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Glucocorticoids have a strong influence on growth and maturation of fetal organ systems, but overexposure to exogenous glucocorticoids may retard fetal growth and alter developmental processes in sensitive tissues. The aim of this study was to specifically determine whether prenatal exposure to dexamethasone (Dx) altered normal development and function of pituitary gonadotropic cells in neonatal, infant and peripubertal female offspring. On day 16 of pregnancy, rat dams received 1.0 mg Dx/kg body weight (BW) s.c., followed by 0.5 mg Dx/kg BW on days 17 and 18 of gestation. Control gravid females received the same volume of saline. Female offspring were sacrificed on days 5, 16 and 38 after delivery. The volume of the pituitary gland estimated using Cavalieri's principle was significantly reduced (p < 0.05). Using a fractionator-physical disector method, we found reduced total numbers of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) cells (p < 0.05), accompanied by a decrease (p < 0.05) in serum concentrations of FSH and LH, while the relative intensity of FSH and LH immunofluorescence remained unchanged in neonatal, infant and peripubertal female offspring prenatally exposed to Dx. The data document that overexposure to Dx during fetal development evokes developmental programming of the female reproductive system at the pituitary cellular level, which may be associated with impaired reproductive function. (C) 2016 S. Karger AG, Basel Ministry of Education, Science and Technological Development of the Republic of Serbia {[}173009]; Laviefarm d.o.o., Belgrade, Serbia; Promedia d.o.o., Kikinda, Serbia

Pulsatile secretion of hypothalamic gonadotropin-releasing hormone (GnRH) is indispensable for controlling proper pituitary gonadotrope functions; however, the mechanism underlying GnRH pulse generation remains largely unknown. It is important to understand the cellular oscillator in individual GnRH neurons and temporal synchronization among GnRH neurons. In this brief review, we summarize our recent findings on episodic GnRH gene transcription at the single GnRH neuron level and in synchronized multicellular burst in relation to the temporal pattern of GnRH secretion. We also detail the effects of kisspeptin on ultradian rhythmic GnRH gene transcription and secretion. We extend our discussion to the hierarchical interaction between circadian and ultradian rhythms. Taken together, the current review elucidates the genomic control of GnRH pulse generation in hypothalamic neurons.

Context: Early pubertal luteinizing hormone (LH), and by inference gonadotropin-releasing hormone (GnRH), pulse secretion is marked by high nocturnal but low daytime frequency; however, the underlying mechanisms remain unclear. Plasma concentrations of progesterone, the major regulator of GnRH frequency in women, increase in the early morning in early pubertal girls and may help slow daytime GnRH frequency. Objective: To evaluate the effect of progesterone on LH pulse frequency in early to mid-pubertal girls. Design: Controlled interventional study. Setting: General clinical research center. Participants: Eighteen non-obese, non-hyperandrogenemic Tanner 1–3 girls. Intervention: Twelve-hour (19:00–07:00 h) blood sampling with or without oral progesterone administration (25–50 mg at 16:00 and 20:00 h). Main Outcome Measure: LH pulse frequency. Results: Girls receiving progesterone (n = 5) exhibited lower 12-hour LH pulse frequency than controls (n = 13), but this difference was not statistically significant (average interpulse intervals 196.0 ± 61.9 and 160.4 ± 67.1 min, respectively; p = 0.2793). In contrast to controls, however, girls receiving progesterone exhibited no LH pulses during waking hours (19:00–23:00 h; estimated interpulse interval 326.0 ± 52.7 vs. 212.0 ± 120.9 min; p = 0.0376), while nighttime (23:00–07:00 h) interpulse intervals were similar (174.8 ± 62.0 vs. 167.5 ± 76.9 min, respectively; p = 0.7750). Conclusions: Exogenous progesterone acutely suppressed daytime, but not nocturnal, LH pulse frequency in early to mid-pubertal girls, suggesting that GnRH pulse frequency is differentially regulated by progesterone depending on sleep status.

Passive immunization against inhibin with an anti-inhibin serum (AIS) during the diestrous phase in cycling rats increased follicle-stimulating hormone secretion, stimulated the ovaries and reduced the magnitude of the luteinizing hormone (LH) surge in the afternoon of proestrus. The involvement of gonadotrope progesterone receptor (PR) expression/action in the inhibitory effects of the follicle-stimulating hormone-dependent putative ovarian factor gonadotropin surge-attenuating factor on preovulatory LH secretion was studied in the absence of circulating free inhibin. Proestrous pituitaries from rats injected with AIS or a non-immune serum (NIS) were studied for determination of PR-AB and PR-B mRNAs by RT-PCR and PR-B and PR-A isoform proteins by Western blot. In addition, pituitaries from AIS- and NIS-injected rats were incubated and studied for PR-dependent LH secretion parameters: LH-releasing hormone (LHRH)-stimulated LH secretion, progesterone-potentiated LHRH-stimulated LH secretion and LHRH self-priming. Also, the effects of the antiprogestagen RU486 on these LH secretion parameters were evaluated and compared with those of AIS. Finally, gonadotrope PR phosphorylation was evaluated by immunohistochemistry. Results showed that the hyperstimulated ovaries of AIS-injected rats produce a factor, different from inhibin, that blocked LHRH self-priming and P-potentiation of LHRH-stimulated LH secretion. These effects were not due to decreased pituitary PR mRNAs, PR protein expression or PR protein B/A ratio. The inhibitory effect of AIS on PR-dependent LH secretion seemed to be due to gonadotrope PR dephosphorylation. Taken together, the findings indicated that the putative gonadotropin surge-attenuating factor affected LH surge through an inhibition of PR phosphorylation/action but not PR expression.

Women harbouring inactivating mutations in luteinizing hormone (LH) beta subunit (LHB) or LH receptor (LHCGR) genes have similar clinical manifestations characterized by female external genitalia, spontaneous breast and pubic hair development at puberty, and normal or late menarche followed by oligo-amenorrhea and infertility. Oestradiol and progesterone levels are normal for the early to midfollicular phase, but do not reach ovulatory or luteal phase levels, confirming lack of ovulation. Notably, serum LH levels are low in patients with LHB mutations and high in those with LHCGR mutations, whereas follicle-stimulating hormone levels are normal or only slightly increased. Pelvic ultrasound has demonstrated a small or normal uterus and normal or enlarged ovaries with cysts. Women with LHB mutations may be treated with hCG (human chorionic gonadotropin) or LH, whereas those with mutations in LHCGR are resistant. Lhb and Lhcgr knockout female mice are close phenocopies of the respective human mutations, and confirm that early follicular development, low levels of oestrogen production and theca cell development are independent of LH action, which is necessary for ovulation. Although inactivating mutations in LHB and LHCGR are rare in comparison to other genetic and non-genetic causes of hypogonadism, they should be considered in the differential diagnosis of oligo-amenorrhea and infertility.

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) play key roles in vertebrate gametogenesis and steroidogenesis. They are mainly synthesized in the pituitary gland. While investigating the ontogeny of FSH and LH cells in the cichlid fish Cichlasoma dimerus by immunohistochemistry (IHC), we unexpectedly found immunoreactive neurons in the preoptic area, sending their projections through different brain areas and neurohypophysis. Our previous work using Western blot and IHC techniques applied to the adult brain confirmed these findings. To further demonstrate the extrapituitary expression of these hormones, we performed RT-PCR detecting sequences coding for β-FSH and β-LH subunits in the C. dimerus pituitary and brain (preoptic-hypothalamic area). The expression of these transcripts in both organs was consistent with their peptide expression showing a high sequence homology when compared with other phylogenetically related fish. An individual pituitary in vitro culture system was utilized to study the possible modulatory effect of brain-derived gonadotropins on pituitary hormone secretion. Pituitary explants were cultured with different concentrations of LH or FSH, and the culture media were analyzed by Western blot. Exogenous LH produced a dose-dependent increase in pituitary β-LH, β-FSH and somatolactin (SL) releases. No effect was observed on growth hormone (GH). The effect on prolactin (PRL) was not consistent among treatments. Exogenous FSH produced an inhibition in β-LH release, dose-dependent increases in β-FSH and SL releases, and no effect on PRL and GH releases. These findings support the concept of regulation of pituitary trophic hormones by brain-derived gonadotropins.

We have shown that cortisol infusion reduced the luteinizing hormone (LH) response to fixed hourly GnRH injections in ovariectomized ewes treated with estradiol during the non-breeding season (pituitary-clamp model). In contrast, cortisol did not affect the response to 2 hourly invariant GnRH injections in hypothalamo-pituitary disconnected ovariectomized ewes during the breeding season. To understand the differing results in these animal models and to determine if cortisol can act directly at the pituitary to suppress responsiveness to GnRH, we investigated the importance of the frequency of GnRH stimulus, the presence of estradiol and stage of the circannual breeding season. In experiment 1, during the non-breeding season, ovariectomized ewes were treated with estradiol, and pulsatile LH secretion was restored with i.v. GnRH injections either hourly or 2 hourly in the presence or absence of exogenous cortisol. Experiments 2 and 3 were conducted in hypothalamo-pituitary disconnected ovariectomized ewes in which GnRH was injected i.v. every 2 h. Experiment 2 was conducted during the non-breeding season and saline or cortisol was infused for 30 h in a cross-over design. Experiment 3 was conducted during the non-breeding and breeding seasons and saline or cortisol was infused for 30 h in the absence and presence of estradiol using a cross-over design. Samples were taken from all animals to measure plasma LH. LH pulse amplitude was reduced by cortisol in the pituitary clamp model with no difference between the hourly and 2-hourly GnRH pulse mode. In the absence of estradiol, there was no effect of cortisol on LH pulse amplitude in GnRH-replaced ovariectomized hypothalamo-pituitary disconnected ewes in either season. The LH pulse amplitude was reduced in both seasons in experiment 3 when cortisol was infused during estradiol treatment. We conclude that the ability of cortisol to reduce LH secretion does not depend upon the frequency of GnRH stimulus and that estradiol enables cortisol to act directly on the pituitary of ovariectomized hypothalamo-pituitary disconnected ewes to suppress the responsiveness to GnRH; this effect occurs in the breeding and non-breeding seasons.

Background: A number of peptides regulate luteinizing hormone (LH) release. In some cases, these peptides exert an effect at the pituitary, to directly regulate LH release and/or to modulate the effects of gonadotropin-releasing hormone (GnRH). A link between nutrition and reproductive function is well established. In this study we investigated the effects of two peptides associated with appetite control, galanin and leptin, on the regulation of LH release from the pituitary. Methods: Using perifused anterior pituitary tissue from freely fed rats, we investigated the effect of galanin on basal LH release and GnRH-stimulated LH release from pituitaries in a high-estrogen (proestrus) and a low-estrogen (metestrus) environment. In addition, we examined the effect of galanin on LH release following GnRH self-priming. The effect of inhibiting protein synthesis, with cycloheximide, was also studied under these conditions. Finally, the effects of leptin alone and on a galanin-modulated LH response were investigated. Results: There was no detectable effect of leptin, either alone or in conjunction with galanin. We observed that galanin enhanced GnRH stimulation of LH secretion, an effect that was dependent on protein synthesis and on an estrogenized environment. In addition, importantly, galanin also enhanced the LH response in GnRH self-primed pulses. Conclusion: Our results provide further details on the effect of galanin on the LH surge, particularly the effect on the response to pulsatile GnRH and the effect of protein production, and thereby indicate a means by which appetite- and nutrition-related peptides may act on the ovulatory cycle at the pituitary.

Several hypotheses have been proposed about the pathogenesis of polycystic ovarian syndrome (PCOS), however, the fundamental physiological interactions that initiate the development of follicular cysts have not yet been elucidated. Hence, in this study the proliferation, density and population of gonadotrophs, mammotrophs, somatotrophs and corticotrophs of the pituitary glands of rats with induced follicular cysts have been investigated by 2 experimental models (continuous light exposition and estradiol valerate-treated rats). Specific immunoreactivity associated with follicle-stimulating hormone, luteinizing hormone, prolactin, growth hormone and adrenocorticotropic hormone was evaluated by immunohistochemistry with specific hormone antibodies and proliferation of secretory cells by their colocalization (double-labeling) with proliferating cell nuclear antigen. The results indicate a reduction in the density and proliferation of gonadotrophs in both experimental groups. A reduction in the average density, proliferation and population of lactotrophs and corticotrophs was also observed in estradiol valerate-treated animals. However, no significant differences were found in somatotrophs. The present study contributes to the information about alterations of some cell populations that occur in the pituitary gland of rats with polycystic ovaries, and will enhance our understanding of the pathogenesis of this disease. Fil: Francou, María Manuela. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Departamento de Ciencias Morfológicas. Laboratorio de Biología Celular y Molecular; Argentina Fil: Durdos, Mauro. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Departamento de Ciencias Morfológicas. Laboratorio de Biología Celular y Molecular; Argentina Fil: Salvetti, Natalia Raquel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; Argentina Fil: Baravalle, Celina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; Argentina Fil: Rey, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; Argentina Fil: Ortega, Hugo Hector. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; Argentina

Gonadotropin-releasing hormone (GnRH) receptor activation has been demonstrated to inhibit cell proliferation in vitro and in vivo. These effects are dependent on the degree of receptor expression and the intracellular signaling protein milieu. The physiological and pathophysiological relevance is largely undefined, and its potential for exploitation in the treatment of specific malignancies is the subject of ongoing investigations. GnRH receptors are expressed in embryonic, juvenile and adult tissues, including brain, pituitary, gonads, accessory reproductive organs and placenta. The levels of receptor expression vary, from high in pituitary gonadotropes to low in peripheral tissues, although quantification of functional receptor protein has been determined in relatively few cell types. Roles for GnRH receptor signaling at different stages of animal development and its influence on reproductive health remain largely unexplored, except in cases of hereditary hypogonadal infertility. In addition to regulating hormone secretion, GnRH is postulated to act as a chemokine or a growth- and differentiation-inducing factor. Hence, receptor activation may influence the function of neuronal networks in the brain and the maturation of reproductive tissue epithelia. GnRH may also potentially influence the biology of cancerous cells in reproductive tissue since receptor activation may signal terminal differentiation, cell cycle arrest or apoptosis. In this context, the cell surface expression of GnRH receptor is important since it influences the intensity of intracellular signaling, and correlates with the ability to inhibit proliferation in transformed cells in vitro. Here, we review data on the effects of GnRH agonists on cell proliferation and apoptosis, and put forward hypotheses for investigation to determine whether the GnRH receptor acts as a tumor suppressor in neuroendocrine or epithelial cells.

Background: The combined growth hormone-releasing hormone and growth hormone-releasing peptide-6 (GHRH + GHRP-6) test is most potent in evaluating GH secretion. Aims: To assess its capability in children with GH deficiency and low spontaneous GH secretion (GH neurosecretory dysfunction). Methods: 35 children with GH Results: Six were nonresponders to the combined test, with significantly lower peak GH 20.7 (7.8–31.8) than C and the rest of the patients (responders). Peak GH was similar between prepubertal (PP) controls 167 ± 88, GHD 202 ± 110 and GHND 155 ± 83. Pubertal (P) controls had higher peak GH 328 ± 149 than P-GHD 203 ± 105 and P-GHND 186 ± 105. While P-C had higher peak GH than PP-C, PP and P children had similar responses within the GHD and GHND groups. Conclusions: The GHRH + GHRP-6 test detects children with severe GH insufficiency. Patients with GHD respond similarly to those with GHND, indicating a possible hypothalamic GH neuroregulatory dysfunction in GHD. Responders to the combined test may be eligible for treatment with a synthetic GH secretagogue.

The current review highlights how hormone antibodies, characterized for specificity and bioneutralization capability, can be used as specific hormone antagonists to study the physiological requirement and biochemical action of gonadotropins FSH and LH as well as estrogen

Luteinizing hormone and follicle-stimulating hormone are called gonadotropins, because they stimulate the gonads – in males the testes and in females the ovaries. They are not necessary for life, but are essential for reproduction. In addition, the association of these hormones with prostate cancer has been the interest of many researchers. Their detection in the human prostate has been investigated using different methods, including immunologic and RT-PCR techniques. In addition, the increasing evidence of paracrine/autocrine functions of the gonadotropic glycoprotein hormones, their allocation to the superfamily of cystine knot growth factors, and luteinizing hormone/chorionic gonadotropin receptor gene expression in non-gonadal tissues led many researchers to investigate intraprostatic glycoprotein hormones and their receptor gene expression. We aim in this review to shed light on the physiology of the gonadotropins and their association with prostate cancer and highlight the future possibilities of their use as targets in treating this disease.

Objective: Interferon-γ (IFN-γ) is a cytokine produced by T helper cells on antigenic challenge that may affect the release of several pituitary hormones. However, in vitro or in vivo studies have yielded disparate results with stimulatory, inhibitory or absent effects of IFN on pituitary hormone release. One of the reasons for these discrepancies could be that hormone changes were commonly assessed at a single time point in the day-night cycle. In this study we measured the circadian pattern of plasma ACTH, growth hormone (GH), prolactin, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) at 6 different time points within a 24-hour cycle in adult male Wistar rats. Methods: Groups of 6–8 rats kept under light from 08:00 to 20:00 h daily received 5 daily injections intraperitoneally of human IFN-γ (105 IU/kg body weight) or saline at 08:30 h. Plasma ACTH, GH, prolactin, LH and FSH levels were measured by a homologous specific double antibody RIA. Results: A factorial ANOVA for main effects indicated a significant 43% increase of circulating prolactin in IFN-γ-treated rats. Time of day changes were significant for the five hormones examined and these diurnal variations became altered by IFN-γ administration, with a phase advance of ACTH peak, a suppression of the rest phase peak of GH, the appearance of a second peak of prolactin at an early phase of daily photoperiod, and the blunting of the 24-hour variations of plasma FSH. Conclusion: The data point out an effect of IFN-γ on the mechanisms responsible for the circadian organization of pituitary hormone release.

We investigated the kinetics of gonadotropin-releasing hormone (GnRH)-induced activation of the protein kinase C (PKC) δ isoform in αT3-1 gonadotrope cells. Results were evaluated in subcellular fractions and whole-cell lysates using specific antibodies recognizing either non- or (trans- and auto-)phosphorylated forms of the kinase at Thr505 and Ser643 residues modulating stability and/or activation of the enzyme. Under basal conditions, and in contrast to PKC Ε, PKC δ was mainly associated with the membrane compartment. GnRH (10–7M) elicited further and rapid membrane translocation and time-dependent phosphorylation at both sites of PKC δ. The neuropeptide’s effects did not show a refractory period after short but successive GnRH stimulation and were abolished by the GnRH antagonist, antide. Sustained GnRH stimulation (2–6 h) provoked rapid down-regulation of PKC δ. Antide, by inhibiting the initial processes (translocation, phosphorylation), counteracted the degradation of the enzyme. Proteolytic processing of PKC δ was shown to mainly involve proteasome activity. Indeed, specific proteasome inhibitors prevented GnRH-elicited kinase depletion and induced membrane accumulation of the enzyme in a phosphorylated (Thr505, Ser643) form. Thus, GnRH may regulate time-dependent cell responses by modulating the phosphorylation/activation state of its signal transduction effector proteins, and by maintaining their appropriate expression balance via proteolytic processes involving the proteasome system.

Objectives: The 10-µg gonadotropin-releasing hormone (GnRH) test assesses pituitary gonadotroph responsiveness, whereas the 100-µg dose assesses maximal secretory capacity. Our aims were to establish normative data for the low-dose test in children and to evaluate the test in diagnosing common pubertal disorders. Methods: We retrospectively classified 107 children who underwent 10-µg GnRH tests into normal prepubertal (20 boys, 10 girls), normal early pubertal (10 boys, 16 girls), constitutional delay of puberty (CDP, 13 prepubertal boys >12 years), hypogonadotropic hypogonadism (HH, 5 prepubertal boys >12 years), central precocious puberty (CPP, 19 girls) or premature thelarche/variant (13 girls). Results: Peak LH response was higher in prepubertal boys >12 years compared with younger boys (p < 0.01) but showed no further change in early puberty. CDP boys had LH responses similar to prepubertal boys >12 years. HH boys showed an absent LH response which diagnosed HH with 100% sensitivity and 96% specificity. Thelarche girls had LH:FSH peak ratios lower than normal prepubertal (p = 0.001), pubertal (p < 0.05) or CPP (p = 0.001) girls. Conclusions: We have established normative values for the low-dose GnRH test in children. The test successfully differentiated HH from CDP in boys, and contributed to the differential diagnosis of CPP and premature thelarche in girls.

Objective: The LH response of pituitary gland to gonadotropin-releasing hormone (GnRH) stimulation is not well defined in patients with mutant β-subunit (Trp8 to Arg8 and Ile15 to Thr15). Here we compared the relative activities and dynamics of LH secretion in patients with wild-type and variant LH following injection of GnRH. Methods: A GnRH stimulation test was performed in 33 patients with ovulatory disorders (patient group) and 29 women with normal ovulatory cycles (control group) heterozygous for the variant LHβ allele. Blood samples were obtained up to 120 min after GnRH injection. Serum LH response was determined by comparing the results of LH immunoassays using a monoclonal antibody that recognizes wild-type LH only with those of another assay using a polyclonal antibody that recognizes equally both variant and wild-type LH (total LH). The ratio of variant LH to total LH (LH ratio) was used to determine the serum LH status. Results: The LH ratio in the control group showed the peak 15 min after GnRH injection, while that in the patient group showed the peaks 30–60 min after injection. The LH ratio in the patient group at 120 min after injection was significantly lower than that in the control group. The percent increases in LH ratio in both groups showed the peak 15 min after injection. The patient group had significantly lower changes of LH ratio at 15, 60, 90 and 120 min after GnRH injection compared with that in the control group. Conclusion: Differences in circulatory kinetics of the two types of LH may explain the differences in LH function between patients with ovulatory disorders and women with normal ovulatory cycles.

Genetic targeting of the cAMP-specific phosphodiesterase 4D1 (PDE4D1) to gonadotropin-releasing hormone (GnRH) neurons in the GPR-4 transgenic rat resulted in decreased luteinizing hormone (LH) pulse frequency in castrated female and male rats. A similar decrease in the intrinsic GnRH pulse frequency was observed in GT1 GnRH cells expressing the PDE4D1 phosphodiesterase. We have extended these findings in ovariectomized (OVX) GPR-4 rats by asking what effect transgene expression had on pulsatile LH and follicle-stimulating hormone (FSH) secretion, plasma and pituitary levels of LH and FSH, and levels of the alpha-glycoprotein hormone subunit (alpha-GSU), LH-beta and FSH-beta subunit mRNAs. In OVX GPR-4 rats the LH pulse frequency but not pulse amplitude was decreased by 50% compared to wild-type littermate controls. Assaying the same samples for FSH, the FSH pulse frequency and amplitude were unchanged. The plasma and anterior pituitary levels of LH in the GPR-4 rats were significantly decreased by approximately 45%, while the plasma but not anterior pituitary level of FSH was significantly decreased by 25%. As measured by real-time RT-PCR, the mRNA levels for the alpha-GSU in the GPR-4 rats were significantly decreased by 41%, the LH-beta subunit by 38% and the FSH-beta subunit by 28%. We conclude that in the castrated female GPR-4 rats the decreased GnRH pulse frequency results in decreased levels of LH and FSH and in the alpha- and beta-subunit mRNA levels.

Previous studies have indicated that arachidonic acid and its lipoxygenase (LO) metabolites play a role in the post-receptor effects of gonadotropin-releasing hormone (GnRH) but the exact role and nature of these putative eicosanoids remain unclear. The potential role of arachidonic acid and LO in GnRH receptor-mediated signaling was investigated in the LβT2 gonadotrope cell line, which expresses gonadotropins (LH and FSH) and GnRH-receptor mRNAs. Western immunobloting of LβT2 cell extracts, performed with a murine leukocyte polyclonal antibody against 12-LO, showed a 70-kD band, suggesting the presence of 12-LO protein in these cells. GnRH nearly doubled the release of 12-hydroeicosatetraenoic acid, a product of the 12-LO enzyme, within 10 min. A specific reverse transcriptase polymerase chain reaction with a set of primers based on the reported sequence of rat brain 12-LO yielded a 170-bp band which showed 100% homology with the expected rat brain 12-LO sequence. Exposure of LβT2 cells to pulsatile GnRH treatment (10 nM, 90-min interpulse, one and three pulses) led to a ∼3-fold increase in 12-LO mRNA levels. In conclusion, we provide evidence for the presence of a 12-LO enzyme in LβT2 cells, the expression and activity of which are increased by short-term/pulsatile exposure to GnRH. LβT2 cells represent a potential model to further study the involvement of 12-LO in GnRH receptor signaling.

We have suggested that gamma-aminobutyric acid (GABA) in the hypothalamus plays a tonic inhibitory role in the control of the luteinizing hormone (LH) release in intact male rats. To assess whether feedback from the testis alters the inhibitory GABAergic tone in the medial preoptic area (MPO) of male rats, an in vivo microdialysis study was performed in gonadally intact (n = 10), castrated (n = 12) and castrated testosterone-primed (n = 10) male rats. The microdialysis samples were collected and sequential blood samples were also obtained at 1-hour intervals. GABA in the dialysate was determined by high-performance liquid chromatography system and serum LH concentration was determined by radioimmunoassay. Episodic GABA release in the MPO was observed in all three groups of male rats, although castrated male rats showed lower GABA release (2.3 ± 0.3 ng/h) than intact and castrated testosterone-primed male rats (4.0 ± 0.5 and 4.6 ± 1.0 ng/h, respectively). Conversely, castrated male rats showed higher serum LH concentration (7.31 ± 0.46 ng/ml) than intact and castrated testosterone-primed male rats (0.71 ± 0.04 and 0.53 ± 0.07 ng/ml, respectively). In addition, intravenous infusion of bicuculline significantly increased serum LH in intact male rats, whereas bicuculline did not alter serum LH concentrations in castrated male rats. These results are consistent with the hypothesis that the feedback of testosterone stimulates GABA release in the region of the GnRH cell bodies and dendrites in male rats.

Ghrelin is an endogenous ligand for the growth hormone secretagogue (GHS) receptor, expressed in the hypothalamus and pituitary. Ghrelin, like synthetic GHSs, stimulates food intake and growth hormone (GH) release following systemic or intracerebroventricular administration. In addition to GH stimulation, ghrelin and synthetic GHSs are reported to stimulate the hypothalamo-pituitary-adrenal (HPA) axis in vivo. The aims of this study were to elucidate the hypothalamic mechanisms of the hypophysiotropic actions of ghrelin in vitro and to assess the relative contribution of hypothalamic and systemic actions of ghrelin on the HPA axis in vivo. Ghrelin (100 and 1,000 nM) stimulated significant release of GH-releasing hormone (GHRH) from hypothalamic explants (100 nM: 39.4 ± 8.3 vs. basal 18.3 ± 3.5 fmol/explant, n = 49, p < 0.05) but did not affect either basal or 28 mM KCl-stimulated somatostatin release. Ghrelin (10, 100 and 1,000 nM) stimulated the release of both corticotropin-releasing hormone (CRH) (100 nM: 6.0 ± 0.8 vs. basal 4.2 ± 0.5 pmol/explant, n = 49, p < 0.05) and arginine vasopressin (AVP) (100 nM: 49.2 ± 5.9 vs. basal 35.0 ± 3.3 fmol/explant, n = 48, p < 0.05), whilst ghrelin (100 and 1,000 nM) also stimulated the release of neuropeptide Y (NPY) (100 nM: 111.4 ± 25.0 vs. basal 54.4 ± 9.0 fmol/explant, n = 26, p < 0.05) from hypothalamic explants in vitro. The HPA axis was stimulated in vivo following acute intracerebroventricular administration of ghrelin 2 nmol [adrenocorticotropic hormone (ACTH) 38.2 ± 3.9 vs. saline 18.2 ± 2.0 pg/ml, p < 0.01; corticosterone 310.1 ± 32.8 ng/ml vs. saline 167.4 ± 40.7 ng/ml, p < 0.05], but not following intraperitoneal administration of ghrelin 30 nmol, suggesting a hypothalamic site of action. These data suggest that the mechanisms of GH and ACTH regulation by ghrelin may include hypothalamic release of GHRH, CRH, AVP and NPY.

The pulsatile release of growth hormone (GH) by the anterior pituitary is stimulated by small synthetic molecules termed GH secretagogues (GHS). The receptor for GHS (GHS-R) belongs to the family of G-protein-coupled receptors. An endogenous specific ligand of 28 amino acids has recently been purified from rat stomach, it has been termed ‘ghrelin’. Ghrelin demonstrates potent and reproducible GH-releasing activity, as well as significant prolactin-, ACTH- and cortisol-releasing activity. However, its major physiological relevance may relate to energy homeostasis. Peripheral daily administration of ghrelin caused weight gain by reducing fat utilization in mice and rats. In man, intravenous ghrelin was shown to stimulate food intake. The pathophysiological role and the potential clinical use of ghrelin are reviewed.

Estrogen affects apoptotic cell death in estrogen-responsive tissues. The purpose of the present study was to examine dynamic changes in apoptotic cell death in the anterior pituitary gland during the estrous cycle and to investigate neuroendocrine regulation of these changes in cycling female rats. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) for in situ detection of DNA strand breaks revealed that the number of TUNEL-positive anterior pituitary cells was changing during the estrous cycle, with a maximum in the morning of proestrus and a minimum in the morning of estrus. A similar pattern was observed with Bax immunostaining; however, no difference was observed in Bcl-2 immunostaining. Most of Bax-immunoreactive cells were identified as a subpopulation of gonadotropes. Pentobarbital administered in the afternoon of proestrus attenuated the decrease in TUNEL-positive or Bax-immunoreactive cells in the morning of estrus, although estradiol treatment failed to affect it. This action of pentobarbital was reduced by simultaneous treatment with an ovulation-inducing dose of gonadotropin-releasing hormone (GnRH), but not with progesterone, an ovarian steroid also released after GnRH treatment. These results suggest that anterior pituitary cells, mostly gonadotropes, undergo a cyclic change in apoptotic cell death during the estrous cycle and that the inhibition of apoptosis on estrus is due, at least in part, to the proestrous surge of GnRH secretion.

Specific blockade of the androgen receptor by the nonsteroid antiandrogens flutamide and Casodex has proven to be a useful tool for studying androgens in vivo. The aim of the present study was to investigate the effect of antiandrogen administration at the pituitary level by evaluating the ultrastructural changes in gonadotrophs, in correlation with the quantitative immunohistochemical findings, and by comparing these alterations with the effect of androgen deprivation by castration either with or without subsequent androgen replacement. Male Sprague-Dawley rats (23 days old) were grouped as follows: (1) controls, (2) flutamide-injected (10 mg/rat/day), (3) Casodex-injected (10 mg/rat/day), (4) castrated, and (5) castrated plus androgen-replaced (dihydrotestosterone propionate; 40 µg/rat/day). Groups were sacrificed after 10 days of maintenance under each condition. Pituitaries were processed for both light and electron microscopy. Serial sections (4 µm) were obtained at different levels and immunostained by means of the primary murine monoclonal antibodies anti-FSH and anti-LH and a peroxidase-mediated EnVision System (Dako). Volume density, cell density and mean cell area were measured with an image analysis system (Imaging Technology, Software Optimas 5.2). The mean cell area (p < 0.001) and the volume density (p < 0.05) increased significantly in the flutamide- and Casodex-treated groups as well as the castrated group of FSH and LH cells. On the other hand, androgen replacement in the castrated rats, however, reduced in both parameters related to control animals. The cell density of FSH-secreting cells was increased (p < 0.05) in the Casodex and flutamide treatment as well as castrated group. The cell density of LH-secreting cells was augmented (p < 0.05) in the Casodex-treated group, while there was no increase in such parameter with flutamide and castration. The ultrastructure of all groups showed two types of gonadotrophs. Type I cells contained large (300–500 nm) and small (150–200 nm) secretory granules, while type II cells were smaller, and exhibited only small granules (100–200 nm). Flutamide-treated, Casodex-treated and castrated groups presented a decreased number of secretory granules with some exocytotic profiles, well-developed rough endoplasmic reticulum and an expanded Golgi complex of both types of cells. The gonadotrophs from the castrated group exhibited numerous mitochondria with electron-dense ring-shaped laminar figures, while in the castrated plus androgen-replaced rats only a few mitochondria had similar changes to those observed in castrated animals, as a possible residual alteration. Finally, the gonadotrophs from flutamide-treated rats showed mitochondrial alterations with clear areas and isolated electron-dense laminar figures. In summary, we conclude that lack of androgen reaction through the effects of nonsteroid antiandrogens and castration on prepubertal rats produced a hypertrophia-hyperplasia of the FSH cells, and hypertrophia of LH-secreting cells, with marked alterations at the ultrastructural level suggestive of a hyperstimulation stage.

It has repeatedly been suggested that galanin acts within the anterior pituitary (AP) in an auto/paracrine manner to modulate luteinizing hormone (LH) release. Except for one recent report in the rat, evidence for this notion is absent. The purpose of this study was to investigate in the pig the effects of galanin on LH and growth hormone (GH) release and to evaluate putative local effects using various AP culture systems (monolayer, perifusion, reaggregates). Independent of age galanin dose dependently (0.05, 0.2, 1 µM) stimulated basal but not gonadotropin-releasing hormone (GnRH; ≧0.01 nM)-induced LH release. Neither basal nor GH-releasing hormone (GHRH)-stimulated GH release was affected at any age. Of 4 galanin receptor antagonists (0.2, 1 µM) tested C7 proved to have agonistic effects, whereas M40 and M15 (galantide) were ineffective in blocking galanin (0.2 µM)-induced LH secretion or affecting basal or GnRH-induced LH release. M32 [galanin (1–13) NPY (25–36) amide] inhibited (p ≤ 0.05) GnRH-induced LH release at doses of ≧2 µM, an effect which could be totally compensated by 1 µM galanin. However, the neuropeptide (NPY) antagonist BIBP 3226 (1 µM) partially overcame the effect of M32 (M32 is known to also bind to NPY receptors and NPY is inhibitory in the pig). In further studies using APs from preovulatory gilts a specific well-characterized galanin antiserum diluted 1:20 or 1:50 attenuated GnRH-induced LH release (p ≤ 0.05). However, an NPY antiserum (also affinity purified and at the same dilution) used as control unexpectedly inhibited GnRH (and galanin)-induced LH release as well, thus suggesting that attenuation of GnRH-induced LH release by galanin antiserum might be at least partly nonspecific. Furthermore 96-hour exposure of AP reaggregates to two types of porcine preprogalanin antisense oligodeoxynucleotides neither affected basal nor GnRH-induced LH release. In line with the failure to unequivocally prove paracrine effects of galanin, concentrations of galanin in AP cultures and AP culture medium were very low (≤2 pg galanin/105 AP cells). In conclusion the present study provides some evidence to ascribe a hypophysiotropic role to galanin in regulating LH but not GH secretion in the pig. The study also points to the critical role of appropriate controls when trying to prove auto/paracrine control mechanisms within the anterior pituitary. Our findings do not provide convincing evidence to support the notion that intrapituitary galanin is involved in the fine tuning of LH secretion, at least in the preovulatory pig.

In neonatal rat gonadotropes, melatonin inhibits gonadotropin-releasing-hormone (GnRH)-stimulated increase in intracellular Ca2+ concentration ([Ca2+]i); in cells transfected with the Mel1a melatonin receptor, however, melatonin has been shown to potentiate agonist-stimulated [Ca2+]i increase. To elucidate this discrepancy, we investigated the effects of melatonin in neonatal gonadotropes over a wide range of melatonin concentrations. Nystatin perforated patch recording of Ca2+-dependent potassium currents was used to monitor GnRH-induced [Ca2+]i changes. In 32% of cells, increasing melatonin concentrations in the range of 1 pM to 100 nM prolonged the latency of, and inhibited GnRH (10 nM)-stimulated [Ca2+]i increases in a concentration-dependent manner. In the remaining 68% of cells, the Ca2+ increase elicited by exposure to 10 nM GnRH was also inhibited by picomolar concentrations of melatonin, but at nanomolar concentrations the inhibitory effect disappeared and melatonin was only able to prolong the latency of the response. This dual effect of melatonin however was not observed in cells stimulated with lower (2 nM) GnRH concentrations; in that case, melatonin was inhibitory at all concentrations tested with an IC50 of about 30 pM. In contrast, application of nanomolar concentrations of melatonin resulted in potentiation of the GnRH-induced Ca2+ increase in a small population of gonadotropes which did not respond by inhibition or prolonged latency. These results indicate that in neonatal gonadotropes, melatonin has both inhibitory and potentiating effects on GnRH-stimulated [Ca2+]i increases. Ranges of concentrations needed to produce either effect suggest that two distinct G proteins may be involved, as already observed in transfected cells.

Normal action of the female ovulatory cycle is dependent on a surge of luteinizing hormone (LH) from the pituitary. Regulation of the levels of LH is therefore vital to reproductive function. It has long been established that gonadotropin-releasing hormone (GnRH) is an important component of the regulatory processes. Other peptides, including oxytocin and neuropeptide Y (NPY), have also been observed to affect LH activities. However, the possibility of the concurrent actions of these peptides has rarely been considered despite their documented presence in the hypophyseal blood during pro-oestrus. In this study, the direct effects of oxytocin and NPY on LH release were studied, as well as the effects of both peptides simultaneously. Also, pituitaries were stimulated with GnRH, and the effects of pre-exposure of the pituitary tissue to oxytocin or NPY were investigated. Further, the effect of oxytocin and NPY together on GnRH stimulation of LH release was determined. Anterior pituitaries were collected from adult female rats on the morning of pro-oestrus. Hemipituitaries were cut in two and placed in a chamber of a perifusion system. The pituitary tissue was perifused with medium alone, oxytocin and NPY, separately or in combination, for 2 h after an initial 100-min equilibration period with no peptide present. Fractions of eluate were collected and LH was measured by radioimmunoassay. LH output was stimulated by both oxytocin (p < 0.01) and NPY (p < 0.02). Furthermore, the addition of NPY to oxytocin during the perifusion elicited a further increase in LH release (p < 0.05). The pituitary tissue was exposed to a 4-min pulse of GnRH after 220 min. Stimulation of LH release by GnRH was synergistically augmented by exposure of the tissue to either oxytocin (p < 0.01) or NPY (p < 0.05) for the immediately preceding 2 h. When NPY was added to oxytocin in the perifusion medium, stimulation of LH release by GnRH was increased even further (p < 0.05). Oxytocin also synergistically enhanced the effect of a second, primed GnRH pulse, whereas the effect of NPY was less robust. This study therefore demonstrated that LH release is modified in the presence of oxytocin, NPY or GnRH alone, and also that combinations of the peptides produce further variations in LH output. Therefore, the concentrations of LH that are present in vivo are likely to be at least partly the result of the co-ordinated effects of combinations of peptides acting on the pituitary.

The development of the catfish gonadotropin-releasing hormone (cfGnRH) fiber network in the pituitary of male African catfish (Clarias gariepinus) was investigated in relation to puberty. Double immunolabeling studied by confocal laser scanning microscopy revealed a concomitant development of gonadotropes and of pituitary cfGnRH innervation during the first wave of spermatogenesis. Catfish GnRH-immunoreactive fibers in the proximal pars distalis (PPD) of the pituitary were initially observed at the age of 10 weeks (onset of spermatogonial proliferation) and gradually reached the adult pattern at the age of 20 weeks (spermatozoa present in the testis). The content of cfGnRH-associated peptide (cfGAP, part of the prohormone) in the pituitary similarly increased during puberty. At the electron microscopical level, fibers containing cfGAP-ir granules came into close proximity of the gonadotropes at 18 weeks of age. In vitro studies indicated a progressively increasing basal and cfGnRH-stimulated luteinizing hormone (LH) secretion during pubertal development. The LH secretion patterns were similar in response to exogenous cfGnRH (0.1 µM) or to endogenous cfGnRH, the release of which was induced by forskolin (1 µM). Castration experiments demonstrated that the innervation of the pituitary with cfGnRH fibers continued after surgery, accompanied by an increase in the cfGAP levels. However, gonadotrope development was retarded, suggesting a differential regulation of the two maturational processes. Since testosterone stimulates both processes, other testicular factors may also be involved. Puberty-associated changes in LH release patterns appear to reflect changes in the GnRH sensitivity and in the pool of releasable LH, while availability of cfGnRH does not appear to be a limiting factor.

According to recent reports, substance P (SP) is localized in the anterior pituitary gland within subsets of thyrotropes and somatotropes, although earlier electron-microscopic studies described the presence of this tachykinin in mammotropes and gonadotropes. Transgenic mice overexpressing the growth-hormone-releasing hormone (GHRH) gene have markedly enlarged pituitary glands, due to hyperstimulation of the somatotropes. Therefore, we speculated that if somatotropes are able to synthesize tachykinins, these peptides should be greatly increased in the anterior pituitary of transgenic GHRH mice. We found that, in accordance with our working hypothesis, both SP and neurokinin A (NKA) were markedly increased in the anterior pituitary gland of male and female transgenic mice, compared with their respective normal controls. In male transgenic mice, NKA was 13.6- and SP 20.2-fold higher than in the anterior pituitary from normal mice. In female transgenic mice, NKA was 40- and SP 100-fold higher than in the anterior pituitary from normal female mice. In male transgenic mice, NKA and neuropeptide K (NPK) contents in the anterior pituitary showed no significant changes between 26 and 50 days of age but significantly increased between 50 days and 5 months of age. The concentration of NKA in the anterior pituitary did not show significant differences between 26 days and 5 months of age, but NPK concentrations in the anterior pituitary significantly decreased with age. In female transgenic mice, NKA content and concentration in the anterior pituitary increased after 35 days of age, but NPK concentrations significantly decreased after 26 days of age. Triiodothyronine markedly decreased anterior pituitary tachykinins, but ovariectomy and estrogen administration failed to significantly affect tachykinin concentrations in the anterior pituitary of transgenic mice. Tachykinin immunostaining was detected in some somatotropes, but tachykinins were also present in cells that were not GH positive. These findings indicate that hyperstimulated somatotropes contain increased stores of tachykinins and that these cells are a source of tachykinins in the anterior pituitary. Tachykinin stores in the anterior pituitary of transgenic mice were affected by thyroid hormones but seem to be insensitive to estrogens. The GHRH transgenic mice may be an interesting model to study the regulation of tachykinin stores in the anterior pituitary gland.

A site-directed polyclonal antipeptide antibody was generated in rabbits against segment 392–404 of the rat pituitary growth hormone-releasing hormone receptor (GHRH-R), using a multiple antigenic peptide system strategy of immunization. This C-terminal intracellular region of the rat GHRH-R exhibits 85% sequence identity with the human GHRH-R. The purified anti-GHRH-R(392–404) IgGs were characterized in cell lines expressing the human GHRH-R and in rat and human anterior pituitary, using immunoblotting. The polyclonal antibody recognized a 45-kD protein in human GHRH-R-transfected BHK 570 cell membrane preparations but not in wild-type cells. A 45-kD Nα-tagged human GHRH-R was immunodetected with both antitag and anti-GHRH-R antibodies in human GHRH-R-transfected HEK 293 cells. Cross-linking of [125I-Tyr10]hGHRH(1–44)NH2 to GHRH-R-transfected BHK cells led to the detection of a major and specific 45-kD radioactive complex. Its probing with the anti-GHRH-R(392–404) IgGs led also to the detection of a 45-kD entity. In rat anterior pituitary homogenates or membrane preparations, immunoblotting led to the detection of 44-, 47- and 65-kD proteins. In human anterior pituitary membrane preparations, immunoblotting led to the detection of 52- and 55-kD proteins. No immunoreactive signal was observed in the rat liver. Cross-linking of [125I-Tyr10]hGHRH(1–44)NH2 to rat anterior pituitary homogenates revealed the presence of specific 28-, 47- and 65-kD radioactive complexes. Probing of these radioactive complexes with the anti-GHRH-R(392–404) IgGs resulted in the visualization of 28-, 47- and 65-kD entities and of an additional immunoreactive 44-kD protein. To assess the usefulness of this GHRH-R antibody, estimation of changes in the concentration of rat anterior pituitary GHRH-R was performed by immunoblotting and compared to binding data after a 3-week antithyroid treatment. The treatment known to depress the 2.5- and 4-kb GHRH-R mRNA transcripts by at least 1.7-fold decreased the apparent maximal concentration of high (Bmax1) and low (Bmax2) affinity binding sites by 4.6- and 15.2-fold, respectively, and the 47- and 65-kD GHRH-R proteins by 3.5- and 1.25-fold, respectively. Altogether, the characteristics of the anti-GHRH-R(392–404) polyclonal antibody indicate that it specifically recognizes the human and rat GHRH-R. It also represents an additional valuable tool to estimate variations of GHRH-Rs in physiopathological conditions known to affect GHRH-R mRNA and/or GHRH binding site concentrations.

In the male rat, androgens are involved in the feedback regulation of gonadotropin synthesis and secretion. Specific androgen-receptor blockade by the nonsteroidal antiandrogens, flutamide and Casodex, has proven to be a valid tool for studying androgen effects in vivo. The aim of the present study was to investigate the effect of antiandrogen administration at the pituitary level by evaluating the changes in gonadotropes through quantitative immunohistochemistry, and by comparing these alterations with the effect of androgen deprivation by castration either with or without subsequent androgen replacement. Male Sprague-Dawley rats (23 days old) were randomly divided into 5 groups for the following treatments: (a) controls; (b) flutamide-injected (10 mg/rat/day in a gelatin vehicle); (c) Casodex-injected (10 mg/rat/day in an oil vehicle); (d) castrated, and (e) castrated and dihydrotestosterone propionate-replaced (40 µg/rat/day in an oil vehicle). Groups were then sacrificed after 10 days of maintenance under each condition. Pituitaries were fixed in Bouin’s fluid and embedded in paraffin. Serial sections (4 µm) were obtained at different levels and immunostained by means of the primary murine monoclonal antibodies anti-FSH and anti-LH and a peroxidase-mediated EnVision System (Dako). Measurements of volume density (VD) and individual mean cell area were made by means of an image-analysis system (Imaging Technology, Optimas). Serum FSH and LH levels were determined by radioimmunoassay (RIA). Serum gonadotropin levels, VD, and mean cell area increased significantly in the flutamide-treated, Casodex-treated, and castrated groups (p < 0.05). Androgen replacement in the castrated rats, however, reduced VD, mean cell area, and serum gonadotropins to levels comparable to those of controls. We conclude that either androgen blockade by antiandrogens or castration produce an enhancement in the gonadotrope cell population in prepubertal rats, as shown by an increase in both VD and mean cell area, as well as an elevation in FSH- and LH-immunoreactive cells. These observations correlate well with the changes found in the levels of circulating gonadotropins as measured by RIA.

Recent studies have shown that gonadotropin-releasing hormone (GnRH) can exert various effects on the rat ovary by acting through its specific receptors. To determine the cell types responsive to the action of GnRH under physiological conditions in the ovary, distribution of the GnRH receptor mRNA was studied histologically by in situ hybridization in neonatal and adult rats. Expression of the luteinizing hormone receptor mRNA was also examined to judge the growing state of follicles and the corpora lutea. In neonatal rat ovaries, no significant GnRH receptor mRNA signal was detected until 5 days after birth. The expression was first observed at 10 days in the interstitial cells. At 15 days of age, the receptor mRNA was expressed in the granulosa cells of most preantral and early antral follicles, while no hybridization signal was detected in oocyte and theca cells. In adult cycling rats, GnRH receptor mRNA was detected mainly in the granulosa cells of most follicles and luteal cells. The granulosa cells of atretic follicles showed a very high level of the mRNA expression throughout their degenerating process. A strong hybridization signal was also detected in the mural granulosa cells of mature follicles. Newly formed (developing) corpora lutea exhibited signals with moderate intensity in the luteal cells, and the older ones showed weaker signals. The finding that the initial expression of GnRH receptor mRNA was seen in the interstitial cells of neonatal ovaries implies an unknown function of the ovarian GnRH receptor in ovarian development. The high level expression of GnRH receptor mRNA in atretic and mature follicles supports the putative roles of GnRH in the induction of follicular atresia and ovulation in rat ovaries.

To assess whether fetal luteinizing hormone releasing hormone (LH-RH) neurosecretory neurons have the capacity to respond to an exogenous stimulus, a synthetic excitatory amino acid analogue, N-methyl-D-L-aspartate (NMDA; 15 mg/kg), was given rapidly intravenously to 8 chronically catheterized fetuses (130–142 days of gestation; term 147 ± 3 days). All 8 fetuses exhibited a rise in plasma ovine luteinizing hormone (oLH) and ovine follicle-stimulating hormone (oFSH) within 5 min. The mean maximal increments of oLH (2.25 ± 0.36 ng/ml) and oFSH (1.21 ± 0.32 ng/ml) were significantly greater than in 6 normal saline-injected controls (oLH p < 0.0002; oFSH p < 0.03). The secretion of ovine prolactin (oPRL) and ovine growth hormone (oGH) was unaffected. LH-RH (5 μg) evoked a greater oLH response (p < 0.0009) and a greater oFSH response (p < 0.03) than NMDA (n = 6). Desensitization of the fetal gonadotrope by a potent LH-RH agonist, D-Trp6Pro9NEt-LH-RH (10 μg/day i.v. × 4 days), abolished the fetal oLH and the oFSH response to NMDA (n = 5). Moreover, D,L-2-amino-5-phosphonovalerate, a specific competitive antagonist for the NMDA receptor, completely inhibited the fetal oLH and oFSH response to NMDA, whereas D-L-2-amino-5-phosphonovalerate alone did not affect the plasma oLH or oFSH levels, the gonadotropin response to LH-RH, or the release of oGH or oPRL (n = 3). In primary ovine fetal pituitary cell cultures, NMDA (10–10 to 10–6 M) had no effect on oLH, oFSH, oGH, or oPRL secretion, whereas LH-RH stimulated oLH (10–8 M; p < 0.0004) and oFSH (10–8 M; p < 0.0001) release, evidence that NMDA did not have a direct pituitary effect. The results suggest that NMDA induces oLH and oFSH secretion by stimulation of the fetal LH-RH pulse generator and is mediated by central NMDA receptors. Fetal LH and FSH secretion and the response to LH-RH decrease in late gestation in the ovine and human fetus. The relative importance of sex steroid dependent and sex steroid independent central nervous system inhibition in this developmental change is unclear. It appears that central neural inhibition in addition to sex steroid negative feedback contributes to the decrease in fetal gonadotropin concentrations in late gestation. NMDA did not affect fetal oGH or oPRL secretion.

Recent evidence suggests that nitric oxide (NO), a free radical gas, plays an important role in regulating the function of a variety of neuroendocrine systems. With respect to the hypothalamo-pituitary-gonadal axis, a stimulatory effect of NO on the release of gonadotropin-releasing hormone (GnRH) from rat hypothalamus has been demonstrated in vitro. However, no previous study has reported NO-stimulated secretion of GnRH from in vivo hypothalamus, and also the precise cellular site of action of NO within the GnRH neuronal system remains to be elucidated. In the present study, utilizing the push-pull perfusion technique of rat hypothalamus, we examined the effect of L-arginine (L-Arg), an NO donor, on the release of GnRH, neuropeptide Y and cyclic GMP (c-GMP), which is a pivotal second messenger molecule of the NO system. For comparison, we also examined the effect of carbon monoxide (CO), another putative gaseous neurotransmitter, using hematin, a CO donor. During the period of 11.00–18.00 h, we collected blood and hypothalamic perfusates from ovariectomized adult rats that had been implanted with an estradiol capsule 2 days before. During the entire period of observation, L-Arg (1.0 or 10 mM), hematin (10 or 100 µM) or artificial cerebrospinal fluid alone (as the control) was infused into the medial preoptic area (MPOA) where there are cell bodies of GnRH neurons, or the median eminence-arcuate nucleus complex (ME-ARC) where axon terminals of GnRH neurons are localized. Although 10 mM of L-Arg significantly stimulated GnRH and c-GMP, but not neuropeptide Y, levels in both the MPOA and ME-ARC, GnRH and c-GMP in the ME-ARC were already increased by 1.0 mM of L-Arg. By contrast, both concentrations of hematin were without effect at either site of the hypothalamus. This study is the first to demonstrate that NO is capable of stimulating GnRH release from rat hypothalamus in vivo. Our data also suggests that both cell bodies and axon terminals of GnRH neurons may be sites of action of NO. Our data do not support a previous study by other investigators that reported a stimulatory effect of CO on the GnRH release.

Most electrical and ionic properties of anterior pituitary cells are common to all pituitary cell types; only gonadotropes exhibit a few cell specific features. Under basal conditions, the majority of pituitary cells in vitro, irrespective of their cell type, display spontaneous action potentials and [Ca2+]i transients that result from rhythmic Ca2+ entry through L-type Ca2+ channels. The main function of these action potentials is to maintain cells in a readily activable responsive state. We propose to call this state a ‘pacemaker mode’, since it persists in the absence of extrinsic stimulation. When challenged by hypothalamic releasing hormones, cells exhibit two distinct response patterns: amplification of pacemaker activity or shift to internal Ca2+ release mode. In the internal Ca2+ release mode, [Ca2+]i oscillations are not initiated by entry of external Ca2+, but by release of Ca2+ from intracellular stores. In somatotropes and corticotropes, GHRH or CRH triggers the pacemaker mode in silent cells and amplifies it in spontaneously active cells. In contrast, in gonadotropes GnRH activates the internal Ca2+ release mode in silent cells and switches already active cells from the pacemaker to the internal Ca2+ release mode. Interestingly, homologous normal and tumoral cells display the same type of activity in vitro, in the absence or presence of hypothalamic hormones. Pacemaker and internal Ca2+ release modes are likely to serve different purposes. Pacemaker activity allows long-lasting sequences of [Ca2+]i oscillations (and thus sustained periods of secretion) that stop under the influence of hypothalamic inhibitory peptides. In contrast, the time during which cells can maintain internal Ca2+ release mode depends upon the importance of intracellular Ca2+ stores. This mode is thus more adapted to trigger secretory peaks of large amplitude and short duration. On the basis of these observations, theoretical models of pituitary cell activity can be proposed.

Pulsatile gonadotropin-releasing hormone (GnRH) release from the median eminence is critical for the appropriate function of the pituitary gonadotropes and for the generation of a preovulatory gonadotropin surge. The mechanisms by which many GnRH axon terminals are synchronized to release GnRH in a coordinated fashion into the capillaries of the primary plexus are unknown as are the anatomical sites at which the regulation of GnRH neurons takes place. While many neurotransmitters have been shown to influence GnRH release, it is not clear if such neurotransmitters regulate GnRH neurons directly through synaptic interactions or through intermediate neurons. An alternative mechanism of interneuronal communication is provided by gap junctions which allow a rapid, bidirectional exchange of signals. In order to explore if GnRH neurons synthesize the appropriate proteins to form gap junctions with adjacent cells we used double immunohistochemistry for GnRH and connexins-26, -32 or -43 as well as dual in situ hybridization to identify GnRH mRNA and connexin-32 mRNA. The results show that all GnRH neurons contain connexin-32 immunoreactive puncta at their perikarya and, occasionally, at their axon terminals in the median eminence while connexin-26 and -43 immunoreactivity was absent in GnRH neurons. In addition, connexin-32 mRNA was detected in GnRH mRNA containing neurons. However, gap junctional connections between adjacent GnRH neurons were not observed. The data suggest that gap junctional coupling of GnRH neurons with neighboring non-GnRH containing cells may occur and may represent a mechanism by which GnRH neurons can be synchronized or by which hormonal or neurotransmitter signals can be conveyed to the GnRH neurons.

Recent work has demonstrated that glutamate functions as a major transmitter involved in the regulation of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion in female animals, although the specific receptors and mechanisms mediating its effects have not been completely worked out. The purpose of the present study, therefore, was to examine the role of the AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid)-type glutamate receptor in the control of GnRH and LH secretion in female animals. Toward this end, the dose- and steroid-dependent effects of AMPA on GnRH and LH secretion in female rats were investigated using both in vitro and in vivo approaches, and the role of AMPA receptors in the production of the steroid-induced LH surge was also assessed. The results of the study revealed that central administration of AMPA resulted in a stimulation of LH release in the estrogen-primed ovariectomized adult rat. AMPA was also found to potently stimulate GnRH release in vitro from mediobasal hypothalamic (MBH) fragments obtained from estrogen-primed ovariectomized adult rats, and this effect was blocked by the selective AMPA receptor antagonist, NBQX. The mechanism of action of AMPA appeared to differ from that of N-methyl-D-aspartate (NMDA) as AMPA, in contrast to NMDA, failed to elevate nitric oxide synthase activity in the hypothalamus. The effect of AMPA on LH secretion was demonstrated to be steroid dependent, as central administration of AMPA stimulated LH release in estrogen-primed ovariectomized rats, but was inhibitory to LH release in non-estrogen-primed ovariectomized rats. In contrast, AMPA stimulated GnRH release equally well from MBH fragments obtained from estrogen-primed or non-estrogen-primed ovariectomized rats. The different effects of AMPA on LH release may be due to different pituitary sensitivities between the two models, or alternatively, AMPA may stimulate the release of LH inhibitory factors in the ovariectomized rat in the absence of estrogen. Finally, a physiological role for AMPA receptors in the production of the steroid-induced LH surge was suggested, based on the finding that central administration of the selective AMPA receptor antagonist, NBQX, into the third cerebroventricle significantly attenuated the steroid-induced LH surge in the ovariectomized adult female rat.

The ability of cannabinoids to affect anterior pituitary luteinizing hormone (LH) secretion has been largely attributed to a central nervous system site of action, however, the mechanism(s) by which cannabinoids alter LH release remains unclear. In the present study, the acute administration of delta-9-tetrahydrocannabinol (THC) produced a dose-related suppression of plasma LH and stimulation of adrenocorticotropin (ACTH) levels in ovariectomized female rats. To determine if activation of the hypothalamic-pituitary-adrenal axis was involved in the ability of THC to inhibit LH release, female rats were either pretreated with the corticotropin-releasing hormone (CRH) receptor antagonist, alpha-helical CRH, or were adrenalectomized prior to acute THC administration, in order to assess the roles of CRH and corticosterone in the ability of THC to suppress LH secretion. A low dose of THC (0.5 mg/kg b.w., iv) produced a decrease in plasma LH levels at 20 and 40 min posttreatment in ovariectomized, sham adrenalectomized rats. However, in adrenalectomized animals, plasma LH levels were suppressed at 40 min and remained decreased at 80 min following THC administration. Thus, the duration of LH suppression following THC treatment was significantly increased in adrenalectomized versus sham adrenalectomized rats (p0.05). Furthermore, pretreatment with the CRH receptor antagonist, alpha-helical CRH (100 micrograms/5 microliters, icv), 30 min before THC administration, attenuated the ability of a high THC dose (1.0 mg/kg) to inhibit LH release in ovariectomized rats. Together, these results demonstrate that THC has significant effects on LH and ACTH secretion in ovariectomized rats and suggest that THC-induced CRH activation, but not corticosterone release, plays a role in the suppression of LH release by cannabinoids.

In neonatal rat gonadotrophs, melatonin acts through the high-affinity membrane-bound receptors to inhibit GnRH-induced [Ca2+]i increase. GnRH increases [Ca2+]i primarily by mobilization from the inositol trisphosphate-sensitive pool followed by Ca2+ influx through the voltage-sensitive channels. Melatonin inhibits the GnRH-induced [Ca2+]i increase. When added after the GnRH-induced spike, melatonin decreases [Ca2+]i in 52% of the gonadotrophs. The effect of melatonin is dependent on extracellular Ca2+ and may be mimicked by Ca2+-free medium or verapamil. When added before GnRH, melatonin inhibits the [Ca2+]i spike. This effect of melatonin is independent of extracellular Ca2+ as it persists in Ca2+-free medium. These findings indicate that melatonin blocks Ca2+ mobilization as well as Ca2+ influx in the gonadotrophs.

The effect of melatonin on the gonadotropin-releasing-hormone (GnRH)-induced oscillatory rises in intracellular calcium concentration, [Ca2+]i, was studied in cultured cells from

The physiological mechanism underlying the enormously elevated fetal plasma GH concentrations in mammalian species is not well understood. We postulated that a decreased sensitivity to the inhibitory effects of somatostatin (SRIH) and insulin-like growth factor-1 (IGF-1) at the pituitary level during porcine fetal life might be one factor in the high plasma GH levels. Therefore, the acute effects of GH-releasing hormone (GHRH), SRIH, and IGF-1 on GH release from the porcine anterior pituitary (AP) were studied using a perifusion system. AP tissue pieces from male and female fetuses (95 days postcoitum) and piglets (10-12 days postpartum) were perifused (at least 6 replicates per treatment and sex) at a rate of 0.1 ml/min and fractions were collected at 10-min intervals. Tissue was subjected to various treatments in random order: a 10-min GHRH pulse (1 nM) with or without concomitant 40-min exposure to SRIH (1, 10 or 100 nM) or IGF-1 (1 or 10 nM) or exposure to SRIH or IGF-1 alone (10 nM each). None of the treatments revealed sex differences, therefore data from males and females were pooled. Exposure to GHRH resulted in a rapid stimulatory GH response both in fetuses (from 61.1 +/- 4.1 to 125.6 +/- 4.7 ng GH.80 min-1.mg-1 AP, p0.05) and neonates (from 58.1 +/- 5.3 to 104.6 +/- 4.5 ng GH.80 min-1.mg-1 AP, p0.05). Exposure to the lowest dose of SRIH or IGF-1 (1 nM) during GHRH pulse inhibited (p0.05) the GH response to GHRH in neonates (74.3 +/- 3.9 and 72.5 +/- 6.3 ng GH.80 min-1.mg-1 AP, respectively), but not in fetuses (115.9 +/- 5.8 and 118.7 +/- 5.4 ng GH.80 min-1.mg-1 AP, respectively). When high doses of SRIH (10 or 100 nM) or IGF-1 (10 nM) were used, the GHRH-induced GH release was totally blocked in neonates (p0.05). However, in fetuses the GH response was inhibited only during the first 40 min after GHRH, and a rebound or delay effect occurred during the next 40 min. Irrespective of these general findings, some individual profiles from fetuses (4 profiles) showed that 1 nM SRIH or IGF-1 could inhibit GHRH-induced GH secretion. In contrast, individual profiles from neonates indicated that high doses of SRIH (7 profiles) or IGF-1 (5 profiles) were unable to block GH release stimulated by GHRH. On the other hand, a paradoxical stimulation of GH was observed during treatment with SRIH alone in three individual profiles from fetuses. These data confirm our hypothesis that the highly elevated plasma GH levels in the porcine fetus in vivo (compared to neonates) may be explained, at least in part, by a decreased sensitivity to the inhibitory effects of SRIH and IGF-1 at the pituitary level during fetal life. Results from some profiles indicated individual variations in the maturation of the GH control system, and moreover, even a 'paradoxical' enhancement of GH release in some fetal GH profiles.

Gonadotropin-releasing hormone (GnRH) controls all aspects of reproductive function. GnRH is secreted by hypothalamic neurons and exerts its effects on the endocrine system through pituitary gonadotropes, while its effects on sexual receptivity are mediated by the central nervous system. The electrophysiological responses of central neurons to GnRH have shown both excitatory and inhibitory responses, but little is known about the mechanisms by which GnRH can change neuronal excitability. The present study addresses the mechanisms whereby stimulation of the human GnRH receptor changes neuronal excitability by using a combination of electrophysiological and heterologous expression techniques. Microinjection of in vitro transcribed cRNA coding for the human GnRH receptor into enzymatically dissociated adult rat superior cervical ganglion neurons resulted in GnRH receptor expression. Activation of the GnRH receptor inhibited both M-type K+ and N-type Ca2+ channels. Inhibition of M-type K+ channels was insensitive to pertussis toxin pretreatment and blocked by intracellular GDPbetaS. Inhibition of Ca2+ channels was slow in onset, voltage independent and insensitive to pertussis toxin. Wash-out of GnRH resulted in an unusual transient reversal of tonic G-protein-mediated Ca2+ channel inhibition. Block of the N-type Ca2+ channel with omega-conotoxin GVIA decreased Ca2+ current inhibition from 43 to 15%, indicating that the N-type Ca2+ channel is an effector target. Ca2+ channel inhibition was completely abolished by including a Ca2+ chelator in the patch pipette. Cell-attached macropatch experiments indicated that Ca2+ channel inhibition is mediated by a diffusible second messenger. These results demonstrate that the human GnRH receptor can inhibit M-type K+ and N-type Ca2+ channels when heterologously expressed in adult rat neurons. Modulation of M-type K+ and N-type Ca2+ channels in central neurons which contain GnRH receptors is likely to contribute to the changes in neuronal excitability elicited by GnRH.

The presence of multiple monomeric forms has been described for the estrogen receptor (ER) in the pituitary gland. We analyzed ER mRNA forms in male and female rat pituitary. A single 6.2-kb ER mRNA species was detected in the male rat pituitary, whereas the female rat pituitary exhibited two ER mRNA forms of 6.2 and 5.5 kb, respectively. The 6.2-kb mRNA was present throughout the different stages of the estrous cycle, while the 5.5-kb mRNA appeared to be restricted to proestrus, suggesting an acute regulation of ER transcription at this stage. The 5.5-kb ER mRNA could be rapidly induced either by 17 beta-estradiol replacement in ovariectomized adult female rats or by priming immature rats with pregnant-mare serum gonadotropin. Using enriched cell populations, an inverse and strong correlation was established between the presence of the 5.5-kb ER mRNA form and the number of gonadotropes. Conversely, the localization of the 5.5-kb mRNA form was demonstrated in lactotrope populations. In order to elucidate the structural modifications in the transiently expressed ER mRNA, a series of reverse-transcriptase polymerase chain reaction amplifications was carried out using several pairs of primers corresponding to the entire ER-coding region. The data showed that no alternative splicing was occurring in the ER-coding region involving a potential role of either 3'- or 5'-untranslated regions. Thus, ER presents a 17 beta-estradiol-dependent transcriptional mechanism triggered on proestrous day and specific to the female lactotropes.

In order to determine the processing of growth hormone (GH) by its pituitary target cells, male rats were injected intracardially with 125I-bGH and their pituitaries removed at specific time intervals (2-120 min). Autoradiographic analysis performed at the electron-microscopic level showed that only three cell populations specifically took up 125I-bGH: somatotropes, lactotropes and gonadotropes. Specificity was demonstrated by concomitant injection with an excess of unlabeled bGH. A time course study indicated that eight compartments had distinct labeling patterns. The plasma membrane was highly labeled after as little as 2 min, and showed biphasic labeling 2 and 60 min after injection. The secretory granules of the somatotropes were more intensely labeled than those of the other cell populations. The rough endoplasmic reticulum was more intensely labeled in the gonadotropes. The Golgi apparatus was specifically labeled only in the gonadotropes. The mitochondria showed the highest degree of labeling at 15 and 120 min after injection. The lysosome compartment showed triphasic labeling, with maxima at 2, 30 and 120 min after injection. The labeling of the nuclear membrane showed a biphasic pattern, firstly at 15 min, then at 120 min after injection, except in the gonadotropes, and the labeling in the nuclear matrix showed similar biphasic pattern and maxima. These results show that GH is specifically taken up in the anterior pituitary by the somatotropes, lactotropes and gonadotropes, where, after binding to the plasma membrane, it is internalized into several cellular compartments, including the nucleus. The differences in cellular localization and processing between these cell types may reflect different paracrine and autocrine roles for GH.