Progesterone modulation of gonadotropin secretion by dispersed rat pituitary cells in culture. I. Basal and gonadotropin-releasing hormone-stimulated luteinizing hormone release

Dispersed, estradiol-treated, rat pituitary cells were cultured to characterize the influences of a physiologic concentration of progesterone (P, 10(-7) M) on gonadotroph responsiveness to gonadotropin-releasing hormone (GnRH). Acute (less than 6 h) P treatment enhanced and chronic (greater than 12 h) treatment suppressed both basal and GnRH-stimulated luteinizing hormone (LH) release. This modulation took place without any change in intracellular LH stores, indicating that the secretory changes are not attributable to changes in LH synthesis, and were not accompanied by similar alterations in basal or thyrotropin-releasing hormone-stimulated prolactin secretion. Moreover, the timing of these responses was fixed since a 10-fold lower P concentration produced only smaller and briefer alterations in LH release. Analyses of the temporal characteristics of effective P stimuli indicated that a brief 6 h exposure to P inhibited GnRH-stimulated LH secretion 18 h later. In contrast, P's acute actions rapidly dissipated following removal of the steroid from the culture medium. Finally, P-induced enhancement and suppression of GnRH-stimulated LH release could be blocked by appropriately timed treatments with protein synthesis inhibitors. Our findings are consistent with the hypothesis that P influences gonadotroph secretory function via the production of specific proteins.     

DOPAMINERGIC CONTROL OF GONADOTROPHIN SECRETION IN NORMAL WOMEN AND IN PATIENTS WITH PATHOLOGICAL HYPERPROLACTINAEMIA

The role of dopaminergic mechanisms in the control of gonadotrophin secretion in normal and hyperprolactinaemic subjects is controversial. Whilst bromocriptine, a potent dopamine agonist, has been used to restore normal gonadotrophin secretion in subjects with pathological hyperprolactinaemia (PHP), dopamine and dopamine agonists have been reported to suppress basal and stimulated gonadotrophin release. We therefore investigated the importance of dopaminergic control of gonadotrophin secretion by studying LH, FSH and PRL responses in normal and PHP subjects to central dopamine synthesis inhibition using monoiodotyrosine (MIT) and to a 4 h dopamine infusion designed to elevate peripheral plasma dopamine concentration to levels reported for pituitary portal plasma (1-6 ng/ml). MIT administration resulted in a significant release of PRL (peak increment 520 +/- 84% above basal) but not of LH or FSH in normal subjects. In PHP subjects there was a blunted PRL response (peak 13.3 +/- 3.5%) to MIT administration and significant LH (P less than 0.05) but not FSH release. Dopamine infusion (0.5 microgram/kg/min) resulted in suppression of PRL (min 19 +/- 3% of basal) but not of LH or FSH. A rebound of PRL (peak 188 +/- 68% of basal) but not LH or FSH occurred on cessation of dopamine. There was an apparent rise in LH (P less than 0.02 vs. normals) but not FSH in PHP patients during dopamine infusion. Plateau dopamine levels achieved during the infusion were 2.9 +/- 0.3 ng/ml and 5.9 +/- 0.8 ng/ml in normal and PHP subjects respectively. The responses to MIT show that dopamine functions as an inhibitor of PRL but not of LH or FSH in normal subjects. In PHP patients the responses suggest increased dopaminergic inhibition of LH release but loss of inhibitory control of PRL release. Physiological concentrations of plasma dopamine do not significantly inhibit LH or FSH release in normal subjects but paradoxically results in an apparent release of LH in PHP patients. We conclude that dopamine mechanisms do not play a significant role in modulating gonadotrophin release in normal subjects. In PHP patients, PRL feedback results in increased hypothalamic dopamine activity which in turn inhibits LH release. We conclude that the inhibitory action of dopamine on PRL release restores LH secretion by removing central dopaminergic inhibition through hypothalamic feedback of PRL.     

Estrogen regulates the gonadotropin-releasing hormone-stimulated secretion of biologically active luteinizing hormone

Estrogen produces time-dependent bidirectional effects on the GnRH-stimulated release of immunoactive LH in various species. To examine estrogen's regulation of biologically active LH secretion in response to pulsatile stimulation by GnRH, we studied estrogen-deficient postmenopausal women basally and during treatment with diethlystilbesterol (DES; 1 mg, orally, daily). Basal and GnRH-stimulated plasma concentrations of bioactive LH were assayed by the in vitro rat interstitial cell testosterone bioassay. GnRH-promoted LH secretory bursts in response to two consecutive stimuli were quantitated by multiple parameter deconvolution analysis. Basal half-lives of LH averaged 171 +/- 17 min (immunoactive) and 223 +/- 10 min (bioactive). Analysis of variance revealed a significant decrease in mean basal plasma bioactive LH concentrations on days 10 and 30 of DES treatment. Mean serum immunoactive LH concentrations fell similarly. DES significantly increased the half-life of immunoactive LH (days 5 and 10), but did not change that of bioactive LH. GnRH self-priming of bioactive LH secretion (increased LH secretory peak 2 compared to peak 1) was demonstrated, with a maximal value on day 10 of DES treatment. In addition, the ratio of the mass of bioactive to immunoactive LH secreted in response to the first GnRH pulse was significantly enhanced by estrogen on day 5, whereas that after the second pulse of GnRH was significantly suppressed on day 30 of DES. The self-priming action of GnRH on bioactive LH release evident in the presence of oral DES was corroborated in a separate group of six women, who were treated for 30 days with 17 beta-estradiol via an intravaginally placed Silastic ring. In conclusion, we infer that estrogen exerts a highly selective effect on the gonadotroph secretory process, such that successive GnRH stimuli result in an increase in the maximal rate and mass of secretion of biologically active LH.     

In vitro basal and GnRH-stimulated secretion of gonadotrophins reflects long-lasting modulatory effects, and peripheral levels are not predicted by pituitary responsiveness to GnRH

OBJECTIVE: Production of the appropriate pattern of gonadotrophin levels is crucial to proper functioning of the female reproductive system. We aimed to establish whether the pituitary has invariant secretory characteristics when isolated from in vivo controls. We aimed to obtain information during both the rising and declining phases of the gonadotrophin surge. DESIGN: This study investigated factors that are directed at the pituitary by isolating it from the acute influences of the in vivo environment and studying gonadotrophin secretion in vitro. METHODS: Pituitaries of adult female rats were collected at selected times during the day of pro-oestrus and incubated in vitro, and at the same time blood was collected. Peripheral levels of LH and FSH were measured over the whole day of pro-oestrus, basal in vitro secretions of LH and FSH from pituitaries were measured, GnRH-stimulated LH and FSH secretion were assessed, and the responsiveness of LH and FSH secretion to GnRH were calculated. RESULTS: Peripheral levels of LH peaked at 1800 h (P<0.02) followed by a subsequent decline. In contrast, although FSH had a peak at 1800 h (P<0.01), serum levels were also high at the end pro-oestrus. The profile of basal LH and FSH secretion from the pituitary in vitro, in the absence of added secretagogue, resembled that of the peripheral blood levels of each gonadotrophin. Pituitaries collected at 1800 h secreted most LH (P<0. 02). FSH secretion was low early on the day of pro-oestrus and then increased to and was maintained at high levels in the last quarter of the day (P<0.01).When the pituitaries were stimulated with GnRH the patterns of LH release and FSH release approximated those observed for basal release. Responsiveness of the pituitaries to GnRH was calculated by determining the ratio of GnRH-stimulated release to basal release. However, low levels of gonadotrophin were secreted even from pituitaries which were highly responsive as determined from consideration of percentage increase in secretion induced by GnRH. CONCLUSIONS: The secretory activity was dependent on the time of day the pituitaries were collected. Since the secretion occurred after the tissue had been removed from the direct influence of the in vivo environment, the variations in secretion must reflect long-lasting components of the mechanism that regulate gonadotrophin concentrations. There were changes in both LH and FSH responsiveness to GnRH stimulation over the day of pro-oestrus. Delineation of the time courses and changing predominance of multiple processes is needed to assist understanding the mechanisms underlying the female reproductive cycle.     

Interaction of endogenous chicken gonadotrophin-releasing hormone-I and -II on chicken pituitary cells

The presence of two endogenous forms of gonadotrophin-releasing hormone (GnRH) in the chicken hypothalamus (chicken GnRH-I ([Gln8]GnRH) and chicken GnRH-II ([His5, Trp7, Tyr8]GnRH)), and the stimulation of gonadotrophins by both forms, suggests the possible existence of GnRH receptor subtypes and gonadotroph subtypes in the chicken pituitary. This question was investigated by assessing the effects of various combinations of the two known forms of chicken hypothalamic GnRH and antagonist analogues of GnRH on LH release from dispersed chicken anterior pituitary cells in both static and perifused systems. The relative inhibition of chicken GnRH-I-stimulated and chicken GnRH-II-stimulated LH release by 12 GnRH antagonists did not differ significantly, suggesting a single GnRH receptor type. Chicken GnRH-II was approximately sixfold more potent than chicken GnRH-I in releasing LH. Release of LH in response to maximal doses of chicken GnRH-I and chicken GnRH-II and to a mixture of both was similar and the two peptides were not additive in their effects, consistent with the presence of a single type of LH gonadotroph and a GnRH receptor which binds both forms of GnRH. Each form of GnRH desensitized cells to subsequent stimulation with the other form, providing additional evidence for a single type of LH gonadotroph. These findings suggest that chicken GnRH-I and -II stimulate gonadotrophin release through a single GnRH receptor type on a single class of LH gonadotroph in the chicken pituitary.     

Estrogenic modulation of the gonadotropin-releasing hormone-stimulated secretory activity of the gonadotrope and lactotrope in prepubertal females with Turner's syndrome

The nature of estrogen's modulation of GnRH-stimulated secretion of the female prepubertal gonadotrope and lactotrope was studied in nine girls with primary gonadal failure (Turner's syndrome; mean age, 10.0 +/- 0.25 yr). LH, FSH, and PRL release was evaluated by sampling blood every 20 min from 2000-0800 h. Hormone secretion was stimulated by one of two randomized doses of GnRH (50 or 750 ng/kg) delivered at fixed intervals of every 90 min in an attempt to replace the function of the endogenous GnRH pulse generator with an exogenous GnRH clamp. To evaluate the time dependency of estrogen action, studies were conducted at baseline and after 1 and 5 weeks of oral administration of ethinyl estradiol (EE; 100 ng/kg.day). In vivo gonadotropin secretory dynamics were quantitated by deconvolution mathematical modeling. We found a suppression of total LH secretion in response to repeated fixed doses of GnRH after 1 and 5 weeks of EE exposure, viz. a 10% (1 week) and 60% (5 weeks) reduction in the total mass of LH released after six consecutive GnRH pulses. Before estrogen exposure, patients manifested a decreasing mass of LH secreted per burst (slope of mass/burst vs. GnRH injection number was -3.3 +/- 1.44), suggesting down-regulation of the LH secretory response. However, after 5 weeks of EE treatment, the same series of GnRH doses elicited a progressive increase in the mass of LH secreted per burst (slope, 1.06 +/- 0.036; P = 0.041). Such serial amplification of LH secretory responses (despite overall suppression of the mean serum LH concentrations by EE) is consistent with the emergence of priming of GnRH actions. This phenomenon was specific, since the half-life of LH and the LH secretory burst duration were not altered. FSH responses to GnRH were significantly suppressed after 5 weeks of EE exposure (mean serum FSH concentrations, 61.9 +/- 11.4 IU/L at baseline vs. 14.4 +/- 6.9 at week 5; P = 0.003). However, in contrast to the LH responses on a given study day, there was increased FSH responsivity to successive doses of GnRH, suggesting a priming effect of serial GnRH exposure on GnRH-stimulated FSH secretion regardless of the estrogen milieu. PRL secretion was stimulated by GnRH at baseline (16.8 +/- 0.88 micrograms/L), but release was reduced at week 5 on estrogen (11.6 +/- 0.4 micrograms/L). This may represent withdrawal of the paracrine effects of endogenous GnRH and/or increased dopaminergic tone induced by estrogen.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dissociation between pituitary GnRH binding sites and LH response to GnRH in vitro

Experiments were performed to study gonadotroph responsiveness to gonadotrophin releasing hormone (GnRH) in vitro in dispersed pituitary cells from ovariectomised rats and mice when GnRH binding sites were increased or reduced, respectively. Maximal/basal LH release after GnRH treatment of intact female rat pituitary cells was 4.7 to 9.7-fold (range n = 3 expts.) compared to 3.4 to 5.0-fold for cells from ovariectomised rat donors. Both basal and maximal GnRH-stimulated LH release from ovariectomised (OVX) rat pituitary cells were 1.5 to 3-fold greater than from intact rat cells, which corresponded to increased LH content of the cells. There was no significant change in the GnRH ED50 concentration (intact = 2.3 +/- 0.03 X 10(-10) M; OVX = 3.3 +/- 0.08 X 10(-10) M (mean +/- SEM, n = 3 expts.)), despite a 57-88% increase in GnRH binding sites in ovariectomised rat pituitary cells. Basal and maximal LH release from ovariectomised mouse pituitary cells was 1.5 to 3-fold greater than that from intact mouse pituitary cells. There was no change in the GnRH ED50 concentration (intact = 4.3 +/- 2.3 X 10(-9) M; OVX = 3.4 +/- 0.9 X 10(-9) M (mean +/- SEM, n = 3 expts.)), even though GnRH binding sites were reduced by 40-73% in the cells from ovariectomised mice. These data indicate that changes in GnRH binding sites of the magnitude observed after ovariectomy play no part in the regulation of gonadotroph responsiveness to GnRH, which is determined by changes in post-receptor events, one of which is an increase in cellular LH.     

Gonadotropin-releasing hormone differentially regulates expression of the genes for luteinizing hormone alpha and beta subunits in male rats.

Gonadotropin-releasing hormone (GnRH) and gonadal steroids regulate synthesis and release of luteinizing hormone (LH). GnRH is secreted intermittently by the hypothalamus, producing pulsatile LH release, and a pulsatile GnRH stimulus is required to maintain LH secretion. We report the regulatory effects of GnRH pulse injections on pituitary concentrations of LH alpha and beta subunit mRNAs in a castrated/testosterone-replaced male rat model. Replacement with physiologic amounts of testosterone decreased concentrations of both LH subunit mRNAs. GnRH pulse injections (10-250 ng per pulse given every 30 min for 48 hr) increased both mRNA concentrations, but the dose response patterns were markedly different. alpha subunit mRNA was increased by all GnRH doses but not the levels seen after castration alone. In contrast, LH beta subunit mRNA concentrations showed a marked dependence on GnRH dose. Maximal responses, to values similar to those in castrates, occurred after 25-ng GnRH pulses, and larger doses produced a smaller increase in LH beta subunit mRNA. Both the acute LH secretory response to GnRH and the number of GnRH receptors followed a pattern similar to the LH beta subunit mRNA concentration and were maximal after the 25-ng GnRH dose. These results show that GnRH can differentially regulate LH subunit mRNAs and suggest that concentrations of LH beta subunit mRNA may be a limiting factor in GnRH-stimulated LH release.     

Development of gonadotrope desensitization to gonadotropin-releasing hormone (GnRH) and recovery are not coupled to inositol phosphate production or GnRH receptor number.

After initial GnRH pretreatment (10 nM, 5 h), subsequent GnRH-stimulated LH release from the gonadotrope was diminished (1 microM GnRH stimulated release of 36.4 +/- 1.4% total cellular LH over 3 h in cells initially pretreated with medium alone compared to 27.4 +/- 1.2% in GnRH-pretreated cells); however, inositol phosphate (IP) production in response to the releasing hormone remained unaffected (1 microM GnRH provoked IP accumulation of 161 +/- 9% above basal levels after 45 min in control cells and 162 +/- 11% in GnRH-pretreated cells). Pretreatment of pituitary cell cultures with NaF (a guanyl nucleotide binding protein activator, 10 mM, 3 h) also decreased subsequent GnRH-stimulated LH release, and in addition, provoked a decrease in GnRH receptor number, an increase in GnRH receptor affinity, reduction of GnRH-stimulated IP production to basal levels, and an increase in the amount of LH released in response to stimulation with the calcium ionophore A23187. In order to determine if the changes in LH release were a result of decreased IP production and/or decreased GnRH receptor binding, the time course of recovery to control levels of these processes was assessed. GnRH receptor binding continued to decrease after NaF pretreatment, reaching a nadir (62% of control) at 6 h after the pretreatment period and recovering at 48 h (90% of control). In contrast, GnRH-provoked IP accumulation did not return to control levels even after 48 h of recovery after NaF pretreatment (1 microM GnRH-stimulated IP accumulation in NaF-pretreated cells was 57% compared to control cells after 48 h of recovery). GnRH-stimulated LH release was inhibited immediately after NaF pretreatment (1 microM GnRH-stimulated LH release in NaF-pretreated cells was 65% of control levels). Cells began to recover within 3 h (80% of control) and were almost completely recovered by 6 h (90% of control). A23187-provoked LH release was enhanced immediately after NaF pretreatment (30 microM A23187-stimulated LH release in NaF-pretreated cells was 170% of control levels). Responsiveness to ionophore was 133% of control by 0.5 h, and complete recovery was measured within 1 h (100% of control). Furthermore, both NaF and GnRH pretreatment still provoked a decrease in gonadotrope responsiveness when IP production was inhibited by the phospholipase C inhibitor U-73122. The results suggest that the development of gonadotrope desensitization (by either NaF or GnRH pretreatment) can be uncoupled from changes in IP production.(ABSTRACT TRUNCATED AT 400 WORDS)     

Gonadotrophin-releasing hormone modulation of gonadotrophins in the ewe: evidence for differential effects on gene expression and hormone secretion.

While the regulation of gonadotrophin secretion by gonadotrophin-releasing hormone (GnRH) has been well documented in both rats and sheep, its role in the synthesis of gonadotrophin subunits remains unclear. We have investigated the effects of the specific inhibition of GnRH by a GnRH agonist on the expression of gonadotrophin subunit genes and the subsequent storage and release of both intact hormones and free alpha subunit. Treatment with GnRH agonist for 6 weeks abolished pulsatile LH secretion, reduced plasma concentrations of FSH and prevented GnRH-induced release of LH and FSH. This was associated with a reduction of pituitary LH-beta mRNA and FSH-beta mRNA levels (to 5 and 30% of luteal control values respectively), but not alpha mRNA which was significantly increased (75% above controls). While there was a small decrease in the pituitary content of FSH (30% of controls), there was a drastic reduction in LH pituitary content (3% of controls). In contrast to the observed rise in alpha mRNA, there was a decrease in free alpha subunit in both the pituitary and plasma (to 30 and 80% of control levels). These results suggest that, while GnRH positively regulates the expression of both gonadotrophin beta-subunit genes, it can, under certain circumstances, negatively regulate alpha-subunit gene expression. Despite the complete absence of LH and FSH in response to GnRH, there remained a basal level of beta-subunit gene expression and only a modest reduction (50%) in the plasma levels of both FSH and LH, suggesting that there is a basal secretory pathway.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Gonadal steroid modulation of signal transduction and luteinizing hormone release in cultured chicken pituitary cells

The mechanism whereby gonadal steroids modulate GnRH-stimulated LH secretion by primary cultures of chicken pituitary cells was investigated. Estradiol (10(-8) M), testosterone (10(-7) M), and progesterone (10(-7) M) inhibited LH release stimulated by GnRH (10(-7) M) by 56%, 61%, and 53%, respectively, and the inhibitory effects required prolonged preincubation (24-48 h) with the steroids. The steroids inhibited the spike (0-3 min) and plateau (9-30 min) phases of LH release to a similar degree. The ED50 values of estradiol, testosterone, and progesterone for inhibition of GnRH-stimulated LH release were 7 x 10(-11), 2 x 10(-9), and 1 x 10(-9) M, respectively. Estradiol, testosterone, and progesterone inhibited the maximal LH response to GnRH, but the ED50 of GnRH (4 x 10(-9) M) was not altered by steroid pretreatment. Steroid pretreatment did not cause a change in cellular LH content, suggesting that the steroids do not inhibit LH synthesis. Combinations of two or three of the steroids were not additive, suggesting that all three steroids affect GnRH-stimulated LH release via the same mechanism. In experiments investigating their mechanism of action, the steroids inhibited LH release stimulated by GnRH and Ca2+ ionophore A23187, but generally had no effect on the responses to phorbol ester (12-O-tetradecanoylphorbol-13-acetate), forskolin, K+, Bay K8644, or veratridine. Estradiol inhibited GnRH-stimulated 45Ca2+ efflux, but its inhibitory effect on GnRH-induced inositol phosphate production was not significant. Estradiol had no effect on binding of 125I-[His5,D-Tyr6]GnRH to a pituitary cell preparation. These findings suggest that the site of steroid modulation of GnRH action is distal to binding of GnRH to its receptor, and that the inhibitory effects are exerted at two intracellular sites: 1) the coupling events linking receptor activation to mobilization of Ca2+, and 2) a site distal to Ca2+ mobilization.     

The self-priming effect of gonadotropin-releasing hormone on luteinizing hormone release: observations using rat anterior pituitary fragments and dispersed cells continuously perifused in parallel

To study in vitro the self-priming effect of GnRH on LH release, rat anterior pituitaries were prepared either as fragments or dispersed cells and continuously perifused in parallel chambers. The experimental groups consisted of rats killed at 0800 h on diestrus day 1, diestrus day 2, proestrus or estrus, or at 1400 h on proestrus. To insure truly independent observations, each experimental preparation was tested on three occasions. After basal LH release had stabilized, the tissue preparations were exposed to 10 nM GnRH as two 30-min challenges separated by 1 h. LH secretory rates (nanograms per min/pituitary for fragments; nanograms per min/10(7) cells for dispersed cells) were calculated 1) for basal release (during the 20-min period immediately preceding each GnRH challenge), 2) in response to GnRH, and 3) as the sum of basal and GnRH-stimulated release. Comparison of the two preparations revealed that basal and GnRH-stimulated LH release by pituitary fragments was more variable than LH release by dispersed cells. In addition, while dispersed cells responded promptly to the addition/withdrawal of stimuli, fragments did so more gradually. With respect to GnRH self-priming, the second mean secretory rate for basal LH release by fragments (range, 28.8-46.5) was significantly (0.1 greater than P greater than 0.01) higher than the first rate (range, 14.4-22.0) on diestrus day 1, diestrus day 2, proestrus at 0800 h, and estrus. With dispersed cells, the first and second basal rates were similar to each other on diestrus day 1 and estrus, but on diestrus day 2 and on proestrus at 0800 and 1400 h, the second basal rate (range, 36.8-93) was significantly (P less than 0.001) higher than the first range (range, 17.7-31.7). When fragments received GnRH, the second mean secretory rate (range, 35.2-64.2) was significantly (0.1 greater than P greater than 0.03) higher than the first rate (range, 13.4-34.1) on diestrus day 2 and proestrus at 0800 h. With dispersed cells, the mean secretory rate in response to the second GnRH challenge was higher only on diestrus day 2 (37.0 +/- 4.1 vs. 60.3 +/- 3.8; P less than 0.05). When considered as the total of basal plus GnRH-stimulated LH release, the second secretory rate by fragments (range, 54.5 - 110.8) was significantly (0.1 greater than P greater than 0.02) higher than the first rate (range, 27.9 - 51.4) on diestrus day 1, diestrus day 2, and proestrus at 0800 h.(ABSTRACT TRUNCATED AT 400 WORDS)     

BROMOCRIPTINE AND OESTROGEN MODULATION OF GONADOTROPHIN RELEASE IN NORMO-AND HYPERPROLACTINAEMIC PATIENTS WITH AMENORRHOEA

Effects of oestradiol, bromocriptine, and bromocriptine plus oestradiol, on basal and GnRH stimulated gonadotrophin concentrations were studied in normo-(group 1, n= 7) and hyperprolactinaemic (group 2, n= 6) patients with secondary amenorrhoea. Before drug administration, hyper-responsiveness of LH, but normal FSH responses to GnRH were observed in most patients. Oral administration of 2 mg oestradiol for 4 days resulted in increased 17β-oestradiol levels in plasma in normal women (n= 6) in the early follicular phase of the cycle, and in groups 1 and 2. During oestradiol administration plasma LH concentration increased significantly and there was an increase of LH and FSH responses to GnRH in normal subjects, but not in amenorrhoeic women. In groups 1 and 2 basal FSH levels were suppressed but no change in GnRH stimulated gonadotrophin responses was seen. Bromocriptine (5 mg per day for 5 days) significantly decreased prolactin concentrations and increased 17β-oestradiol levels in plasma in group 2 but not in group 1. The mean plasma 17β-oestradiol concentration had increased to levels similar to those obtained during oestradiol administration alone. The mean LH response to GnRH was suppressed in group 2, but not in group 1. Basal and GnRH-stimulated plasma FSH concentrations were not changed by bromocriptine treatment. Compared with the GnRH induced LH response during bromocriptine alone, bromocriptine treatment plus oestradiol administration resulted in a significantly increased LH response in group 2. This was not found in group 1. The present results suggest that there is an increased dopamine activity and inhibition of GnRH at the hypothalamic level and a relative dopamine deficiency at the pituitary level in hyperprolactinaemic patients. Normoprolac-tinaemic patients with hypothalamic amenorrhoea have increased dopaminergic activity at the hypothalamic, as well as the, pituitary level, or alternatively that the LH release is not influenced by dopamine in these patients. Finally bromocriptine sensitizes LH-secreting cells to GnRH in hyperprolactinaemic, but not in normoprolactinaemic, patients.     

The relationship between gonadotropin-releasing hormone-stimulated luteinizing hormone release and inositol phosphate production: studies with calcium antagonists and protein kinase C activators

The coupling between GnRH-stimulated phosphoinositide (PI) turnover and LH release has been investigated in rat pituitary cell cultures. Accumulation of [3H]inositol phosphates ([3H]IPs) formed by hydrolysis of PIs was measured in cells that had been preloaded with [3H]myo-inositol. GnRH stimulated both LH release and incorporation of [3H]inositol into total [3H]IPs with similar dose and time dependencies. [3H] IP production in response to GnRH could be blocked by a GnRH antagonist, but was stimulated by a compound that provokes receptor microaggregation. GnRH-stimulated IP production persisted in the presence of either the Ca2+ channel blocker D600 or the calmodulin antagonist pimozide at concentrations that reduced LH release to 60% and 20% of control, respectively. Stimulated [3H]IP production was inhibited at higher concentrations of D600. In 1-h incubations, GnRH-stimulated [3H]IP production, but not LH release, was markedly inhibited by the protein kinase C activators phorbol myristate acetate and 1,2-dioctanoylglycerol. These findings indicate that in the gonadotrope, GnRH-stimulated LH release and [3H]IP production are closely coupled to receptor activation by an agonist; Ca2+ antagonists uncouple stimulated LH release from [3H]IP production; and protein kinase C activators uncouple stimulated PI turnover from LH release. Thus, GnRH-stimulated production of PI metabolites, as measured by [3H]IP accumulation, is apparently not sufficient to support LH release in the absence of Ca2+. In addition, GnRH-stimulated LH release is apparently not dependent on full expression of the PI response.     

Effects of bovine 35 kDa FSH-suppressing protein on FSH and LH in rat pituitary cells in vitro: comparison with bovine 31 kDa inhibin

The mode of action of a recently isolated gonadal protein, termed FSH-suppressing protein (FSP) or follistatin, on basal and gonadotrophin-releasing hormone (GnRH)-stimulated release of FSH and LH and on pituitary cell content of FSH and LH was examined in rat pituitary cell cultures and compared with the previously reported effects of inhibin. Pituitary cells were cultured for 3-9 days in the presence of graded doses of FSP and the basal release rates and changes in cell contents of FSH and LH determined during this period. FSP suppressed both the basal release rate and the cell content of FSH with median inhibitory concentrations (IC50) of 135 and 161 pmol/l respectively. The corresponding effects of FSP on LH basal release rate and LH cell content (IC50 = 200 pmol/l) were limited compared with the effects on FSH. The effect of FSP on GnRH-stimulated release of FSH and LH during 4 h was determined in cells which had been preincubated with FSP for 3 days, and the GnRH-stimulated release of FSH and LH analysed as a percentage of the respective gonadotrophin available for release. FSP antagonized GnRH action with dose-related increases in the GnRH median effective (stimulatory) concentrations for FSH and LH release (EC50 values = 56 and 400 pmol/l respectively) and a suppression in the maximum release of FSH and LH by excess GnRH (IC50 values = 142 and 150 pmol/l respectively). The effect of FSP on FSH cell content after 3 days in culture was insensitive to the neutralizing effects of an inhibin antiserum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of Neurokinin B and Dynorphin A in pituitary gonadotroph and somatolactotroph cell lines

The role of Neurokinin B (NKB) and Dynorphin A (Dyn) in the regulation of the hypothalamic pituitary axis is an important area of recent investigation. These peptides are critical for the rhythmic release of GnRH, which subsequently stimulates the secretion of the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The present study utilized the gonadotroph cell line LβT2 and the somatolactotroph GH3 cell line to examine the possible role of these peptides in pituitary hormone secretion. The NKB receptor (NK3R) and the Dyn receptor (the κ-opiate receptor (KOR)) were both detected in LβT2 cells and GH3 cells. NKB, by itself, failed to increase gonadotropin LHβ and FSHβ promoter activities and did not modulate the effects of GnRH on gonadotropin promoter activity. In GH3 cells, NKB significantly increased TRH-induced PRL promoter activity although NKB alone did not have an effect on basal PRL promoter activity. Dyn had no effect on gonadotropin promoters alone or in combination with GnRH stimulation. PRL promoters stimulated by TRH were not significantly changed by Dyn. TRH-induced PRL promoter activity was further increased in the presence of higher concentrations of NKB, whereas Dyn did not have a significant effect on the PRL promoter even at a high concentration. In addition, TRH-induced ERK (Extracelluar signal-regulated kinase) activation was enhanced in the presence of NKB. Our current study demonstrated that NKB had a stimulatory effect on PRL expression in a PRL-producing cell, but had no effect on gonadotropin secretion from a gonadotroph cell line.     

The nature of the gonadotropin-releasing hormone stimulus-luteinizing hormone secretory response of human gonadotrophs in vivo

To examine the stimulus-secretion response of human pituitary gonadotrophs in vivo, we applied a new multiple parameter deconvolution technique to analyze (1) exogenous GnRH-stimulated LH secretory responses in 10 men with isolated hypogonadotropic hypogonadism (IHH), and (2) endogenous and exogenous GnRH-stimulated LH secretory responses in 8 normal men. The GnRH-deficient men were given 4 bolus doses of synthetic GnRH (7.5, 25, 75, and 250 ng/kg) iv at 2-h intervals in randomized order after long term pulsatile GnRH administration. The normal men were studied by sampling blood at 10-min intervals for 12 h basally and after 2 consecutive 10-micrograms iv GnRH doses. The serum LH peaks in both groups were subjected to quantitative deconvolution to resolve underlying LH secretory and clearance rates simultaneously. Such analyses revealed that exogenous GnRH-induced LH secretory episodes in GnRH-deficient men with IHH could be modeled as algebraically Gaussian distributions of instantaneous LH secretory rates with a mean half-duration of 14 +/- 2 min. The simultaneously resolved half-life of endogenous LH disappearance was 71 +/- 5 min. The log dose-response relationship for GnRH dose vs. maximal LH secretory rate or vs. calculated mass of LH released per secretory burst was linear. In contrast, varying GnRH doses did not alter the duration of LH secretory bursts, the half-time of LH disappearance, or the latency of LH secretory bursts after iv GnRH injections (viz. 7.6 min). Deconvolution analysis of the spontaneous (endogenous GnRH-stimulated) LH peaks in normal men revealed a mean half-duration of secretory bursts of 9.9 +/- 1.5 min, and a mean half-time of endogenous LH disappearance of 76 +/- 5 min. These values were not significantly different from those in the GnRH-treated normal or GnRH-deficient men. In summary, deconvolution analysis of LH release in men with IHH revealed a significant linear relationship between iv doses of pulsed GnRH and computer-resolved LH secretory rate and/or the mass of LH released per secretory event. In contrast, varying doses of GnRH did not alter the lag time between the GnRH stimulus and the LH secretory burst, the duration of LH secretion, or the calculated half-life of the LH released. We conclude that GnRH exerts dose-dependent effects on specific attributes of the secretory response of human gonadotrophs in vivo.     

Influence of steroids and GnRH on biosynthesis and secretion of secretogranin II and chromogranin A in relation to LH release in LbetaT2 gonadotroph cells

The granin proteins secretogranin II (SgII) and chromogranin A (CgA) are commonly found associated with LH and/or FSH within specialised secretory granules in gonadotroph cells, and it is possible that they play an important role in the differential secretion of the gonadotrophins. In this study we have examined the regulation of the biosynthesis and secretion of SgII and CgA, in relation to LH secretion, in the LbetaT2 mouse pituitary gonadotroph cell line. Three experiments were carried out to investigate the effects of oestradiol (E2) and dexamethasone (Dex) in the presence and absence of GnRH (experiment 1), differing GnRH concentrations (experiment 2) and alterations in GnRH pulse frequency (experiment 3). In experiment 1, exposure to E2, Dex or E2+Dex, either with or without GnRH treatment, resulted in increased LH secretion. Steroids alone had no effect on LHbeta mRNA levels, but in the presence of GnRH LHbeta mRNA levels were increased in Dex- and E2+Dex-treated cells. GnRH receptor (GnRH-R) mRNA levels were up-regulated by Dex and E2+Dex, but were unaffected by GnRH. There were no steroid-induced changes in SgII or CgA mRNA, but increased levels of CgA mRNA were observed after GnRH treatment in cells cultured in the presence of Dex. In experiment 2, increasing concentrations of GnRH resulted in increases in LH secretion that were inversely dose-dependent. No changes in LHbeta, GnRH-R or SgII mRNA levels were observed, but there were dose-dependent increases in CgA mRNA levels. In experiment 3, GnRH was given as either 1 pulse/day or 4 pulses/day for 3 days. Both pulse regimes resulted in increased LH, SgII and CgA secretion compared with controls during the first 15 min pulse on day 3. Exposure to GnRH at 4 pulses/day increased LH and SgII secretion compared with controls during all 4 pulses, but secretion of both proteins was reduced during pulses 2-4 compared with pulse 1. CgA secretion also increased due to GnRH in pulse 1, but was decreased by GnRH treatment during pulse 2, and unchanged by GnRH during pulses 3 and 4. Total daily secretion of LH and SgII from cells given 1 pulse/day of GnRH increased compared with controls on all three treatment days, while total CgA secretion increased in response to GnRH on days 2 and 3 only. Intracellular levels of SgII, but not LH, decreased after GnRH treatment. In contrast, intracellular CgA was increased, but only after 4 pulses/day of GnRH. Levels of LHbeta, but not SgII, mRNA were increased by both pulse regimes, while CgA mRNA levels increased after 1 pulse/day of GnRH. These results indicate that there is a close correlation between the GnRH-stimulated release of LH and SgII from LbetaT2 cells, suggesting that SgII may have an influential role in the regulated secretion of LH, possibly by inducing LH aggregation to facilitate trafficking into secretory granules. CgA secretion does not appear to be closely associated with that of LH, but CgA expression does appear to be regulated by GnRH, which may indicate involvement in the control of LH secretion, possibly by influencing the proportion of LH in the different types of secretory granules.