GnRH-stimulated LH Release Research Articles

A novel agent for induced spawning using a combination of GnRH analog and an FDA-approved dopamine receptor antagonist

In certain fish species, endogenous dopamine has a significant inhibitory effect on LH release and spawning induced by externally applied GnRH. Preovulatory LH surge and spawning in these fish species are caused by the simultaneous release of GnRH and exclusion of the dopaminergic inhibition. To induce spawning, a potent dopamine D2 receptor (DRD2) antagonist and a GnRH analog are concomitantly applied to fish with solid dopaminergic inhibition. However, the currently used dopamine antagonists are not approved by the FDA for veterinary use. This project was conducted in order to find a novel FDA-approved dopamine antagonist that will be efficient in spawning induction in fish species and will not be lethal to the fish. First, we studied the inhibitory effect of the novel dopamine antagonist, azaperone, on tilapia DRD2, transiently expressed in COS-7 cell line. Azaperone was more effective than metoclopramide in antagonizing the effect of quinpirole on taDRD2 both in terms of EC50 and maximal response. Next, we tested the ability of the combination of the dopamine antagonist and sGnRHa on GTH secretion, in adult tilapia. The novel dopamine antagonist significantly increased the GnRH-stimulated LH release in vivo, on both LH release and gene expression. We next aimed to test the novel combination in carp. The combination of the novel dopamine antagonist together with GnRHa, increased the release of estradiol and LH, in a dose-dependent manner. A higher hatching rate and spawning efficiency were observed than in the positive control that contained metoclopramide as a dopamine antagonist.

OR16-5 Feedback on LH in Testosterone-Clamped Men Depends on the Mode of Testosterone Administration and Body Composition

Context. Investigations of testosterone (T)-mediated feedback on LH secretion are few and contradictory. Discrepancies may reflect the choice of androgen, as well as dose, route and/or pattern (continuous vspulsatile) of administration. Moreover, for controlled T feedback studies, endogenous T secretion must be shut down and replaced by iv T infusion. Objective. The goal of this study was to quantify feedback in relation to the infused T pattern (pulsatile vs nonpulsatile) with the hypotheses that: 1) feedback by T pulses is less inhibitory, 2) T feedback operates partly at the pituitary level, 3) T feedback is controlled in part by body composition. Design. This was a placebo-controlled, blinded, and prospectively randomized crossover study comprising 16 men (age range 23-54 yr and BMI between 22.3-34.2 kg/m2). Subjects received ketoconazole (1600 mg over 22 h to block endogenous T secretion: serum T below 85 ng/dL) followed by pulsatile or continuous iv T addback (total T dose 5 mg/22 h). Cortisol inhibition was treated with low-dose dexamethasone. Setting. The study was performed in a Clinical Translational Research Unit. Interventions. Subjects underwent 14 h of blood sampling at 10-min intervals, starting at 0600 h. T was given continuously starting at 0900 h or as 45-min squarewave pulses every 90 min. To test pituitary feedback, a bolus iv injection of 33 ng/kg GnRH was given at 1630 h. Outcome measures. Log-transformed LH and T concentration ratios before and after GnRH administration. In addition, a four-parameter logistic regression between serum T concentration and LH secretion rate (calculated by deconvolution analysis) was used to independently estimate feedback of T on LH. Results. Despite higher T (total, bioavailable and free T) concentrations during pulsatile feedback, LH concentrations and secretion rates, whether driven by endogenous or exogenous GnRH, were similar to those during continuous infusion, indicating diminished pulsatile T feedback. This interpretation was corroborated by the right-shift (i.e. towards higher T concentrations) of the T-LH inhibitory dose-response curve. During continuous and pulsatile T infusion, LH concentrations and LH:T ratios depended positively on BMI to a similar extent before and after GnRH administration. Under both feedback regimens, basal but not pulsatile LH secretion correlated negatively with CT-estimated abdominal visceral fat mass. Age was not a significant covariate over the limited range explored. Conclusion. In summary, pulsatile T is less effective as a negative feedback signal on LH secretion than continuous T administration, as demonstrated by two independent methods. This difference in feedback strength was evident for exogenous GnRH-stimulated LH release as well, and thus occurs probably in part at the pituitary level, while not excluding additional hypothalamic involvement.

Effect of orexin A on the release of GnRH-stimulated gonadotrophins from cultured pituitary cells of immature and mature female rats

Orexin A (OxA), also known as hypocretin 1, is a regulatory neuropeptide involved in the control of various autonomic and neuroendocrine functions. It appears to have a significant impact on the regulation of trophic hormones secretion by influencing the hypothalamus and the pituitary. Orexin A acts through two types of receptor found in the pituitary. This suggests the possibility of direct action of OxA at the adenohypophysis level.The aim of this study was to investigate the direct effect of OxA on GnRH (gonadotrophin-releasing hormone)-stimulated LH and FSH secretion from cultured pituitary cells of sexually immature and mature female rats.Anterior pituitary cells obtained from immature and mature female rats (ovariectomized, and ovariectomized and treated with estradiol) were incubated with 10−10M or 10−7M orexin A for 1hour and 4h and the effect on GnRH-stimulated (10−9M or 10−6M) LH and FSH release was examined. The concentrations of secreted gonadotrophins in the culture media were determined by RIA methods.Orexin A significantly inhibited GnRH-stimulated FSH release from pituitary cells isolated from immature female rats, whereas in cells of mature ovariectomized animals, the effect of OxA was dependent on the stimulatory dose of GnRH. When the cells were stimulated with a low dose of GnRH, orexin A inhibited the secretion of gonadotrophins, but when a high dose of GnRH was used, orexin A increased mainly the release of LH. In cultured pituitary cells from ovariectomized, estrogenized mature rats, orexin A inhibited the secretion of LH if the cells were stimulated with a high dose of GnRH.In conclusion, the results of this study revealed that orexin A may modify the sensitivity of gonadotrophic cells to GnRH, and its effect depends on the maturity and estrogen status of the rats from which the cells are isolated.

Differential Involvement of Phosphoinositide 3-Kinase in Endogenous Gonadotropin-Releasing Hormone Signalling in Gonadotropes and Somatotropes of Goldfish, Carassius auratus.

Event Abstract Back to Event Differential Involvement of Phosphoinositide 3-Kinase in Endogenous Gonadotropin-Releasing Hormone Signalling in Gonadotropes and Somatotropes of Goldfish, Carassius auratus. Joshua G. Pemberton1, James L. Stafford1, Yi Yu1 and John P. Chang1* 1 University of Alberta, Biological Sciences, Canada Two endogenous gonadotropin-releasing hormones (salmon (s)GnRH and chicken (c)GnRH-II) stimulates LH and GH secretion via Ca2+-dependent intracellular signalling pathways in goldfish, Carassius auratus. Whether phosphoinositide 3-kinase (PI3K) mediates GnRH-stimulated LH and GH release and associated intracellular Ca2+ increases ([Ca2+]i) were investigated using hormone release studies with primary cultures of dispersed goldfish pituitary and Fura2 fluorescence Ca2+ imaging in morphologically identified goldfish pituitary somatotropes and gonadotropes. Western blot showed the presence of immunoreactive PI3K p85α, the regulatory subunit for conventional Class IA PI3Ks, in goldfish pituitary and brain tissue extracts and both endogenous GnRH isoforms increased phosphorylation of PI3K p85α in excised pituitary fragments. Selective PI3K inhibitors wortmannin (100 nM) and LY294002 (10 µM) significantly reduced sGnRH- and cGnRH-II-elicited LH release responses from primary cultures of mixed pituitary cells and [Ca2+]i increases in identified gonadotropes. Surprisingly, both PI3K inhibitors reduced the GH release response to the two GnRHs but only attenuated the [Ca2+]i response to cGnRH-II. The effects of PI3K inhibition on hormone release responses were specific to GnRHs and not the result of general decreases in the releasable hormone pool or sensitivity to [Ca2+]i changes as both wortmannin and LY294002 had no effect on Ca2+ ionophore-evoked LH and GH secretion. These results indicate that PI3K is involved upstream of [Ca2+]i increases in mediating LH and GH responses in a cell- and GnRH isoform-specific manner and is the first to implicate PI3K in GnRH-induced LH and GH release in any primary pituitary cell system. Acknowledgements This research was supported by Natural Sciences and Engineering Research Council of Canada (NSERC) individual Discovery Grants to J.P. Chang and J.L. Stafford. Financial support for J.G. Pemberton was provided by the Province of Alberta Queen Elizabeth II Graduate Scholarship, NSERC Alexander Graham Bell Canada Graduate Scholarship (CGS M), and the University of Alberta as a recipient of the Dr. Richard E. Peter Memorial Scholarship, Walter H. Johns Graduate Fellowship and Department of Biological Sciences Teaching Assistantship. Y. Yu was a recipient of a F.S. Chia PhD Recruitment Scholarship from the University of Alberta. We thank Alan Mar and Herman Cortes for their assistance with this study. Keywords: fura2 fluorescence calcium imaging, gonadotropes and somatotropes, gonodotropin-releasing hormone, luteinizing and growth hormone secretion, Phosphoinositide 3-kinase, wortmannin and LY294002 Conference: NASCE 2011: The inaugural meeting of the North American Society for Comparative Endocrinology, Ann Arbor, United States, 13 Jul - 16 Jul, 2011. Presentation Type: Poster Topic: Intracellular signaling Citation: Pemberton JG, Stafford JL, Yu Y and Chang JP (2011). Differential Involvement of Phosphoinositide 3-Kinase in Endogenous Gonadotropin-Releasing Hormone Signalling in Gonadotropes and Somatotropes of Goldfish, Carassius auratus.. Front. Endocrinol. Conference Abstract: NASCE 2011: The inaugural meeting of the North American Society for Comparative Endocrinology. doi: 10.3389/conf.fendo.2011.04.00079 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 19 Jul 2011; Published Online: 09 Aug 2011. * Correspondence: Prof. John P Chang, University of Alberta, Biological Sciences, Edmonton, Alberta, T6G2E9, Canada, john.chang@ualberta.ca Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Joshua G Pemberton James L Stafford Yi Yu John P Chang Google Joshua G Pemberton James L Stafford Yi Yu John P Chang Google Scholar Joshua G Pemberton James L Stafford Yi Yu John P Chang PubMed Joshua G Pemberton James L Stafford Yi Yu John P Chang Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Age disrupts androgen receptor-modulated negative feedback in the gonadal axis in healthy men

Testosterone (T) exerts negative feedback on the hypothalamo-pituitary (GnRH-LH) unit, but the relative roles of the CNS and pituitary are not established. We postulated that relatively greater LH responses to flutamide (brain-permeant antiandrogen) than bicalutamide (brain-impermeant antiandrogen) should reflect greater feedback via CNS than pituitary/peripheral androgen receptor-dependent pathways. To this end, 24 healthy men ages 20-73 yr, BMI 21-32 kg/m2, participated in a prospective, placebo-controlled, randomized, double-blind crossover study of the effects of antiandrogen control of pulsatile, basal, and entropic (pattern regularity) measurements of LH secretion. Analysis of covariance revealed that flutamide but not bicalutamide 1) increased pulsatile LH secretion (P = 0.003), 2) potentiated the age-related abbreviation of LH secretory bursts (P = 0.025), 3) suppressed incremental GnRH-induced LH release (P = 0.015), and 4) decreased the regularity of GnRH-stimulated LH release (P = 0.012). Furthermore, the effect of flutamide exceeded that of bicalutamide in 1) raising mean LH (P = 0.002) and T (P = 0.017) concentrations, 2) accelerating LH pulse frequency (P = 0.013), 3) amplifying total (basal plus pulsatile) LH (P = 0.002) and T (P < 0.001) secretion, 4) shortening LH secretory bursts (P = 0.032), and 5) reducing LH secretory regularity (P < 0.001). Both flutamide and bicalutamide elevated basal (nonpulsatile) LH secretion (P < 0.001). These data suggest the hypothesis that topographically selective androgen receptor pathways mediate brain-predominant and pituitary-dependent feedback mechanisms in healthy men.

Mechanisms of FSH synthesis: what we know, what we don't, and why you should care

The pituitary gonadotropin hormones, FSH and LH, are key regulators of reproductive physiology. Though the two hormones are produced by the same cell type, often in response to the same endocrine and paracrine regulators, they sub-serve different biological functions and their synthesis and secretion are differentially regulated. This stems largely from differences in transcriptional, post-transcriptional, and post-translational regulation of their unique beta subunits. That is, both hormones are dimeric glycoproteins and share a common alpha subunit. Their unique beta subunits, however, derive from different genes encoding distinct proteins. Past and recent research indicates synthesis and release of the two hormones are subject to extensive and independent regulation. LH appears to be secreted predominantly via the regulated secretory pathway, whereas FSH release is largely constitutive. As such, investigations of FSH-beta subunit synthesis may lend direct insight into mechanisms underlying patterns of secreted FSH, more so than investigations of the LHbeta subunit. Here, we review recent investigations of transcriptional regulation of the FSH-beta subunit gene from different mammalian species, including humans. The results reveal both conserved and species-specific regulatory mechanisms that might contribute to inter-species variation in FSH release.

Cells Expressing RFamide-Related Peptide-1/3, the Mammalian Gonadotropin-Inhibitory Hormone Orthologs, Are Not Hypophysiotropic Neuroendocrine Neurons in the Rat

An RFamide peptide named gonadotropin-inhibitory hormone, which directly inhibits gonadotropin synthesis and secretion from the anterior pituitary gland, has recently been discovered in the avian hypothalamus. It is not known whether the mammalian orthologs of gonadotropin-inhibitory hormone and RFamide-related peptide (RFRP)-1 and -3 act in the same way. We used a newly generated antibody against the rat RFRP precursor combined with retrograde tract tracing to characterize the cell body distribution and fiber projections of RFRP-1 and -3 neurons in rats. RFRP-1/3-immunoreactive cell bodies were found exclusively within the dorsomedial hypothalamus. Immunoreactive fibers were observed in the septal-preoptic area, hypothalamus, midbrain, brainstem, and hippocampus but not in the external zone of the median eminence. Intraperitoneal injection of the retrograde tracer Fluoro-Gold in rats resulted in the labeling of the majority of GnRH neurons but essentially no RFRP-1/3 neurons. In contrast, intracerebral injections of Fluoro-Gold into the rostral preoptic area and CA2/CA3 hippocampus resulted in the labeling of 75 +/- 5% and 21 +/- 8% of RFRP-1/3 cell bodies, respectively. To assess actions at the pituitary in vivo, RFRP-3 was administered as an iv bolus to ovariectomized rats and plasma LH concentration measured at 0, 2.5, 5, 10, and 30 min. RFRP-3 had no effects on basal secretion, but GnRH-stimulated LH release was reduced by about 25% at 5 min. Together these observations suggest that RFRP-3 is not a hypophysiotropic neuroendocrine hormone in rats.

Effects of non-steroidal gonadal factors on LH secretion in female common carp during the reproductive cycle

The aim of this study was to evaluate the effects of recombinant human inhibin A, recombinant human activin A and desteroidized ovarian extract on LH secretion &lt;I&gt;in vitro&lt;/I&gt; and &lt;I&gt;in vivo&lt;/I&gt; in female common carp during different stages of reproductive cycle. Inhibin stimulated spontaneous as well as GnRH-stimulated LH release &lt;I&gt;in vivo&lt;/I&gt; in fish during gonadal recrudescence. This hormone did not have an influence on spontaneous LH secretion in the periovulatory period, but had a slightly inhibitory effect on GnRH-stimulated LH release in this stage of gonad maturity. Activin decreased spontaneous LH secretion during gonadal recrudescence and increased LH secretion before ovulation, having no effects on GnRH-stimulated LH release during both stages of gonad maturity. The desteroidized ovarian extract failed to modify spontaneous LH secretion, but decreased GnRH-stimulated LH release during recrudescence and especially before ovulation. It is to conclude that these data suggest the differential role of inhibin/activin as substances in the regulation of LH secretion in common carp females.

下垂体前葉におけるアネキシン５（AX5）の性周期に伴う変化と黄体形成ホルモン（LH）放出への関与

下垂体前葉におけるアネキシン５（AX5）の性周期に伴う変化と黄体形成ホルモン（LH）放出への関与

Insulin augments GnRH-stimulated LHβ gene expression by Egr-1

Previous studies have shown that insulin augments GnRH-stimulated LH synthesis and release from primary gonadotrophs. In this study, regulation of LHβ gene expression by GnRH and insulin was examined in LβT2 cells. Endogenous LHβ mRNA is stimulated 2.4-fold by insulin alone, 2.6-fold by GnRH alone, and 4.7-fold by insulin together with GnRH. This effect of insulin, like GnRH, mapped to sequences −140 to +1 in the mouse LHβ gene. Insulin together with GnRH stimulates activity of an LHβ-reporter gene 7.1-fold; whereas, GnRH alone or insulin alone stimulates the reporter activity 2.8- and 3.1-fold, respectively. Blocking the binding of Egr-1 to sequences −51 to −42 in the LHβ gene inhibits effects of insulin and GnRH. Insulin together with GnRH increases Egr-1 mRNA levels and total Egr-1 binding to LHβ DNA. These findings indicate that insulin may impact regulation of the reproductive axis at the level of the pituitary.

Dopamine inhibits luteinizing hormone synthesis and release in the juvenile European eel: a neuroendocrine lock for the onset of puberty.

In various adult teleost fishes, LH ovulatory peak is under a dual neurohormonal control that is stimulatory by GnRH and inhibitory by dopamine (DA). We investigated whether DA could also be involved in the inhibitory control of LH at earlier steps of gametogenesis by studying the model of the European eel, Anguilla anguilla, which remains at a prepubertal stage until the oceanic reproductive migration. According to a protocol previously developed in the striped bass, eels received sustained treatments with GnRH agonist (GnRHa), DA-receptor antagonist (pimozide), and testosterone (T) either alone or in combination. Only the triple treatment with T, GnRHa, and pimozide could trigger dramatic increases in LH synthesis and release as well as in plasma vitellogenin levels and a stimulation of ovarian vitellogenesis. Thus, in the prepubertal eel, removal of DA inhibition is required for triggering GnRH-stimulated LH synthesis and release as well as ovarian development. To locate the anatomical support for DA inhibition, the distribution of tyrosine hydroxylase (TH) in the brain and pituitary was studied by immunocytochemistry. Numerous TH-immunoreactive cell bodies were observed in the preoptic anteroventral nucleus, with a dense tract of immunoreactive fibers reaching the pituitary proximal pars distalis, where the gonadotrophs are located. This pathway corresponds to that mediating the inhibition of LH and ovulation in adult teleosts. To our knowledge, this is the first demonstration of a pivotal role for DA in the control of LH and puberty in a juvenile teleost. These data support the view that DA inhibition on LH secretion is an ancient evolutionary component in the neuroendocrine regulation of reproduction that may have been partially maintained throughout vertebrate evolution.

Temperature effects along the reproductive axis during spawning induction of grass carp ( Ctenopharyngodon idella)

For grass carp ( Ctenopharyngodon idella) raised in the Ivory Coast (with water temperatures of 26–31 °C), induced spawning is obligatory for fry production. However, ovulation rates following hormonal treatment are often low. We hypothesized that high temperatures are an inhibiting factor for the reproductive axis (brain–pituitary–gonad) in these conditions. By in vivo and in vitro experiments, we tried to determine the thermosensitive steps during spawning induction. We compared gonadotropin and maturation-inducing steroid (MIS) profiles during a spawning induction at controlled temperatures of 24 and 28 °C in relation to ovulation success. We performed pituitary cell cultures and ovarian fragment incubations at controlled temperatures. The ovulation rate was lower at 28 °C (10%) than at 24 °C (36%). At the pituitary level, we found only minor thermal impacts on GnRH-stimulated LH release, but our data suggest an increase of the dopaminergic inhibition by high temperatures. The main effects were found at the ovary level, where ovary responsiveness to gonadotropin by MIS synthesis was disturbed, as well as oocyte responsiveness to MIS triggering final maturation, and probably ovulation. These results show the importance of regulating temperature during spawning induction to ensure a high rate of ovulation.

Characterization of the inhibitory dopamine receptor from the pituitary of tilapia

The study was aimed to characterize the inhibitory dopamine (DA) receptor in the pituitary of tilapia. DA D2 and D3 agonists inhibited GnRH-stimulated LH release from tilapia pituitary cells more efficiently than D4 agonists. However, no significant differences were observed between the efficiencies of D2 and D3 drugs. In order to elucidate the signal transduction of the tilapia DA receptor we performed transfection of COS-7 cells with tilapia D2 DA receptor that resulted in the inhibition of forskolin-stimulated cAMP accumulation. Moreover, the tilapia D2 dopamine receptor inhibits adenylate cyclase activity via coupling to pertussis toxin-sensitive G proteins of the Gi/Go family. Our results also show that estradiol up-regulates taDA-R mRNA.

Interactions of insulin-like growth factor-I, insulin and estradiol with GnRH-stimulated luteinizing hormone release from female rat gonadotrophs.

It is well established that ovarian steroids modulate gonadotropin secretion from anterior pituitary cells. It has been speculated that insulin and IGF-I might influence gonadotropin secretion. To investigate the effects of IGF-I and estradiol alone, or combinations of IGF-I with insulin and estradiol on GnRH-stimulated LH release from female rat pituitary cells in serum-supplemented and serum-free culture conditions. Pituitary cells were incubated for 24 h or 48 h with a series of increasing concentrations of IGF-I or estradiol and stimulated with 1 nmol/l GnRH for 3 h. To determine the interaction of IGF-I and estradiol on GnRH-stimulated LH secretion, cells were exposed to increasing concentrations of IGF-I and 100 pmol/l estradiol for 24 h. We also investigated the effects of combined treatment with IGF-I and insulin on GnRH-stimulated LH secretion. Our findings indicate that long-term IGF-I treatment (24 h) alone has a significant augmenting effect on GnRH-stimulated LH release in serum-free medium only, with a maximum at low concentrations (10 and 100 pmol/l). Estradiol significantly increased GnRH-induced LH release in a dose-dependent manner. The extent of GnRH-stimulated LH secretion by long-term estradiol treatment (24 h) was significantly greater in serum-supplemented (+42%) medium than in serum-free medium. Estradiol facilitated IGF-I-primed LH responses to GnRH in serum-free medium. In contrast, in serum-supplemented medium, the facilitating potential of estradiol was lower. We also found that, in GnRH-stimulated cells, LH release was augmented by insulin treatment, in contrast to quiescent cells that had been pretreated with 100 pmol/l IGF-I alone and 1 nmol/l insulin alone. IGF-I and to a lesser extent insulin stimulate GnRH-induced LH secretion from pituitary gonadotrophs. This action is enhanced by estradiol treatment of the cells. However, the well known stimulatory action of estradiol on LH secretion is dependent on the presence of growth factors.

Regulation of G(q/11)alpha by the gonadotropin-releasing hormone receptor.

Evidence from use of pertussis and cholera toxins and from NaF suggested the involvement of G proteins in GnRH regulation of gonadotrope function. We have used three different methods to assess GnRH receptor regulation of G(q/11)alpha subunits (G(q/11)alpha). First, we used GnRH-stimulated palmitoylation of G(q/11)alpha to identify their involvement in GnRH receptor-mediated signal transduction. Dispersed rat pituitary cell cultures were labeled with [9,10-(3)H(N)]-palmitic acid and immunoprecipitated with rabbit polyclonal antiserum made against the C-terminal sequence of G(q/11)alpha. The immunoprecipitates were resolved by 10% SDS-PAGE and quantified. Treatment with GnRH resulted in time-dependent (0-120 min) labeling of G(q/11)alpha. GnRH (10(-12), 10(-10), 10(-8), or 10(-6) g/ml) for 40 min resulted in dose-dependent labeling of G(q/11)alpha compared with controls. Cholera toxin (5 microg/ml; activator of G(i)alpha), pertussis toxin (100 ng/ml; inhibitor of G(i)alpha actions) and Antide (50 nM; GnRH antagonist) did not stimulate palmitoylation of G(q/11)alpha above basal levels. However, phorbol myristic acid (100 ng/ml; protein kinase C activator) stimulated the palmitoylation of G(q/11)alpha above basal levels, but not to the same extent as 10(-6) g/ml GnRH. Second, we used the ability of the third intracellular loop (3i) of other seven-transmembrane segment receptors that couple to specific G proteins to antagonize GnRH receptor-stimulated signal transduction and therefore act as an intracellular inhibitor. Because the third intracellular loop of alpha1B-adrenergic receptor (alpha1B 3i) couples to G(q/11)alpha, it can inhibit G(q/11)alpha-mediated stimulation of inositol phosphate (IP) turnover by interfering with receptor coupling to G(q/11)alpha. Transfection (efficiency 5-7%) with alpha1B 3i cDNA, but not the third intracellular loop of M1-acetylcholine receptor (which also couples to G(q/11)alpha), resulted in 10-12% inhibition of maximal GnRH-evoked IP turnover, as compared with vector-transfected GnRH-stimulated IP turnover. The third intracellular loop of alpha2A adrenergic receptor, M2-acetylcholine receptor (both couple to G(i)alpha), and D1A-receptor (couples to G(s)alpha) did not inhibit IP turnover significantly compared with control values. GnRH-stimulated LH release was not affected by the expression of these peptides. Third, we assessed GnRH receptor regulation of G(q/11)alpha in a PRL-secreting adenoma cell line (GGH(3)1') expressing the GnRH receptor. Stimulation of GGH(3)1' cells with 0.1 microg/ml Buserelin (a metabolically stable GnRH agonist) resulted in a 15-20% decrease in total G(q/11)alpha at 24 h following agonist treatment compared with control levels; this action of the agonist was blocked by GnRH antagonist, Antide (10(-6) g/ml). Neither Antide (10(-6) g/ml, 24 h) alone nor phorbol myristic acid (0.33-100 ng/ml, 24 h) mimicked the action of GnRH agonist on the loss of G(q/11)alpha immunoreactivity. The loss of G(q/11)alpha immunoreactivity was not due to an effect of Buserelin on cell-doubling times. These studies provide the first direct evidence for regulation of G(q/11)alpha by the GnRH receptor in primary pituitary cultures and in GGH3 cells.

Oxytocin augmentation of gonadotropin-releasing hormone-stimulated luteinizing hormone release is modulated by cyclic AMP

Oxytocin has been previously shown to augment GnRH-stimulated LH release. However it is currently unknown which intracellular mediators participate in the process. In this study, after preincubation with oxytocin for 3 hours, quartered pituitaries were stimulated for 15 minutes with GnRH. The effects of diBucAMP, a cell permeable analog of cAMP, and DDA, an adenyl cyclase inhibitor, on the augmentation by oxytocin were investigated. Although addition of diBucAMP increased GnRH-stimulated LH release, it inhibited the augmentation by oxytocin of the response to GnRH. On the other hand addition of DDA induced an increased augmentation by oxytocin. These results indicate that intracellular cAMP inhibits the augmentory activity of oxytocin, and suggest that oxytocin modulation of GnRH action in vivo would be optimal when the hormonal milieu results in reduced levels of cAMP.

Ca2+ entry in gonadotrophs and αT3–1 cells: does store-dependent Ca2+ influx mediate gonadotrophin-releasing hormone action?

In pituitary gonadotrophs GnRH causes biphasic (spike and plateau) increases in cytosolic Ca2+ ([Ca2+]i) and gonadotrophin release. The spike phases reflect mobilization of stored Ca2+ and the plateau responses are attributed, in part, to Ca2+ influx via voltage-sensitive Ca2+ channels. In recent years, store-dependent Ca2+ influx (SDCI), in which depletion of the intracellular inositol 1,4,5-trisphosphate-mobilizable pool stimulates Ca2+ influx, has emerged as a major form of Ca2+ entry activated by phosphoinositidase C-coupled receptors in non-excitable cells. More recent evidence also indicates a role for SDCI in excitable cells. We have used dynamic video imaging of [Ca2+]i in alpha T3-1 cells (a gonadotroph-derived cell line) and manipulation of the filling state of the GnRH-mobilizable Ca2+ pool to test the possible role of SDCI in GnRH action. In Ca(2+)-containing medium, GnRH caused a biphasic increase in [Ca2+]i whereas in Ca(2+)-free medium only a transient increase occurred. The response to a second stimulation with GnRH in Ca(2+)-free medium was reduced by > 95% (demonstrating that Ca2+ pool depletion had occurred) and was recovered after brief exposure to Ca(2+)-containing medium (which enables refilling of the pool). Ionomycin (a Ca2+ ionophore) and thapsigargin (which inhibits the Ca(2+)-sequestering ATPase of the endoplasmic reticulum) also transiently increased [Ca2+]i in Ca(2+)-free medium and depleted the GnRH-mobilizable pool as indicated by greatly reduced subsequent responses to GnRH. Pool depletion also occurs on stimulation with GnRH in Ca(2+)-containing medium because addition of ionomycin and Ca(2+)-free medium during the plateau phase of the GnRH response caused only a reduction in [Ca2+]i rather than the transient increase seen without GnRH. To deplete intracellular Ca2+ pools, cells were pretreated in Ca(2+)-free medium with thapsigargin or GnRH and then, after extensive washing, returned to Ca(2+)-containing medium. Pretreatment with thapsigargin augmented the increase in [Ca2+]i seen on return to Ca(2+)-containing medium (to two- to threefold higher than that seen in control cells) indicating the activation of SDCI, whereas pool depletion by GnRH pretreatment had no such effect. To ensure maintained pool depletion after Ca2+ re-addition, similar studies were performed in which the thapsigargin and GnRH treatments were not washed off, but were retained through the period of return to Ca(2+)-containing medium. Return of GnRH-treated cells to Ca(2+)-containing medium caused an increase in [Ca2+]i which was inhibited by nicardipine, whereas the increase seen on return of thapsigargin-treated cells to Ca(2+)-containing medium was not reduced by nicardipine. The quench of fura-2 fluorescence by MnCl2 (used as a reporter of Ca2+ influx) was increased by GnRH and thapsigargin, indicating that both stimulate Ca2+ influx via Mn2+ permeant channels. The GnRH effect was abolished by nicardipine whereas that of thapsigargin was not. Finally, depletion of intracellular Ca2+ pools by pretreatment of superfused rat pituitary cells with GnRH or thapsigargin in Ca(2+)-free medium did not enhance LH release on return to Ca(2+)-containing medium. The results indicate that (a) thapsigargin stimulates SDCI in alpha T3-1 cells via nicardipine-insensitive Ca2+ channels, (b) in spite of the fact that GnRH depletes the hormone-mobilizable Ca2+ pool, it fails to stimulate SDCI, (c) GnRH stimulates Ca2+ entry predominantly via nicardipine-sensitive channels, a route not activated by SDCI and (d) in rat gonadotrophs, GnRH-stimulated LH release is not mediated by SDCI.

Alterations in pulsatile luteinizing hormone and follicle-stimulating hormone secretion in idiopathic oligoasthenospermic men: assessment by deconvolution analysis--a clinical research center study.

To investigate the nature of neuroendocrine disturbances of the hypothalamo-pituitary-gonadal axis in idiopathic male infertility, we studied 14 infertile men with oligoasthenozoospermia (OLIGO) and 15 age-, body mass index-, and community-matched euspermic controls by blood withdrawal at 10-min intervals for 12 h to encompass basal (8-h) and exogenous GnRH-stimulated (4-h) pulsatile release of LH and FSH (by immunoradiometric assay) as well as testosterone (by RIA). Deconvolution analysis was used to estimate endogenous LH and FSH half-lives, secretory burst frequency, amplitude, duration, and mass. OLIGO men exhibited normal serum concentrations of total, free, and percent dialyzable testosterone and estradiol, but distinct dynamic alterations within the LH and FSH axes; namely (P < 0.05), 1) a prolonged half-life of LH (OLIGO, 95 +/- 19 min; control, 80 +/- 9.3 min) and a reduced half-life of FSH (OLIGO, 260 +/- 79 min; control, 320 +/- 93 min); 2) a low LH, but normal FSH, secretory burst frequency (OLIGO, 12 +/- 3.4; control, 15 +/- 3.0 LH pulses/day); 3) a decreased serum testosterone peak frequency (OLIGO, 16 +/- 4.3; control, 21 +/- 3.2 peaks/day); and 4) an amplified mass of LH (1.1- to 1.3-fold higher in OLIGO) and FSH (2.4- to 2.7-fold higher in OLIGO) secreted per burst basally as well as after GnRH injection. These disturbances were readily distinguishable from the neuroendocrine dysregulation described in other states of male hypogonadotropism (e.g. uremia, fasting, and aging).

Inhibition of ovulation by progestin analogs (agonists vs antagonists): Preliminary evidence for different sites and mechanisms of actions

Continuous administration of the antiprogesterone RU486 inhibits ovulation in women and in monkeys; in this regard RU486 may act as a progestin agonist rather than as an antagonist. We compared the site(s) and mechanism(s) of RU486-induced ovulation inhibition with those of levonorgestrel (LNG). Six regularly menstruating cynomolgus monkeys each received placebo, RU486 (1 mg/kg/d) or LNG (2 g/kg/d) im between days (cd) 2–22 of three separate menstrual cycles. Serum levels of estradiol (E 2), progesterone (P 4), androstenedione, LH and FSH were analyzed by RIAs in daily blood samples. Basal and GnRH-stimulated (1 and 50 g of GnRH iv 2 h apart) secretion of LH and FSH was assessed using serial blood samples collected for 12 h on cd 10. Mean cycle length was prolonged by RU486 and LNG treatments from 32 d to 70 d and 52 d, respectively (p < 0.02). Ovulation was inhibited in five of the six primates during RU486, and in all six during LNG treatment. During RU486 treatment, serum E 2 levels were similar to those of the control cycle; despite peaks of E 2 secretion, no LH peaks were seen. In contrast, E 2 concentrations were profoundly suppressed during LNG treatment (p < 0.005). The reduction in serum E 2 was accompanied by lower levels of androstenedione, and suppressed ratio of E 2/androstenedione (p < 0.02) suggesting both reduced synthesis and aromatization of androgen precursors during administration of LNG. Consequently, LNG treatment was associated with higher levels of serum FSH and LH (p < 0.0001; 1-way ANOVA). Similarly, as during the luteal phase of the menstrual cycle, the amplitude of basal LH-pulses was increased during LNG treatment (p < 0.05), whereas RU486 treatment did not affect basal LH secretion. The GnRH-stimulated release of LH was similar during the placebo, RU486 and LNG cycles; enhanced release of FSH was seen during administration of LNG. Thus, in the present model system, RU486 seems to inhibit ovulation mainly at the level of hypothalamus, possibly by interfering with the steroidal positive feedback signals from the ovary. However, LNG inhibits ovulation differently, most likely via direct progesterone-like effects on folliculogenesis and the hypothalamus. The pituitary does not appear to be the major site of action(s) of RU486 or LNG. Thus, the differential mechanisms of ovulation inhibition by RU486 and LNG seem to result from lesser intraovarian impact of RU486 as well as dissimilar influences on tonic gonadotropin secretory levels. We conclude that when inhibiting ovulation, RU486 does not act as a progestin agonist, but rather, functions through a hypothalamic mechanism(s), which might be unique to RU486 as a progesterone antagonist.

Altered regulation of pituitary luteinizing hormone secretion by GnRH and inhibin in the aged persistent-estrous female rat

The physiologic regulation of pituitary LH secretion is under the direct control of hypothalamic GnRH as well as feedback control via ovarian steroid and peptide hormones. Changes within the hypothalamic-pituitary-ovarian axis during reproductive aging lead to the gradual attenuation and the eventual loss of cyclic LH release that is seen in the old persistent-estrous rat. In the following studies, we determined whether changes in the pituitary response to regulation by hypothalamic GnRH and/or ovarian inhibin could be involved in age-related deficits in LH release by examining the direct effect(s) of GnRH and inhibin on LH release in old persistent-estrous (PE) and young female rats using an in vitro pituitary cell culture system. Both pituitary LH content and LH release were significantly lower in PE pituitary cells compared to young pituitary cells. GnRH (10 ng/ml) significantly increased LH release above basal levels in young and PE pituitaries. However, the magnitude of LH release was significantly lower in PE pituitaries compared to young pituitaries. Treatment with recombinant inhibin (0.1, 1, or 5 ng/ml) did not significantly affect basal or GnRH-stimulated LH release from young rat pituitaries under our culture conditions. In contrast, there were significant decreases in the levels of both basal LH and GnRH-stimulated LH release from PE pituitaries after treatment with 1 or 5 ng/ml human recombinant inhibin, as well as with inhibin plus 10 ng/ml GnRH, respectively. These results suggest that there are significant changes in the pituitary response to GnRH and ovarian inhibin regulation that may lead to significant alterations in LH secretion during aging.