obtained in this study indicate that certain portions of the ACTH molecule may affect gonadotropin secretion, perhaps by interacting with the GnRH receptor.     

In vivo and in vitro responses to gonadotropin releasing hormone in the turtle, Chrysemys picta, in relation to sex and reproductive stage

In vivo and in vitro responsiveness to gonadotropin releasing hormone (GnRH) was studied in the turtle, Chrysemys picta, after manipulation of reproductive condition by temperature: Warm temperatures (28 degrees) induced testicular growth and ovarian regression compared to cold (17 degrees) treatment. Only males (and primarily from cold treatment) responded to GnRH injection (40 micrograms/100 g body wt intracardiac); correlated increases occurred in plasma LH and testosterone. Effects of GnRH (10 and 100 ng/ml) on LH and FSH secretion by hemipituitaries were studied in a superfusion system; tissues responded to between 0.1 and 1 ng/ml GnRH. Sex differences were evident in both acute and chronic effects of GnRH. Although both groups of females had significantly (sixfold) higher pituitary LH content, basal secretion rates of gonadotropins were similar, and LH and FSH secretion in males was more responsive to GnRH. Gonadotropin secretion rates by male glands showed high initial increments (approx four- to sevenfold) followed by an attenuation (especially LH) during 5 hr of GnRH superfusion. In contrast, tissues from warm-treated females showed a smaller initial response (approx twofold) followed by a progressive increase in output over time, and glands from cold-treated females did not respond to GnRH. Total LH secretion by superfused male hemiglands represented almost half the total LH recovered (secreted + stored); whereas, females secreted only 5% ("cold-treated") or 10% ("warm-treated") of total LH. Thus, the capacity of the pituitary to respond to GnRH is influenced by both sex and reproductive condition in the turtle. Secretion of both FSH and LH were similarly stimulated by GnRH, but thyrotropin (TSH) secretion was independent of GnRH.     

Effect of recombinant human inhibin on gonadotropin secretion by the male rat

We have examined the effect of recombinant human inhibin-A on basal and GnRH-induced gonadotropin secretion by male rats or cultured anterior pituitary cells. Inhibin, administered sc 6 h before the experiment, induced dose- and time-related decreases in plasma FSH, but not LH, levels in both intact and castrated male rats. Inhibin also significantly interfered with the in vivo stimulatory effect of 20-500 ng GnRH on FSH release, but had inconsistent and usually modest effects on the LH response. While exposure of cultured pituitary cells to inhibin for 72 h has been reported to interfere with GnRH-induced gonadotropin release, we examined here the effects of shorter exposure periods relevant to in vivo experiments. Exposure of the cells to inhibin (31.3-312.5 pM) for 2-6 h measurably (P less than or equal to 0.01) decreased the ability of 10 nM GnRH to stimulate both FSH and LH released by cultured cells. In contrast, lower (3.1 and 9.4 pM) doses of inhibin had little or no effect. Longer exposures to inhibin (10, 24, and 72 h) increased the inhibitory effect of 31.3-312.5 pM inhibin, while 3.1 and 9.4 pM remained ineffective at all times. These results indicate that exposure of the male rat to inhibin for 6 h decreases FSH secretion, and that this effect is at least partially mediated through blunting of the pituitary response to GnRH. In contrast, the ability of inhibin to interfere with LH release, which is readily apparent in cultured pituitary cells, appears to be of lesser importance in the intact male rat.     

Gonadotropin gene expression and secretion in gonadotropin-releasing hormone antagonist-treated male rats: effect of sex steroid replacement.

The possibility of direct pituitary effects of sex steroids on gonadotropin gene expression and synthesis was studied in male rats. The animals were treated with a potent GnRH antagonist, Ac-D-pClPhe-D-pClPhe-D-Trp-Ser-Tyr-D-Arg-Leu-Arg-Pro-D-Ala-+ ++NH2CH3COOH (Org 30276; 0.5 mg/kg BW, sc, twice daily) for 10 days. Groups of the antagonist-treated rats were implanted at the beginning of the injections with Silastic capsules containing testosterone (T), 5 alpha-dihydrotestosterone (DHT), or diethylstilbestrol (DES). Groups treated with the antagonist alone or vehicle served as controls. The antagonist treatment decreased unoccupied pituitary receptors of GnRH by 93% (P less than 0.001), serum LH by 34% (P less than 0.01), and serum FSH by 30% (P less than 0.05), and serum T became undetectable (less than 0.10 nmol/liter). Compared to antagonist treatment alone, no further effects on serum or pituitary LH levels found after steroid replacements. In contrast, the antagonist-induced decreases in serum and pituitary FSH (30% and 70%, respectively; P less than 0.05-0.01) were totally reversed by the T and DHT implants, but not by DES. Pituitary levels of the LH beta-subunit mRNA were decreased by 60% (P less than 0.01) after antagonist treatment. Combination treatment with androgens had no further effect on this mRNA, whereas DES partially reversed this suppression (P less than 0.05). In contrast, the pituitary mRNA level of the FSH beta-subunit, which decreased with antagonist treatment by 90% (P less than 0.01), returned to the control level with T and DHT replacements, but only partially with DES. The pituitary mRNA level of the common alpha-subunit was significantly suppressed only by combined antagonist plus DHT treatment (P less than 0.01). However, combination of DES with the antagonist increased alpha-subunit mRNA levels 2.4-fold (P less than 0.05) compared to antagonist treatment alone. It is concluded that the suppression of gonadotropin secretion by GnRH antagonist treatment is accompanied in male rats by a parallel reduction in mRNA levels of the gonadotropin beta-subunits. Sex steroid replacement of the antagonist-treated animals selectively reverses some of the mRNA changes. Androgens (T and DHT) increase the mRNA of FSH beta-subunit, but have no effect on the LH beta-subunit. Estrogen increases the mRNA levels of common alpha- and LH beta-subunits and slightly increases that of FSH beta.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of gonadal steroids on gonadotropin secretion in males: studies with perifused rat pituitary cells

The feedback effects of testosterone (T) and estradiol (E2) on FSH and LH secretion were compared in dispersed pituitary cells from adult male rats perifused with pulses of GnRH. Cells were stimulated with 10 nM GnRH for 2 min every 1 h. T (10 nM) pretreatment for 24 h reduced the amplitude of FSH and LH pulses to 77 +/- 4% (mean +/- SE) and 47 +/- 3% of control values, respectively (P less than 0.01), whereas 6-h T treatment was without effect. By contrast, interpulse secretion of FSH was increased after 24 h T to 184 +/- 7% of the control value (P less than 0.01), but interpulse LH release was unchanged (104 +/- 5%). E2 (0.075 nM) treatment of pituitary cells reduced GnRH-stimulated FSH and LH release within 2 h to 75 +/- 2% and 73 +/- 3% of control values, respectively (P less than 0.01). E2 pretreatment for 24 h stimulated (P less than 0.025) GnRH-induced FSH (136 +/- 10%) and LH (145 +/- 8%) release and also increased (P less than 0.01) interpulse FSH (127 +/- 5%) and LH (145 +/- 8%) secretion. These data indicate that the suppression of FSH and LH secretion by T in males is due in part to a direct effect on the pituitary. The findings that T suppresses GnRH-stimulated FSH less than LH, and that T stimulates interpulse FSH, but not LH, provide evidence for differential regulation of FSH and LH secretion by T. The dissimilar actions of T on GnRH-stimulated pulses and interpulse gonadotropin secretion suggest that interpulse secretion is unrelated to stimulation by GnRH, although its physiological significance is unknown. Since E2, in physiological levels for males, increased pituitary FSH and LH secretion, the suppression of gonadotropin secretion by E2 in vivo in males may result from an effect on the hypothalamic pulse generator; however, additional studies are needed before extending these conclusions to higher mammals and men.     

Inhibition of gonadotropin-releasing hormone release as the basis of pituitary-gonadal dysfunction during treatment with 4-aminopyrazolo-(3,4-d)-pyrimidine

The inhibitor of hepatic lipoprotein release, 4-aminopyrazolo-(3,4-d)-pyrimidine (4-APP), has been shown to reduce testosterone production via impairment of pituitary gonadotropin secretion rather than through decreased cholesterol availability. It was previously shown that serum LH levels were reduced by more than 75% in male rats treated with 4-APP, but pituitary stores of LH and the gonadotropin response to exogenous GnRH were maintained. Also, there was a reduction in pituitary GnRH receptors which was consistent with hypothalamic GnRH deficiency. The present studies were undertaken to examine the mechanism by which 4-APP inhibits GnRH synthesis and/or release. Intact, adult male or 2-week ovariectomized female rats were treated daily with 25 mg/kg 4-APP for 3 days. Both sexes showed lowered basal serum levels of LH and absence of the elevations in serum LH normally elicited by the opiate antagonist, naloxone. In pituitary portal plasma collected from normal male rats, GnRH was significantly elevated by naloxone treatment, confirming that naloxone acted at the level of hypothalamic GnRH release. However, naloxone stimulation of GnRH secretion into portal blood was absent in rats treated with 4-APP. In vitro, the potassium-induced release of GnRH from perifused medial hypothalami was reduced by 60% in 4-APP-treated male rats while hypothalamic GnRH content remained unchanged. These data indicate that 4-APP has an inhibitory effect on the mechanism of GnRH release, and that analysis of its actions should clarify the processes involved in neurohormone secretion.     

Studies on the feedback regulation of gonadotropin concentrations in male rats. (III). The effects of testosterone and its metabolites on gonadotropin secretion from rat pituitary cells in culture

To determine whether dihydrotestosterone (DHT) or estradiol (E2) exerts negative or positive feedback effects on rat pituitary gland, Testosterone (T) metabolite (T, DHT, 5 alpha-androstane-3 alpha, 17 beta-diol:3 alpha-diol or E2) was added to the cultured pituitary cells. Anterior pituitary glands were obtained from 6-week-old male rats. Pituitary cells were prepared by trypsin digestion and incubated with various concentrations of steroid hormones for 72 h to determine the effects of steroid hormones on basal secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH) after 48 h preculture without steroids. Then 10 nM luteinizing hormone-releasing hormone (LH-RH) with appropriate concentrations of these steroid hormones was added to the pituitary cells in culture and incubated for another 6h to determine the effects of steroid hormones on LH-RH induced gonadotropin release. After the incubation, pituitary cells were lysed with 0.1% Triton X100 to measure the intracellular gonadotropin content. The concentration of LH and FSH was determined by radioimmunoassay. T, DHT and 3 alpha-diol stimulated basal FSH but not basal LH secretion, and inhibited both the release of FSH and LH from cultured pituitary cells during incubations with LH-RH in a dose-dependent fashion. Intracellular content of both FSH and LH were increased, and total FSH and not LH was also increased by the addition of DHT in a dose-dependent manner. E2 did not exert any of such effects on pituitary cells in culture. These studies suggest that 5 alpha-reduced metabolites but not aromatized metabolite of T play an important role on feedback regulation of gonadotropin secretion at pituitary level. DHT directly acts on pituitary gland not only to stimulate the production of FSH but also to suppress FSH and LH secretion induced by LH-RH.     

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

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

Pulsatile GnRH-stimulated LH release from the human fetal pituitary in vitro: sex-associated differences

An in-vitro perfusion system was utilized to examine LH release from human fetal (19-24 weeks gestation) anterior pituitaries during repetitive GnRH stimulations. Pituitaries (five male, four female) were dissected into halves, and one hemipituitary of each pair was stimulated with 10-min pulses of 1 nM GnRH administered at 60-min intervals over 24h, whereas the matching hemipituitary received pulses of medium alone. Basal (no GnRH stimulation) LH release from female hemipituitaries was significantly (P less than 0.01) greater than from male hemipituitaries, and the amplitude of LH release associated with GnRH pulses was sixfold greater (P less than 0.001) with female hemipituitaries. Furthermore, the magnitude of LH release associated with individual GnRH pulses was significantly (P less than 0.001) enhanced during the course of female hemipituitary perfusions, but not during perifusion of male hemipituitaries. These studies demonstrate that LH secretion by the female, but not male, mid-gestational human fetal pituitary is increased in response to a physiological pattern and interval of repeated pulsatile GnRH stimulation in vitro.     

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.     

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.     

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

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

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)     

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.     

Disruption of GnRH pulses by anti-GnRH serum and phentolamine obliterates pulsatile LH but not FSH secretion in ovariectomized rabbits

It has been hypothesized that the secretion of gonadotropins, i.e. luteinizing hormone (LH) and follicle-stimulating hormone (FSH), is driven by a synchronized neural network ('pulse generator'). This network, regulated in part by alpha-adrenergic activity, ultimately generates bursts of hypothalamic gonadotropin-releasing hormone (GnRH) release. In this study, we used the push-pull (PP) perfusion technique in ovariectomized rabbits to investigate three aspects of the ('GnRH/gonadotropin pulse generator') hypothesis. The objectives were to determine: (1) if plasma LH and FSH pulses occur concomitantly with mediobasal hypothalamic (MBH-) GnRH pulses, (2) changes in the patterns of pulsatile LH and FSH secretion when pulsatile MBH GnRH signals are interrupted by either local immunoneutralization of GnRH or intravenous infusion of the alpha-adrenergic antagonist phentolamine (PHEN, 4 mg/kg BW), and (3) whether third cerebroventricular (3VT-) GnRH patterns reflect neuronal GnRH release from the MBH. We found that while both plasma LH and FSH patterns were pulsatile, MBH GnRH pulses were significantly coupled only with LH pulses (94% coincidence). Both the local immunoneutralization of MBH GnRH pulses and the PHEN-induced suppression of MBH GnRH pulses obliterated the pulsatile secretion of LH, but not FSH. Neither MBH GnRH nor plasma LH or plasma FSH pulses were concurrent with 3VT GnRH pulses. However, the PP perfusion of the 3VT appeared to alter the pulsatile release of MBH GnRH and pituitary LH. The results support the hypothesis that in the absence of ovarian signals, the 'pulse generator' is maintained by tonic alpha-adrenergic input and that a 'cellular unity' of MBH GnRH release (GnRH pulses) drives the gonadotrophs to secrete LH in pulses. In contrast, the pulsatile release of FSH appears to involve additional nonovarian regulatory events to those controlling LH secretion.     

The synthesis and release of gonadotropins in response to gonadotropin-releasing hormone of the rat anterior pituitary gland during weight reduction

It is well recognized that weight reduction produces the suppression of serum LH but not FSH level in rodents. In order to clarify the mechanism by which the discrepancy between LH and FSH levels is brought about, the influence of weight loss on the pituitary function was explored using female rats. The changes of the pituitary response to GnRH and the basal secretion of gonadotropins with progressive weight loss were investigated by in vitro short-term incubation of the pituitary gland after prolonged weight loss in female Wistar rats. On the first day of diestrous and until day 14 of the diet, GnRH induced LH and FSH release from the pituitary and a decrease in pituitary content of them, but the total amount of gonadotropin in culture medium and pituitary tissue was not affected. On day 30 of the diet, the decrease in pituitary content disappeared. On day 60 LH release disappeared, whereas pituitary FSH and the total amount of gonadotropins were increased by GnRH. Non-stimulated FSH but not LH secretion per mg of pituitary was augmented during dieting. The data indicate that pituitary responsiveness to GnRH and non-stimulated FSH release were modified by weight loss: the LH-releasing action of GnRH was diminished, the gonadotropin-synthesizing action of GnRH was augmented, and non-stimulated FSH release was increased.     

Effect of an aproteic diet on gonadotropin release response to GnRH and estrogen-progesterone in rats

The fasting-induced gonadotropin function decrease is unspecific, because in this situation there is a lack of all nutrients. We report here the effect of specific protein lack in the diet during 21 days, on pituitary gonadotropin synthesis and response to exogenous GnRH in adult male rats. We also studied the effect of the aproteic diet (AP) on the positive feedback mechanism in adult female castrated rats. The AP diet decreased significantly, both LH and FSH pituitary concentration and also basal gonadotropin plasma levels in male rats. GnRH produced a significantly increment in LH secretion in both treated and control groups, reaching similar levels after stimulation. Nevertheless, the percentile increment from basal levels in the aproteic group was almost four times the controls, suggesting an increased sensitivity in pituitary response to GnRH in rats fed with AP diet. In female castrated rats, the aproteic diet imposed 3 weeks after the surgery was unable to reduce basal gonadotropin secretion, and so also prolactin secretion. Estradiol/progesterone (EP) administration produced the activation of positive feedback mechanism, increasing significantly LH and FSH secretion in both controls and AP groups. Nevertheless, both gonadotropin responses to EP were significantly greater in rats fed with AP diet. Basal prolactin levels and response to EP were not different between both groups. This results suggest that selective protein lack in a diet, reduced pituitary LH and FSH synthesis and secretion. This type of diet also increments pituitary sensitivity to GnRH administration in male rats, and gonadotropin response to positive feedback mechanism in female rats.