Human neurons express type I GnRH receptor and respond to GnRH I by increasing luteinizing hormone expression

Gonadotropin-releasing hormone receptor I (GnRHR I) has been localized to the limbic system of the rat brain, although the functional consequences of GnRH signaling through these receptors is unknown. In this paper, we characterize the expression of GnRHR I in the human hippocampus and cortex, and the functionality of GnRHR I in human neuroblastoma cells. Robust GnRHR I immunoreactivity was detected in the cell body as well as along the apical dendrites of pyramidal neurons in the CA2, CA1, and end plate, but was clearly lower in the subiculum of the hippocampus. Immunolabeling was also evident in cortical neurons, including those located in the entorhinal cortex and occipitotemporal gyrus but was not observed within the granular layer of the dentate gyrus. No differences in immunohistochemical staining were observed between control and Alzheimer's disease brain. GnRHR I mRNA and protein (mature, immature, and other variant) expression was detected in human neuroblastoma cells (M17, SH-SY5Y) and rat embryonic primary neurons and varied with differentiation and GnRH treatment. Since GnRHR I was expressed by extrapituitary cells, and hypothalamic GnRH I secretion markedly increases post-menopause/andropause, we treated human M17 neuroblastoma cells cultured in serum-free conditions with GnRH I for 6 h and measured LH expression. M17 neuroblastoma cells express LHbeta mRNA, while immunoblot analysis indicated the presence of three LH variants (approximately 30, 47, and 60 kDa) that were upregulated by low concentrations of GnRH I, but down-regulated at higher GnRH I concentrations. LH expression was also found to increase in differentiating embryonic rat primary cortical neurons. Our results demonstrate that neurons expressing GnRHR I are functional, responding to GnRH I by upregulating LH production. Post-reproductive surges in GnRH I secretion may explain the accumulation of LH in pyramidal neurons of the aged human and rat.     

Immunolocalization of gonadotropin-releasing hormone (GnRH)-I, GnRH-II, and type I GnRH receptor during follicular development in the human ovary

CONTEXT: GnRH and its receptor have been detected at the mRNA level in different ovarian cell types, implicating an autocrine role of the GnRH system in the human ovary. However, the expression at the protein level of GnRH and its receptor in specific cell types during follicular development has not been documented in humans. OBJECTIVE: We evaluated the immunohistochemical expression of GnRH-I (the classical form of mammalian GnRH), GnRH-II (the novel isoform), and the type I GnRH receptor (GnRHR) that is known to bind both forms of GnRH, in ovaries of premenopausal women. MAIN OUTCOME MEASURES: Immunohistochemistry, immunofluorescence, immunoblot assay, and real-time RT-PCR were performed. RESULTS: GnRH-I, GnRH-II, and GnRHR were not immunostained in the follicles from the primordial to the early antral stage. In preovulatory follicles, both forms of GnRH and their common receptor were localized predominantly to the granulosa cell layer, whereas the theca interna layer was weakly positive. In the corpus luteum, significant levels of GnRH-I, GnRH-II, as well as GnRHR were observed in granulosa luteal cells, but not in theca luteal cells. Both GnRH isoforms and the type I GnRHR were localized also to the ovarian surface epithelium from which over 85% of ovarian cancers are thought to be derived. CONCLUSION: The expression of GnRH-I, GnRH-II, and GnRHR protein in the human ovary is temporally and spatially specific and further supports the physiological role of an autocrine regulatory system involving GnRH-I, GnRH-II, and GnRHR in follicular development and corpus luteal function.     

Tunicate gonadotropin-releasing hormone (GnRH) peptides selectively activate Ciona intestinalis GnRH receptors and the green monkey type II GnRH receptor

In vertebrates, GnRH binds to its receptor and stimulates predominantly G(q/11)-mediated signal transduction in gonadotropes. However, little is known about the GnRH receptor and its signaling pathway in tunicates, a group that arose before the vertebrates. Although tunicates have had duplications of a few genes in the last 600 million years, the early vertebrates had duplications of the full genome. Also unknown is the nature of GnRH signaling in the tunicate, which lacks both a pituitary gland and sex steroids. However, we know that tunicates have GnRH peptides because we previously reported six GnRH peptides encoded within the tunicate genome of Ciona intestinalis. Here we clone and sequence cDNAs for four putative GnRH receptors from C. intestinalis. These are the only invertebrate GnRH receptors found to date. Each Ciona GnRH receptor was expressed in COS-7 cells, incubated with each of the six C. intestinalis GnRHs and assayed for a signaling response. GnRH receptors 1, 2, and 3 responded to Ciona GnRH peptides to stimulate intracellular cAMP accumulation. In contrast, only GnRH receptor 1 activated inositol phosphate turnover in response to one of the Ciona GnRHs. The green monkey type II GnRH receptor cDNA was tested as a comparison and a positive control. In conclusion, the four GnRH receptors encoded within the C. intestinalis genome were all transcribed into messenger RNA, but only three of the Ciona GnRH receptors were biologically active in our assays. The Ciona GnRH receptors almost exclusively activated the cAMP pathway.     

Luteinizing hormone release and gonadotropin-releasing hormone (GnRH) receptor internalization: independent actions of GnRH

Three different approaches are described which provide independent and new evidence that gonadotropin-releasing hormone (GnRH) internalization and GnRH-stimulated LH release are distinct actions of the releasing hormone. 1) Removal of GnRH from medium bathing the pituitary cell cultures resulted in the prompt return of LH release to basal levels. This finding indicated that a continuous supply of externally applied GnRH is required for the stimulation of LH release. 2) Covalent immobilization of D-Lys6-des-Gly10-Pro9-ethylamide GnRH (a GnRH agonist) on agarose beads resulted in a derivative which stimulated LH release with full efficacy. At concentrations of immobilized releasing hormone analog sufficient to evoke gonadotropin release, the quantity of LH release was restricted by the number of beads added. This finding was interpreted as evidence that the attachment of immobilized agonist was stable during the bioassay and indicated that LH release could be stimulated with full efficacy without the requirement for GnRH internalization. 3) Comparative studies using image-intensified microscopy and the cell culture bioassay showed that 100 microM vinblastin markedly inhibited large scale patching and capping of the GnRH receptor (viewed by image-intensified microscopy), but did not alter the EC50 or efficacy of LH release stimulated by GnRH or the agonist described above. These observations indicated that internalization as well as large scale patching and capping of the GnRH receptor are not required for LH release.     

Effects of cytokines on gonadotropin-releasing hormone (GnRH) gene expression in primary hypothalamic neurons and in GnRH neurons immortalized conditionally

Various cytokines produced during the immune reaction can modulate the neuroendocrine reproductive axis, probably by inducing changes in the activity of hypothalamic GnRH neurons. However, the precise cellular and molecular effects of cytokines on these neurons have not been reported yet. To gain a better insight into these regulations, we first examined the pattern of expression of cytokine receptors in a novel neuronal cell line expressing GnRH (Gnv-4 cells). Among others, gp130 is expressed in Gnv-4 cells, together with the ligand receptor subunits specific for IL-6 as well as oncostatin M (OSM). Consistent with the latter observation, we show that OSM stimulates the expression of the immediate early genes c-fos and early growth response-1 in Gnv-4 cells, an effect dependent upon the activation of the MAPK Erk1/2 intracellular signaling pathway. Functional studies performed in parallel in Gnv-4 cells and in primary hypothalamic neuronal cell cultures show that OSM, although devoid of any effect of its own on GnRH gene expression, can inhibit dose-dependently the stimulation of GnRH expression by N-methyl-d-aspartic acid. In conclusion, these data demonstrate that a GnRH-expressing neuronal cell line can be modulated in vitro by cytokines implicated in the regulation of the reproductive axis. Moreover, they provide the first evidence of an involvement of OSM in these regulations.     

Neuropeptide Y modulates the binding of a gonadotropin-releasing hormone (GnRH) analog to anterior pituitary GnRH receptor sites.

Neuropeptide Y (NPY) increases LH secretion in part by enhancing the release of LH in response to GnRH. The present studies examined whether NPY influences the binding of GnRH to its receptors and also assessed whether specific binding sites for NPY exist in rat anterior pituitary membranes. In concentrations from 66-200 nM, NPY dose-dependently enhanced the binding of a 125I-labeled GnRH agonist, [D-Ala6, des-Gly10]GnRH ethylamide (GnRHa; 30 pM) to anterior pituitary membranes of chronically ovariectomized rats; higher concentrations of NPY were ineffective. At 200 nM, NPY significantly increased the high affinity binding of the GnRHa to these membranes, without change in the apparent maximum binding capacity. Further, 200 nM NPY significantly increased the binding of [125I]GnRHa when this tracer was used in concentrations of less than 100 pM, but NPY did not increase the binding of higher concentrations of [125I]GnRHa. Peptide YY, a gastrointestinal peptide structurally and functionally related to NPY, and the hypothalamic/pituitary peptide galanin (both at 200 nM) also increased the binding of [125I]GnRHa (30 pM) to anterior pituitary membranes, but to approximately one third of the extent produced by NPY. Salmon calcitonin, which, similar to NPY, is a strongly hydrophobic neuroendocrine peptide, did not alter GnRHa binding. Mg++ ion dose-dependently reduced the affinity of GnRHa binding, without changing the maximum binding capacity, and also attenuated the increase in GnRHa binding produced by NPY. To further characterize the nature of NPY interaction with anterior pituitary membranes, [125I] peptide YY was used to label NPY binding sites in anterior pituitary and hypothalamic membranes from ovariectomized rats. Specific, and predominantly high affinity, NPY binding sites were demonstrated in hypothalamic membranes, whereas NPY binding to anterior pituitary membranes could be resolved into high and low affinity components. These results show that at low nanomolar concentrations, NPY can enhance the association of GnRHa to receptors in anterior pituitary membranes. This demonstration of increased GnRHa binding in the presence of NPY may explain in part the action of this neuropeptide to potentiate GnRH-induced LH release from anterior pituitary cells.     

Topical absorption of gonadotropin-releasing hormone (GnRH) in goldfish

The studies reported here show that exogenous GnRH can be absorbed by goldfish from the intraperitoneal (ip) cavity, gill surface, or surrounding water. Mammalian rather than teleost GnRH was applied in order to ensure that GnRH measurement in plasma did not reflect the native form. A radioimmunoassay (RIA) specific to mammalian GnRH was used to measure the concentration of absorbed GnRH; validation for this approach was provided by HPLC and cross-reactivity studies in which mammalian GnRH was shown not to be present in control goldfish brain or pituitary extracts. Plasma concentration of GnRH was highest at the first sampling time, 4 min after administration, for all three routes. For intraperitoneal injection, plasma concentration was halved in 12 min, a period comparable with the half-life in rats. The pituitary content of GnRH also increased rapidly during the first 4 min after ip injection and remained high for 60 min. Absorption of GnRH from the gill was equally effective with water or dimethyl sulfoxide (DMSO) as vehicle.     

Molecular cloning and characterisation of the rat pituitary gonadotropin-releasing hormone (GnRH) receptor.

We have isolated the gonadotropin-releasing hormone receptor (GnRH-R) from a rat anterior pituitary cDNA library, determined its sequence and demonstrated receptor function. The 2.2 kb rat GnRH-R clone encodes a protein of 327 amino acids. A 1.3 kb clone encoding the mouse GnRH-R has previously been described (Tsutsumi et al., 1992). Although both the mouse and rat protein share significant homology with molecules belonging to the family of G protein-coupled receptors, they have certain unusual features, an example being the complete absence of a COOH terminal tail. The 3'-untranslated region reported missing in the mouse is present in the rat cDNA, where an extended 1 kb of 3'-untranslated region extending to the poly-A tail is shown. At the amino acid level, the rat GnRH-R shows considerable homology with that of the mouse. Electrophysiological studies with Xenopus oocytes and transfection of the cDNA into COS-1 cells, have shown that the 2.2 kb cDNA clone encodes a functional receptor.     

A role for hypothalamic astrocytes in dehydroepiandrosterone and estradiol regulation of gonadotropin-releasing hormone (GnRH) release by GnRH neurons

Molecules of astrocyte origin influence gonadotropin-releasing hormone (GnRH) release and GnRH neuronal growth and differentiation. Furthermore, type 1 astrocytes express steroid receptors, presenting the possibility that steroid actions on GnRH neurons might occur via astrocytes. Utilizing GT1-7 cells, a GnRH-secreting cell line, the present study demonstrates that astrocytes mediate dehydroepiandrosterone (DHEA) or estradiol (E2) stimulated GnRH secretion. Conditioned media (CM) from astrocytes cultured for 48 h alone, with DHEA (DHEA-CM), or with E2 (E2-CM) were collected, treated with charcoal to remove steroids, and added to GT1-7 cells in culture for 12 h to test the effect on GnRH secretion. DHEA-CM and E2-CM stimulated GnRH secretion by GT1-7 cells by 4- and 3-fold, respectively. The effect of DHEA-CM on GnRH secretion by GT1-7 cells appears to be related to both DHEA and its metabolite, E2, since blocking the metabolism of DHEA into estrogen in the DHEA-treated astrocytes partially reversed the stimulatory effect of DHEA-CM. Addition of transforming growth factor (TGF)-beta1-neutralizing antibody to the astrocyte cultures reversed the stimulatory effects of both DHEA-CM and E2-CM on GnRH secretion by GT1-7 cells, suggesting that TGF-beta1 derived from astrocytes may be the principle mediator of E2 and DHEA effects. These data provide evidence for a novel mechanism by which circulating steroids and/or neurosteroids may modulate GnRH secretion.     

Small-conductance calcium-activated potassium channels control excitability and firing dynamics in gonadotropin-releasing hormone (GnRH) neurons

The cellular mechanisms determining the firing patterns of GnRH neurons are presently under intense investigation. In this study, we used GnRH-green fluorescent protein transgenic mice and perforated-patch electrophysiology to examine the role of small conductance calcium-activated potassium (SK) channels in determining the electrical excitability and burst-firing characteristics of adult GnRH neurons. After establishing an appropriate protocol for examining the afterhyperpolarization potential (AHP) currents in GnRH neurons, the highly selective SK channel blocker apamin was used to demonstrate that all GnRH neurons express functional SK channels (35.7 +/- 2.7 pA, mean decay time constant = 2167 msec, apamin IC(50) = 9.6 nm) and that this channel underlies approximately 90% of the AHP in these cells. Current-clamp experiments showed that apamin-sensitive SK channels were tonically active in the majority (74%) of GnRH neurons, with apamin (100 nm) administration resulting in a mean 6.9 +/- 0.5 mV membrane depolarization. Apamin also elevated the firing rate of GnRH neurons, including increased burst frequency and duration in spontaneously bursting cells as well as the ability of GnRH neurons to fire action potentials in response to current injection. In GnRH neurons activated by current injection, apamin significantly enhanced the amplitude of the afterdepolarization potential after a single action potential and eliminated spike frequency adaptation. Together, these studies show that apamin-sensitive SK channels play a key role in restraining GnRH neuron excitability. Through direct modulation of the AHP and indirect actions on the afterdepolarization potential, the SK channel exerts a powerful tonic influence upon the firing dynamics of GnRH neurons.     

LH down-regulates gonadotropin-releasing hormone (GnRH) receptor, but not GnRH, mRNA levels in the rat testis.

The demonstration of an inhibitory effect of gonadotropin-releasing hormone (GnRH) agonists upon steroidogenesis in hypophysectomized rats and the presence of mRNA coding for GnRH and GnRH receptors (GnRH-R) in rat gonads suggests that GnRH can act locally in the gonads. To assess this hypothesis, we investigated the effects of GnRH analogs, gonadotropins and testosterone on the levels of both GnRH and GnRH-R mRNA in the rat testis. Using dot blot hybridization, we measured the mRNA levels 2 to 120 h after the administration of the GnRH agonist, triptorelin. We observed an acute reduction of both GnRH and GnRH-R mRNAs 24 h after the injection (about 38% of control). However, the kinetics for testis GnRH-R mRNA were different from those previously found for pituitary GnRH-R mRNA under the same conditions. Initially, the concentrations of serum LH and FSH peaked, then declined, probably due to the desensitization of the gonadotrope cells. In contrast, the GnRH antagonist, antarelix, after 8 h induced a 2.5-fold increase in GnRH-R mRNA, but not in GnRH mRNA, while gonadotropins levels were reduced. Human recombinant FSH had no significant effect on either GnRH or GnRH-R mRNA levels. Inversely, GnRH-R mRNA levels markedly decreased by 21% of that of control 24 h after hCG injection. Finally, 24 h after testosterone injection, a significant increase in GnRH-R mRNA levels (2.3 fold vs control) was found, but a reduction in the concentration of serum LH, probably by negative feedback on the pituitary, was observed. In contrast, GnRH mRNA levels were not significantly altered following testosterone treatment. Since LH receptors, GnRH-R and testosterone synthesis are colocalized in Leydig cells, our data suggest that LH could inhibit the GnRH-R gene expression or decrease the GnRH-R mRNA stability in the testis. However, this does not exclude the possibility that GnRH analogs could also affect the GnRH-R mRNA levels via direct binding to testicular GnRH-R. In contrast, the regulation of GnRH mRNA levels appeared to be independent of gonadotropins. Taken together, our results suggest a regulation of GnRH and GnRH-R mRNA specific for the testis.     

Early prepubertal ontogeny of pulsatile gonadotropin-releasing hormone (GnRH) secretion: I. Inhibitory autofeedback control through prolyl endopeptidase degradation of GnRH.

GnRH[1-5], a subproduct resulting from degradation of GnRH by prolyl endopeptidase (PEP) and endopeptidase 24.15 (EP24.15) was known to account for an inhibitory autofeedback of GnRH secretion through an effect at the N-methyl-D-aspartate (NMDA) receptors. This study aimed at determining the possible role of such a mechanism in the early developmental changes in frequency of pulsatile GnRH secretion. Using retrochiasmatic explants from fetal male rats (day 20-21 of gestation), no GnRH pulses could be observed in vitro, whereas pulses occurred at a mean interval of 86 min from the day of birth onwards. This interval decreased steadily until day 25 (39 min), during the period preceding the onset of puberty. Based on GnRH[1-10] or GnRH[1-9] degradation and GnRH[1-5] generation after incubation with hypothalamic extracts, EP24.15 activity did not change with age, whereas PEP activity was maximal at days 5-10 and decreased subsequently until day 50. These changes were consistent with the ontogenetic variations in PEP messenger RNAs (mRNAs) quantitated using RT-PCR. Using fetal explants, the NMDA-evoked release of GnRH was potentiated in a dose-dependent manner by bacitracin, a competitive PEP inhibitor and the desensitization to the NMDA effect was prevented using 2 mM of bacitracin. At day 5, a higher bacitracin concentration of 20 mM was required for a similar effect. Pulsatile GnRH secretion from fetal explants was not caused to occur using bacitracin or Fmoc-Prolyl-Pyrrolidine-2-nitrile (Fmoc-Pro-PyrrCN), a noncompetitive PEP inhibitor. At postnatal days 5 and 15, a significant acceleration of pulsatility was obtained using 1 microM of Fmoc-Pro-PyrrCN or 2 mM of bacitracin. At 25 and 50 days, a lower bacitracin concentration of 20 microM was effective as well in increasing the frequency of GnRH pulsatility. We conclude that the GnRH inhibitory autofeedback resulting from degradation of the peptide is operational in the fetal hypothalamus but does not explain the absence of pulsatile GnRH secretion at that early age. After birth, PEP activity is high and may account for the low frequency of pulsatility. The potency of that effect decreases before the onset of puberty and may contribute to the acceleration of GnRH pulsatility.     

Fanconi anemia A is a nucleocytoplasmic shuttling molecule required for gonadotropin-releasing hormone (GnRH) transduction of the GnRH receptor

GnRH binds its cognate G protein-coupled GnRH receptor (GnRHR) located on pituitary gonadotropes and drives expression of gonadotropin hormones. There are two gonadotropin hormones, comprised of a common alpha- and hormone-specific beta-subunit, which are required for gonadal function. Recently we identified that Fanconi anemia a (Fanca), a DNA damage repair gene, is differentially expressed within the LbetaT2 gonadotrope cell line in response to stimulation with GnRH. FANCA is mutated in more than 60% of cases of Fanconi anemia (FA), a rare genetically heterogeneous autosomal recessive disorder characterized by bone marrow failure, endocrine tissue cancer susceptibility, and infertility. Here we show that induction of FANCA protein is mediated by the GnRHR and that the protein constitutively adopts a nucleocytoplasmic intracellular distribution pattern. Using inhibitors to block nuclear import and export and a GnRHR antagonist, we demonstrated that GnRH induces nuclear accumulation of FANCA and green fluorescent protein (GFP)-FANCA before exporting back to the cytoplasm using the nuclear export receptor CRM1. Using FANCA point mutations that locate GFP-FANCA to the cytoplasm (H1110P) or functionally uncouple GFP-FANCA (Q1128E) from the wild-type nucleocytoplasmic distribution pattern, we demonstrated that wild-type FANCA was required for GnRH-induced activation of gonadotrope cell markers. Cotransfection of H1110P and Q1128E blocked GnRH activation of the alphaGsu and GnRHR but not the beta-subunit gene promoters. We conclude that nucleocytoplasmic shuttling of FANCA is required for GnRH transduction of the alphaGSU and GnRHR gene promoters and propose that FANCA functions as a GnRH-induced signal transducer.     

Hyperprolactinemia inhibits gonadotropin-releasing hormone (GnRH) stimulation of the number of pituitary GnRH receptors

Gonadotropin secretion is diminished in the presence of hyperprolactinemia, and previous studies have shown that PRL can reduce GnRH secretion and impair LH responses to GnRH. To investigate the mechanisms of the inhibitory effects of PRL on the pituitary, we administered intraarterial pulse injections of GnRH (25 ng/pulse every 30 min) to castrate testosterone-implanted male rats placed in restraint cages. Serum PRL, GnRH receptor (GnRH-R), and LH responses to GnRH were measured at intervals over 72 h. In control animals which received saline pulses, serum PRL was transiently elevated to the range of 100-150 ng/ml during the first 24 h, GnRH-R remained stable (approximately 300 fmol/mg protein) and serum LH was low (less than 10 ng/ml) throughout the 72 h. GnRH pulses in castrate testosterone-implanted animals increased GnRH-R to values (approximately 600 fmol/mg) similar to those in castrate controls (no testosterone implant, saline pulses) through 48 h, but GnRH-R declined to baseline values by 72 h in both groups. Serum LH responses to GnRH pulses were only present at 24 h. Administration of bromocriptine throughout the 72 h to immobilized castrate rats or to castrate testosterone-replaced animals treated with GnRH pulses suppressed serum PRL, and GnRH-R concentrations remained elevated through 72 h. Serum LH responses to GnRH pulses were 5- to 20-fold higher in bromocriptine-treated rats, and responses were present throughout the 72 h of the experiment. Delaying the start of bromocriptine treatment until 36 h (after the spontaneous PRL peak) resulted in reduced GnRH-R and LH responses at 72 h. Similarly, administration of ovine PRL (during the first 48 h) to bromocriptine-treated rats produced low GnRH-R concentrations at 72 h. Thus, the transient elevation of PRL seen in immobilized rats can inhibit the GnRH-stimulated increase in GnRH-R and is associated with reduced LH responses to GnRH. These results indicate that PRL has a direct inhibitory effect on the gonadotrope and suggest that impaired GnRH-R responses to GnRH are one of the mechanisms involved in the diminished gonadotropin secretion seen in hyperprolactinemia.     

Autosomal recessive idiopathic hypogonadotropic hypogonadism: genetic analysis excludes mutations in the gonadotropin-releasing hormone (GnRH) and GnRH receptor genes

Failure of the normal pattern of episodic secretion of GnRH from the hypothalamus results in the clinical syndrome of idiopathic hypogonadotropic hypogonadism (IHH), with failure of pubertal development and infertility. The only gene that has been implicated in normosmic IHH is the GnRH receptor gene (GNRHR), which accounts for 10% of cases. This report presents four families with autosomal recessive IHH, including a consanguineous pedigree from the Middle East. Defects within the genomic coding sequence of the GNRHR, and the GnRH gene itself, GNRH1, were excluded by temperature gradient gel electrophoresis, direct sequencing, and haplotypes created from simple sequence polymorphisms flanking the GNRH1 and GNRHR loci. We concluded that: 1) genetic analysis has excluded sequence variations in GNRH1 and GNRHR in four families with recessive IHH, suggesting the existence of a novel, as-yet-undiscovered gene for this condition, and 2) because mutation analysis of genomic coding sequence will fail to detect mutations deep within introns or regulatory regions, haplotype analysis is the preferred genetic methodology to eliminate the role of specific candidate genes.