Interrelationship between estrogen and thyroxine on the release of luteinizing hormone and gonadotropin-releasing hormone in vitro

The present experiments were designed to study the interaction between estradiol benzoate (EB) and thyroxine (T4) given in vivo on the responsiveness of pituitary luteinizing hormone (LH) to gonadotropin-releasing hormone (GnRH) and the release of GnRH in vitro. Ovariectomized-thyroidectomized (Ovx-Tx) rats were injected s.c. with saline or T4 (2 micrograms/100 g b.wt), and oil or EB (0.1 microgram) once daily for 40 days following a 2 x 2 factorial design. All animals were then decapitated and blood samples were collected. Anterior pituitaries (APs) were incubated in vitro with and without 0.1 ng GnRH at 37 degrees C for 4 h. Mediobasal hypothalami (MBHs) were excised and then incubated with and without APs from Ovx donor rats. Concentrations of LH and GnRH in the medium and that of LH in the serum were measured by radioimmunoassay. The LH level in media containing MBHs and donor APs was used as the index of bioactive GnRH release. In Ovx-Tx rats, T4 injections reduced the serum LH concentration, the pituitary LH response to GnRH, and the bioactive as well as the immunoreactive GnRH release. The serum LH levels and the spontaneous as well as the GnRH-stimulated release of LH in vitro were suppressed in Ovx-Tx rats following administration of EB. By contrast, the serum LH concentration, as well as pituitary LH response to GnRH and GnRH release in vitro, were higher in the group treated with both T4 and EB than in that treated with saline and EB. These results suggest that the differential changes in the LH secretion after thyroidectomy of Ovx versus non-Ovx rats are due to an antagonistic effect between T4 and estrogen on the response of pituitary LH to GnRH, and the release of GnRH.     

Regulation of thyroid hormones on the production of testosterone in rats

The effects of a thyroidectomy and thyroxine (T4) replacement on the spontaneous and human chorionic gonadotropin (hCG)-stimulated secretion of testosterone and the production of adenosine 3',5'-cyclic monophosphate (cAMP) in rat testes were studied. Thyroidectomy decreased the basal levels of plasma luteinizing hormone (LH) and testosterone, which delayed the maximal response of testosterone to gonadotropin-releasing hormone (GnRH) and hCG in male rats. T4 replacement in thyroparathyroidectomized (Tx) rats restored the concentrations of plasma LH and testosterone to euthyroid levels. Thyroidectomy decreased the basal release of hypothalamic GnRH, pituitary LH, and testicular testosterone as well as the LH response to GnRH and testosterone response to hCG in vitro. T4 replacement in Tx rats restored the in vitro release of GnRH, GnRH-stimulated LH release as well as hCG-stimulated testosterone release. Administration of T4 in vitro restored the release of testosterone by rat testicular interstitial cells (TICs). The increase of testosterone release in response to forskolin and androstenedione was less in TICs from Tx rats than in that from sham Tx rats. Administration of nifedipine in vitro resulted in a decrease of testosterone release by TICs from sham Tx but not from Tx rats. The basal level of cAMP in TICs was decreased by thyroidectomy. The increased accumulation of cAMP in TICs following administration of forskolin was eliminated in Tx rats. T4 replacement in Tx restored the testosterone response to forskolin. But the testosterone response to androstenedione and the cAMP response to forskolin in TICs was not restored by T4 in Tx rats. These results suggest that the inhibitory effect of a thyroidectomy on the production of testosterone in rat TICs is in part due to: 1) the decreased basal secretion of pituitary LH and its response to GnRH; 2) the decreased response of TICs to gonadotropin; and 3) the diminished production of cAMP, influx of calcium, and activity of 17beta-HSD. T4 may enhance testosterone production by acting directly at the testicular interstitial cells of Tx rats.     

Differential actions of corticosterone on luteinizing hormone and follicle-stimulating hormone biosynthesis and release in cultured rat anterior pituitary cells: interactions with estradiol

Previous in vivo studies from our laboratory suggested that glucocorticoids antagonize estrogen-dependent actions on LH secretion. This study investigated whether corticosterone (B) may have similar actions on gonadotropin biosynthesis and secretion in vitro. Enzymatically dispersed anterior pituitary cells from adult female rats were cultured for 48 h in alpha-modified Eagle's medium containing 10% steroid-free horse serum with or without 0.5 nM estradiol (E2). The cells were then cultured for 24 h with or without B in the presence or absence of E2. To evaluate hormone release, 5 x 10(5) cells were incubated with varying doses of GnRH (0, 10(-11)-10(-7) M) or pulsatile GnRH (10(-9) M; 20 min/h) for 4 h. Cell and medium LH and FSH were measured by RIA. To evaluate LH biosynthesis, 5 x 10(6) cells were incubated for an additional 24 h with 10(-10) M GnRH, 60 microCi 3H-glucosamine (3H-Gln), 20 microCi 35S-methionine (35S-Met), and the appropriate steroid hormones. Radiolabeled precursor incorporation into LH subunits was determined by immunoprecipitation, followed by SDS-PAGE. Continuous exposure to GnRH stimulated LH release in a dose-dependent manner, and this response was enhanced by E2. B by itself had no effect on LH release, but inhibited LH secretion in E2-primed cells at low concentrations of GnRH (10(-10) M or less). Total LH content was not altered by GnRH or steroid treatment. Similar effects of B were observed in cells that were given a pulsatile GnRH stimulus. In contrast to LH, E2 or B enhanced GnRH-stimulated FSH release at the higher doses of GnRH, while the combination of E2 and B increased basal and further augmented GnRH-stimulated release. Total FSH content was also increased in the presence of B, but not E2 alone, and was further augmented in cells treated with both steroids. There were no effects of the steroids on the magnitude of FSH release in response to GnRH pulses, but the cumulative release of FSH was greater in the E2 + B group compared to controls, indicating an increased basal release. Independent of E2, B suppressed the incorporation of 3H-Gln into LH by more than 50% of control, with only subtle effects on the incorporation of 35S-Met.(ABSTRACT TRUNCATED AT 400 WORDS)     

Increase of thyrotropin response to thyrotropin-releasing hormone (TRH) and TRH release in rats during pregnancy

Regulation of thyrotropin (TSH) release by thyrotropin releasing hormone (TRH) in the anterior pituitary gland (AP) of pregnant rats was studied. The pregnant (day 7, 14, and 21) and diestrous rats were decapitated. AP was divided into 2 halves, and then incubated with Locke's solution at 37 degrees C for 30 min following a preincubation. After replacing with media, APs were incubated with Locke's solution containing 0, or 10 nM TRH for 30 min. Both basal and TRH-stimulated media were collected at the end of incubation. Medial basal hypothalamus (MBH) was incubated with Locke's medium at 37 degrees C for 30 min. Concentrations of TSH in medium and plasma samples as well as the cyclic 3':5' adenosine monophosphate (cAMP) content in APs and the levels of TRH in MBH medium were measured by radioimmunoassay. The levels of plasma TSH were higher in pregnant rats of day 21 than in diestrous rats. The spontaneous release of TSH in vitro was unaltered by pregnancy. TRH increased the release of TSH by AP, which was higher in pregnant than in diestrous rats. Maternal serum concentration of total T3 was decreased during the pregnancy. The basal release of hypothalamic TRH in vitro was greater in late pregnant rats than in diestrous rats. After TRH stimulation, the increase of the content of pituitary cAMP was greater in late pregnant rats than in diestrus animals. These results suggest that the greater secretion of TSH in pregnant rats is in part due to an increase of spontaneous release of TRH by MBH and a decrease of plasma thyroid hormones. Moreover, the higher level of plasma TSH in rats during late pregnancy is associated with the greater response of pituitary cAMP and TSH to TRH.     

Effects of hyperprolactinemia on the control of luteinizing hormone and follicle-stimulating hormone secretion in the male rat

Experiments were conducted to determine the effects of acute hyperprolactinemia (hyperPRL) on the control of luteinizing hormone and follicle-stimulating hormone secretion in male rats. Exposure to elevated levels of prolactin from the time of castration (1 mg ovine prolactin 2 X daily) greatly attenuated the post-castration rise in LH observed 3 days after castration. By 7 days after castration, LH concentrations in the prolactin-treated animals approached the levels observed in control animals. HyperPRL had no effect on the postcastration rise in FSH. Pituitary responsiveness to gonadotropin hormone-releasing hormone (GnRH), as assessed by LH responses to an i.v. bolus of 25 ng GnRH, was only minimally effected by hperPRL at 3 and 7 days postcastration. LH responses were similar at all time points after GnRH in control and prolactin-treated animals, except for the peak LH responses, which were significantly smaller in the prolactin-treated animals. The effects of hyperPRL were examined further by exposing hemipituitaries in vitro from male rats to 6-min pulses of GnRH (5 ng/ml) every 30 min for 4 h. HyperPRL had no effect on basal LH release in vitro, on GnRH-stimulated LH release, or on pituitary LH concentrations in hemipituitaries from animals that were intact, 3 days postcastration, or 7 days postcastration. However, net GnRH-stimulated release of FSH was significantly higher by pituitaries from hyperprolactinemic, castrated males. To assess indirectly the effects of hyperPRL on GnRH release, males were subjected to electrical stimulation of the arcuate nucleus/median eminence (ARC/ME) 3 days postcastration. The presence of elevated levels of prolactin not only suppressed basal LH secretion but reduced the LH responses to electrical stimulation by 50% when compared to the LH responses in control castrated males. These results suggest that acute hyperPRL suppresses LH secretion but not FSH secretion. Although pituitary responsiveness is somewhat attenuated in hyperprolactinemic males, as assessed in vivo, it is normal when pituitaries are exposed to adequate amounts of GnRH in vitro. Thus, the effects of hyperPRL on pituitary responsiveness appear to be minimal, especially if the pituitary is exposed to an adequate GnRH stimulus. The suppression of basal LH secretion in vivo most likely reflects inadequate endogenous GnRH secretion. The greatly reduced LH responses after electrical stimulation in hyperprolactinemic males exposed to prolactin suggest further that hyperPRL suppresses GnRH secretion.     

Stimulation of the secretion of luteinizing hormone by ginsenoside-Rb1 in male rats

Ginsenoside-Rb1 is one of the pharmacologically active components of ginseng, the dry root of Panax ginseng C. A. Meyer (Araliaceae), a well-known traditional Chinese medicine. Ginseng enhanced mounting behaviour of male rats and increased sperm counts in rabbit testes. Some experimental results suggested no sex hormone-like function in ginseng but probably gonadotropin-like action. The present study was to explore the effect of ginsenoside-Rb1 on the secretion of luteinizing hormone (LH) both in vivo and in vitro. Male rats were orchidectomized (Orch) for 2 weeks or subjected to swim training for 1 week before catheterization via the right jugular vein. They were intravenously injected with ginsenoside-Rb1 (10 microg/kg) or saline at 15 min prior to a challenge of gonadotropin-releasing hormone (GnRH) or 10 min-swim. Blood samples were collected at several time intervals following intravenous injection of ginsenoside-Rb1. In the in vitro experiment, male rats were decapitated and their anterior pituitary gland (APs) were either bisceted or enzymatically dispersed. The hemi-APs were preincubated with Locke's medium at 37 degrees C for 90 min and then incubated with Locke's medium containing ginsenoside-Rb1 (10(-7) - 10(-4) M) for 30 min. The dispersed AP cells (1 x 1(5) cells per well) were primed with dihydrotestosterone (DHT, 10(-8) M) for 3 days, and then challenged with ginsenoside-Rbl (10(-6) and 10(-5) M, n = 8) for 3 h. The concentrations of LH or testosterone in samples were measured by radioimmunoassays. Administration of ginsenoside-Rb1 did not alter the levels of plasma LH in both intact and Orch rats but significantly increased plasma LH concentration at the termination of the 10 min swimming exercise. Administration of ginsenoside-Rb1 resulted in a lower testosterone response to GnRH challenge or swimming exercise as compared with saline-treated rats. Ginsenoside-Rb1 dose-dependently increased the release of LH from both hemi-AP tissues and the DHT-primed dispersed AP cells in vitro. These results suggest that ginsenoside-Rb1 increases LH secretion by acting directly on rat AP cells.     

Effectiveness of estradiol and progesterone in inducing LH release in different stages in rat estrous cycles

The effectiveness of estradiol (E2) and progesterone (P4) in inducing the release of the luteinizing hormone (LH) in the acutely ovariectomized (OVx) rats was studied. Female rats were Ovx in different stages of the estrous cycles and received a series of injections of E2 and P4. LH dynamic changes in the blood were examined in the afternoon of the following day after Ovx. Intact rats treated with oil vehicle or E2 and P4 were used as controls. The surgical operation and oil treatment did not interfere with the normal reproductive rhythm and LH secretion, but treatment with E2 and P4 did facilitate the LH release in some intact rats. E2 and P4 were very effective in inducing LH release in Ovx rats as compared with controls. Results indicated that E2 and P4 are essential substances in eliciting the LH surge, but their efficacies are dependent on the stage in the estrous cycles.     

Pituitary gonadotropin-releasing hormone receptor content in rats with polycystic ovaries

A single injection of estradiol valerate (EV) induces, after a lag period of 4-6 wk, a chronic anovulatory polycystic ovarian (PCO) condition in adult rats. This condition is associated with a selective compromise of luteinizing hormone (LH) release and/or synthesis reflected in low basal serum LH concentrations, decreased pituitary content of LH, and decreased gonadotropin-releasing hormone (GnRH)-stimulated LH secretion. The present study was undertaken to determine to what extent the aberrant LH release in rats with PCO could be related to alterations in pituitary content of GnRH receptors. Pituitary GnRH-receptor content was assessed by the evaluation of saturation binding of a GnRH analog, [125I]-D-Ala6-des-Gly10-GnRH, to pituitary membrane preparations. The receptor content of pituitaries from rats with PCO was compared to that obtained from intact animals at estrus and diestrus. Receptor levels in ovariectomized normal rats and rats with PCO were also assessed. The pituitary GnRH receptor content in PCO rats was similar to that observed in normal controls at estrus and was significantly lower than that for rats at diestrus. Although a twofold increase in pituitary GnRH receptor content was observed at 28 days following the castration of control rats, GnRH receptor content in the pituitaries of PCO rats, at 28 days following ovariectomy, remained unchanged. Although, castration-induced elevations in mean serum LH and follicle-stimulating hormone (FSH) concentrations were observed in both the PCO and control animals, the rise in both gonadotropins was significantly attenuated in the PCO-castrates when compared to the ovariectomized controls. Since GnRH is a major factor in the regulation of pituitary GnRH receptor content, these findings suggest that hypothalamic GnRH release is impaired in rats with PCO and that this impairment is independent of any influences from the polycystic ovaries.     

Direct kisspeptin-10 stimulation on luteinizing hormone secretion from bovine and porcine anterior pituitary cells

Kisspeptins are peptide hormones encoded by the KiSS-1 gene and act as the principal positive regulator of the reproductive axis by directly stimulating gonadotropin-releasing hormone (GnRH) neuron activity. However, peripheral administration, as well as central administration, of kisspeptin stimulates luteinizing hormone (LH) secretion in some mammalian species. In order to evaluate the direct effects of kisspeptin-10 (the minimal kisspeptin sequence necessary for receptor activation) on LH secretion from bovine and porcine anterior pituitary (AP) cells, LH-releasing effects of kisspeptin-10 on AP cells were compared with GnRH in vitro. The AP cells were prepared from 1-month-old intact male calves, 8-month-old castrated male calves, or 6-month-old barrows, and then the cells were incubated for 2h with the peptides. The 1000 nM and 10,000 nM, but not lower concentrations, of kisspeptin-10 significantly stimulated LH secretion from the bovine AP cells (P<0.05). The 100 nM and 1000 nM, but not lower concentrations, of kisspeptin-10 significantly stimulated LH secretion from porcine AP cells (P<0.05). As 10nM of GnRH strongly stimulated LH secretion from all AP cells tested in this study, the present results suggest that kisspeptin-10 has a direct, but weak, stimulating effect on LH secretion in bovine and porcine AP cells. The present study is the first to examine the direct actions of kisspeptin on the bovine and porcine pituitary gland as far as we know. Kisspeptin might have other actions on the pituitary because the pituitary has multiple roles.     

Estradiol-17beta triggers luteinizing hormone release in the protandrous black porgy (Acanthopagrus schlegeli Bleeker) through multiple interactions with gonadotropin-releasing hormone control.

We investigated the mechanism of estradiol-17beta (E2) action on stimulation of LH (=gonadotropin II) release in the black porgy fish (Acanthopagrus schlegeli Bleeker) using an in vivo approach and primary cultures of dispersed pituitary cells in vitro. In vivo, E2 but not androgens (testosterone [T] and 11-ketotestosterone [11-KT]) significantly stimulated plasma LH in a dose-dependent manner. Estradiol-17beta also increased brain content of seabream GnRH. GnRH antagonist prevented E2 stimulation of LH release in vivo, indicating that the effect of E2 on LH was mediated by GnRH. In vitro, sex steroids (E2, T, 11-KT) alone had no effect on basal LH release in the cultured pituitary cells, but GnRH significantly stimulated LH release. Estradiol-17beta potentiated GnRH stimulation of LH release, an effect that was inhibited by GnRH antagonist, and 11-KT, but not T, also potentiated GnRH stimulation of LH release. The potentiating effect of 11-KT on GnRH-induced LH release in vitro was stronger than that of E2. These data suggest that E2 triggers LH release in vivo by acting both on GnRH production at the hypothalamus and on GnRH action at the pituitary. In contrast, 11-KT may only stimulate GnRH action at the pituitary. The E2) induction of LH release, through multiple interactions with GnRH control, supports a possible central role of E2in the sex change observed in the protandrous black porgy.     

Testosterone selectively increases serum follicle-stimulating hormonal (FSH) but not luteinizing hormone (LH) in gonadotropin-releasing hormone antagonist-treated male rats: evidence for differential regulation of LH and FSH secretion

Both testosterone (T) and gonadotropin-releasing hormone (GnRH)-antagonist (GnRH-A) when given alone lower serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in intact and castrated rats. However, when graded doses of testosterone enanthate (T.E.) were given to GnRH-A-treated intact male rats, a paradoxical dose-dependent increase in serum FSH occurred; whereas serum LH remained suppressed. This surprising finding led us to ask whether the paradoxical increase in serum FSH in GnRH-A-suppressed animals was a direct stimulatory effect of T on the hypothalamic-pituitary axis or the result of a T effect on a testicular regulator of FSH. To test these hypotheses, we treated adult male castrated rats with GnRH-A and graded doses of T.E. In both intact and castrated rats, serum LH remained undetectable in GnRH-A-treated rats with or without T.E. However, addition of T.E. to GnRH-A led to a dose-dependent increase in serum FSH in castrated animals as well, thus pointing against mediation by a selective testicular regulator of FSH. These data provide evidence that pituitary LH and FSH responses may be differentially regulated under certain conditions. When the action of GnRH is blocked (such as in GnRH-A-treated animals), T directly and selectively increases pituitary FSH secretion.     

The frequency of gonadotropin-releasing hormone secretion regulates expression of alpha and luteinizing hormone beta-subunit messenger ribonucleic acids in male rats

The influence of GnRH pulse frequency on LH subunit mRNA concentrations was examined in castrate, testosterone-replaced male rats. GnRH pulses (25 ng/pulse) or saline to controls, were given via a carotid cannula at intervals of 7.5-240 min for 48 h. alpha and LH beta mRNA concentrations were 109 +/- 23 and 30 +/- 5 pg cDNA bound/100 micrograms pituitary DNA, respectively, in saline controls. GnRH pulse intervals of 15, 30, and 60 min resulted in elevated alpha and LH beta mRNAs (P less than 0.01) and maximum responses (4-fold, alpha; 3-fold, LH beta) were seen after the 30-min pulses. Acute LH release to the last GnRH pulse was seen after the 15-, 30-, and 60-min pulse intervals. In contrast, LH subunit mRNAs were not increased and acute LH release was markedly impaired after the rapid (7.5 min) or slower (120 and 240 min) pulse intervals. Equalization of total GnRH dose/48 h using the 7.5- and 240-min intervals did not increase LH subunit mRNAs to levels produced by the optimal 30-min interval. These data indicate that the frequency of the pulsatile GnRH stimulus regulates expression of alpha and LH beta mRNAs in male rats. Further, GnRH pulse frequencies that increase subunit mRNA concentrations are associated with continuing LH responsiveness to GnRH.     

Starvation-induced suppression of pituitary-testicular function in rats is reversed by pulsatile gonadotropin-releasing hormone substitution

This study was carried out to test the hypothesis that reduced hypothalamic GnRH release is responsible for the suppression of reproductive functions during starvation. Adult male rats were kept for 4 days under total fasting (only water allowed) and injected during this time at 2-h intervals with 100 or 500 ng/kg BW of GnRH or vehicle. Serum levels of LH and FSH decreased by 30% during starvation (p less than 0.05), and these effects were fully reversed by either dose of GnRH treatment. Starvation reduced the pituitary mRNA contents of the gonadotropin common alpha- and FSH beta-subunits by 30% and 35% in starved animals (p less than 0.05 for both), but the LH beta-subunit mRNA was unaffected. The GnRH treatments partly or totally reversed these changes, but up-regulation of the mRNA levels by GnRH was seen only in controls fed ad libitum. Starvation reduced the testicular and serum levels of testosterone by 84% (p less than 0.01) and 42% (p less than 0.05), respectively. These changes were fully reversed by the 500-ng/kg dose of GnRH treatment during fasting, but only serum T was completely reversed by the 100-ng/kg GnRH treatment. To elucidate whether fasting per se had direct effects at the gonadal level, we blocked the secretion of gonadotropins by treatment with a GnRH antagonist, and replaced the gonadotropins by injecting of hCG (10 IU/kg BW once daily) and hFSH (75 IU/kg BW once daily). No differences were observed between starved and control animals in either testicular or serum levels of T, or in accessory sex gland weights.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of acute immunoneutralization of endogenous leptin on prolactin and LH secretion during the afternoon of pro-oestrus or in steroid-treated ovariectomized female rats

Recent data indicate that leptin is involved in the control of reproductive function. Experiments were carried out to analyse the role of endogenous leptin in the regulation of LH and prolactin secretion during the afternoon of pro-oestrus and that induced by ovarian steroids in ovariectomized rats. In the first experiment, cyclic female rats were implanted with intra-auricular and intracerebroventricular (i.c.v.) cannulae and, at pro-oestrus, were injected (i.c.v.) with 10 microliters normal rabbit serum or leptin antiserum (at 13:00 and 14:00 h). Blood samples were obtained at 10:00 h and at intervals of 1 h between 13:00 and 20:00 h. In the second experiment, female rats in pro-oestrus were injected with normal rabbit serum or leptin antiserum at 16:00 and 18:00 h and blood samples were taken every 10 min between 18:00 and 20:00 h. In the third experiment, adult female rats that had been ovariectomized 2 weeks before were implanted with intra-auricular and i.c.v. cannulae and treated with oestradiol benzoate (30 micrograms s.c.) at 10:00 h and progesterone (2 mg s.c.) 48 h later. Normal rabbit serum (10 microliters) or leptin antiserum (10 microliters) were injected (i.c.v.) at 13:00 and 14:00 h, and blood samples were obtained at 10:00 h and at intervals of 1 h between 13:00 and 20:00 h. In the fourth experiment, hemipituitaries from ovariectomized steroid-treated female rats were incubated in the presence of leptin116-130 (an active fragment of the native molecule), GnRH or leptin + GnRH. Prolactin and LH secretion during the afternoon of pro-oestrus in females treated with leptin antiserum was similar to that observed in animals injected with normal rabbit serum. In ovariectomized female rats, the steroid-induced LH surge increased slightly after administration of leptin antiserum, whereas the prolactin surge remained unchanged. In vitro, leptin116-130 (10(-5) to 10(-8) mol l-1) inhibited LH secretion and modulated the effect of GnRH on LH release, depending on the concentration of GnRH: leptin116-130 (10(-6) mol l-1) reduced the effectiveness of 10(-7) mol GnRH l-1 and increased that of 10(-9) mol GnRH l-1. In conclusion, these experiments indicate that acute immunoneutralization of endogenous leptin does not interfere with spontaneous or steroid-induced LH and prolactin surges. In addition, the finding that leptin116-130 inhibited LH release and modulated the effectiveness of GnRH in vitro provides evidence of the direct modulatory role of leptin on LH secretion acting at the pituitary.     

Effect of cortisol or adrenocorticotropic hormone on luteinizing hormone secretion by pig pituitary cells in vitro

The effect of cortisol or adrenocorticotropic hormone (ACTH) on basal and gonadotropin-releasing hormone (GnRH)-induced secretion of luteinizing hormone (LH) was studied in vitro using dispersed pig pituitary cells. Pig pituitary cells were dispersed with collagenase and DNAase and then grown in McCoy's 5a medium containing 10% dextran charcoal-pretreated horse serum and 2.5% fetal calf serum for 3 days. Cells were preincubated with cortisol or ACTH before GnRH was added. When pituitary cells were incubated with 400 micrograms cortisol/ml medium for 6 h or longer, increase basal secretion of LH was observed. However, GnRH-induced LH release was reduced by cortisol. The degree of this reduction was dependent on cortisol, and a concentration of cortisol higher than 100 micrograms/ml was needed. Cortisol also inhibited the 17 beta-estradiol-induced increase in GnRH response. ACTH-(1-24), ACTH-(1-39), or porcine ACTH had no influence on GnRH-induced LH secretion. Our results show that cortisol can act directly on pig pituitary to inhibit both normal and estradiol-sensitized LH responsiveness to GnRH.     

Absence of effect of the peptide PHI-27 on LH release in vitro

The effects of PHI-27, a peptide of the glucagon-secretion family, on luteinizing hormone (LH) release and on LH-releasing hormone (LH-RH)- or estradiol-induced LH release were examined in a sequential double chamber perifusion system by perifusing the pituitary alone or in sequence with the mediobasal hypothalamus (MBH) from normal female rats in diestrus. PHI at 10(-7) M had no significant effect on LH release from the pituitary in series with the MBH. Moreover, on perifusion of the pituitary alone with medium containing 10(-7) M PHI, LH release induced by 20 ng/ml LH-RH from the pituitary was not significantly different from that without PHI. Furthermore, PHI had no effect on estradiol-induced LH release from the pituitary in sequence with the MBH. These data indicate that PHI has no effect on LH release in vitro.     

Effects of a potent antagonist to gonadotropin-releasing hormone on male rats: luteinizing hormone is suppressed more than follicle-stimulating hormone

Previous work with female rats showed that serum levels of follicle-stimulating hormone (FSH) are suppressed by gonadotropin-releasing hormone (GnRH) antagonists less than are levels of serum luteinizing hormone (LH), suggesting a lesser dependency of FSH on GnRH stimulation. The differential regulation of LH and FSH is known to have some aspects that are sexually asymmetrical, and it was of interest to see if males also show differential gonadotropin suppressibility after injection of an antagonist to GnRH. Male rats were prepared for serial sampling 4 wk after castration. After a blood sample was removed at Time Zero, [Ac-3-Pro1, pF-D-Phe2, -D-Trp3,6]-GnRH (Antag) was injected subcutaneously in oil; doses were 0, 4, 20, 100, 500, and 2500 micrograms. Blood was sampled at 2, 5, 12, 24 and 36 h postinjection. All doses above 4 micrograms had lowered LH levels by 2 h, and LH remained suppressed for 12 to 24 h at the three higher doses. By contrast, serum FSH was unaffected by any dose at 5 h, and was only marginally suppressed by the highest doses thereafter. As in females, therefore, FSH secretion in male rats appears not to be as dependent on GnRH as is LH secretion.     

Effect of cycloheximide and tunicamycin on the gonadotrophin-releasing hormone stimulated distal glycosylation of luteinizing hormone by rat pituitary cells

We have previously demonstrated that gonadotrophin-releasing hormone (GnRH) induces not only changes in quantity but also in quality on secreted luteinizing hormone (LH), by increasing [14C]Leu (translation) and [3H]Gal (distal glycosylation) incorporation into newly synthesized hormone. In the present report, we have further examined the GnRH-induced [3H]Gal-LH synthesis and release by treating anterior pituitary cells with polypeptide synthesis and glycosylation inhibitors (cycloheximide and tunicamycin, respectively). Pituitary cells from ovariectomized adult rats were cultured for 4 days and then incubated for different periods (0-5 h) in medium containing [14C]Leu plus [3H]Man or [14C]Leu plus [3H]Gal in the absence (basal) or presence of 10 nmol/L GnRH with or without (control) cycloheximide (1.0 and 4.0 microg/mL) or tunicamycin (0.5 and 2.0 microg/mL). At the end of each incubation period, the cells and the medium were separated and processed for DNA uptake and newly synthesized LH (labeled LH, by immunoprecipitation with a-betaLH) determinations. The velocity of synthesis and release (between 0 and 2 h, and between 2 and 5 h) was calculated by regression analysis and the statistical significance of differences was determined by the slope test. GnRH enhanced the rates of synthesis and release of [14C]Leu-, [3H]Man-, and [3H]Gal-LH to 157 and 237; 164 and 190; and 272 and 508% of basal values, respectively. Cycloheximide totally blocked synthesis and release of [14C]Leu-LH and greatly reduced those of [3H]Man-LH, resulting in the loss of cellular responsiveness to GnRH. Addition of tunicamycin to the pituitary cells inhibited the rates of synthesis and release of [3H]Man-LH which had been induced by GnRH, without altering those of [14C]Leu-LH. These findings indicate that glycosylation is not a condition for GnRH-stimulated LH translation. The GnRH-increased [3H]Gal-LH rates of synthesis and release were affected to a lesser extent by the inhibitors. Thus, GnRH stimulation of distal glycosylation can occur, albeit at a reduced rate, even though protein synthesis and glycosylation are blocked. In conclusion, the present results corroborate that GnRH stimulates the addition of galactose residues into LH molecule. This effect is not simply the consequence of stimulating LH polypeptide chain synthesis. In addition, it is shown that GnRH-increased LH translation is independent of glycosylation.     

Capability of ovarian steroids in inducing LH surges in acutely ovariectomized rats.

The capability of estradiol (E2) or E2 and progesterone (P4) in inducing luteinizing hormone (LH) surge in acutely ovariectomized (Ovx) rats was studied. In group I, rats were Ovx on estrus and were implanted with E2 capsules and atrial cannulae immediately after operation for blood samplings. In group II, rats were also Ovx on estrus but were implanted with E2 capsules and sampling cannulae the next day (the expected diestrus day 1, D1). In group III, rats were Ovx on D1, and were implanted E2 with capsules and atrial cannulae immediately after operation. All surgical operations were done around 1000h in the morning. On the expected diestrus day 2(D2) at 0930h, one half of the rats in each group received an oil vehicle or 2mg of P4 subcutaneously. Blood samples were taken from the indwelled cannulae at 1300, 1500, and 1700hrs in the afternoon. Results showed that P4 treatment amplified LH release in all three groups of rats primed with E2, and that the oil vehicle did not assist in LH release in E2 primed rats of group I and group II, but it did in 8 out of 10 rats in group III in the late afternoon of D2. Results suggested that the estradiol alone was capable in inducing LH surge on the expected D2 afternoon, and that under estradiol-primed conditions, P4 can trigger neural initiators to advance LH surge, but that the internal hormonal milieu at the time of ovariectomy may affect the influence of ovarian steroids in inducing LH release.     

Effects of acute ovariectomy on anterior pituitary gland follicle-stimulating hormone and luteinizing hormone secretion in the metestrous rat

Changes at the anterior pituitary gland level which result in follicle-stimulating hormone (FSH) release after ovariectomy in metestrous rats were investigated. Experimental rats were ovariectomized at 0900 h of metestrus and decapitated at 1000, 1100, 1300, 1500, 1700 or 1900 h of metestrus. Controls consisted of untreated rats killed at 0900 or 1700 h and rats sham ovariectomized at 0900 h and killed at 1700 h. Trunk blood was collected and the serum assayed for FSH and luteinizing hormone (LH) concentrations. The anterior pituitary gland was bisected. One-half was used to assay for FSH concentration. The other half was placed in culture medium for a 30-min preincubation and then placed in fresh medium for a 2-h incubation (basal FSH and LH release rates). The basal FSH release rate and the serum FSH concentration rose significantly by 4 h postovariectomy and remained high for an additional 6 h. The basal FSH release rate and the serum FSH concentration correlated positively (r=0.71 with 72 degrees of freedom) and did not change between 0900 and 1700 h in untreated or sham-ovariectomized rats. In contrast, the serum LH concentration and the basal LH release rate did not increase after ovariectomy. Ovariectomy had no significant effect on anterior pituitary gland FSH concentration. The results suggest that the postovariectomy rise in serum FSH concentration is the result, at least in part, of changes which cause an increase in the basal FSH secretion rate (secretion independent of the immediate presence of any hormones of nonanterior pituitary gland origin). The similarities between the selective rises in the basal FSH release rate and the serum FSH concentration in the ovariectomized metestrous rat and in the cyclic rat during late proestrus and estrus raise the possibility that an increase in the basal FSH release rate may be involved in many or all situations in which serum FSH concentration rises independently of LH.