Gonadotropin-releasing Hormone Receptor Protein Research Articles

Gonadotropin-releasing hormone receptor inhibits triple-negative breast cancer proliferation and metastasis.

BackgroundGonadotropin-releasing hormone receptor (GnRHR) is expressed in several malignant tumors and inhibits the proliferation and metastasis of cancer cells, but its role in triple-negative breast cancers (TNBCs) is unclear. This study investigated the biological effects of GnRHR and their influence on TNBC prognosis.MethodsThe GSE21653 database was used to obtain information about GnRHR expression and clinicopathological factors in patients with TNBC. GnRHR was activated in cultured MDA-MB-231 and MDA-MB-468 cells by leuprolide acetate and antagonized by elagolix sodium. Cell proliferation was assessed by the cell counting kit-8 and colony formation assays. Cell metastasis was detected by the wound healing assay and Transwell assay. Apoptosis and the cell cycle were investigated by flow cytometry. GnRHR protein expression was determined by western blotting.ResultsGnRHR mRNA expression was significantly higher in patients with TNBC than in hormone receptor+/human epidermal growth factor receptor (HER)2– and HER2+ patients with breast cancer. Patients with high GnRHR expression had significantly better disease-free survival than those with lower expression. Activated GnRHR significantly inhibited cell proliferation and metastasis, increased apoptosis, and enhanced GnRHR protein expression levels.ConclusionGnRHR inhibits TNBC proliferation and metastasis, suggesting it could be targeted for TNBC treatment.

EVALUATING THE INHIBITORY EFFECT OF ELAGOLIX & RELUGOLIX ON LEIOMYOMA GROWTH IN 2D CELL CULTURE

Elagolix and relugolix, both oral GnRH antagonists, are structurally similar; however their pharmakinetic properties differ. At the GnRH receptor elagolix’s IC50 is 1.5 nM with a half-life of 4-6 hours, while relugolix’s IC50 is 0.33 nM with a half-life of 49 hours. The goal of this study is to compare the effect elagolix and relugolix have on the expression of extracellular matrix (ECM) proteins in human leiomyoma cells in 2D cell culture. We hypothesized that both GnRH antagonists directly impact ECM protein expression relative to untreated leiomyoma cells. Leiomyoma cells were grown 2-dimentionally and exposed to elagolix and relugolix for 24 and 48 hours. Expression of various ECM proteins and GnRHR were analyzed using western blot and immunohistochemistry. At 100 nM relugolix (n=10) resulted in maximal, decreased expression of col1A1 at 24 hours (1.78 ± 0.06-fold; p<0.01) and 48 hours (1.92 ± 0.14-fold; p<0.04) and the difference between the two time points was not significant (p=0.87). Elagolix (n=11) resulted in maximal, decreased expression of col1A1 at 24 hours (1.56 ± 0.14-fold; p<0.03) but suppression was lost by 48 hours; the difference between the time points was significant (p<0.01). At 100 nM relugolix resulted in maximal, decreased expression of fibronectin at 24 hours (1.7 ± 0.07-fold; p<0.01) and 48 hours (1.8 ± 0.07-fold; p<0.01) with the difference between the two time points not being significant (p=0.52); elagolix resulted in maximal, decreased expression of fibronectin at 24 hours (1.7 ± 0.14-fold; p=0.01) and 48 hours (2.0 ± 0.09-fold; p<0.01) with no significant difference between the time points (p=0.19). An upregulation was seen in versican expression when cells were treated with relugolix for 24 hours though no concentration achieved growth to statistical significance when compared to untreated cells. A significant, concentration dependent downregulation was achieved at 48 hours (p<0.01) and at 100 nM relugolix resulted in maximal, decreased expression of versican (1.5 ± 0.07-fold; p<0.01). At 24 hours maximal, decreased expression of versican was seen at 100 nM (1.6 ± 0.10-fold; p<0.01) for cells treated with elagolix; a statistically significant upregulation was seen at 48 hours (p=0.02). In terms of the GnRH receptor, no significant difference between GnRH receptor protein expression in relugolix and elagolix was seen at 24 hours (p=0.57). At 48 hours, GnRH receptor protein expression increased in cells treated with elagolix when compared to cells treated with relugolix (p=0.037) suggesting the suppressive effect of elagolix at the receptor is lost by 48 hours. Our findings suggest that treatment with relugolix and elagolix directly regulated collagen, fibronectin, versican, and GnRH receptor protein expression in our 2D human leiomyoma cell culture model.

SAT-292 Musashi: A Novel Regulator of the Gonadotrope Transcriptome

Sufficient nutrition is critical for reproduction. We have previously shown that leptin, a circulating indicator of fat stores, signals to pituitary gonadotropes to maintain gonadotropin releasing hormone receptor (GnRHR) protein levels in female mice. We hypothesized that this process is post-transcriptional, happening primarily through regulation of the RNA-binding protein Musashi (MSI). We showed that MSI binds to Gnrhr and inhibits translation, and a gonadotrope-specific deletion of Msi1 and Msi2 (Gon-Msi1/2-null) leads to increased GnRHR protein levels. This culminates in dysregulated luteinizing hormone (LH) and follicle-stimulating hormone (FSH). We have recently identified other gonadotrope and pituitary targets of MSI. We therefore suspected that MSI plays a role in both the maturation of gonadotropes and the normal cyclic regulation of gonadotropes. We hypothesized that the deletion of MSI would lead to downstream effects on (1) the composition of the gonadotrope population and (2) the molecular landscape of these cells. Using our adult, diestrous Gon-Msi1/2-null females, we performed single-cell RNA-sequencing on methanol-fixed dispersed pituitary cells. Libraries were made from two control pools and two mutant pools (n=3 pituitaries/pool) using 10x Genomics v3.1 Single-Cell Gene Expression technology and initially sequenced on an Illumina Next-seq mid-output flow-cell, yielding 5,000 reads/cell. Subsequent high-output sequencing obtained 25,000 reads/cell. We recovered single-cell mRNA transcript information from 18,206 control pituitary cells and 16,255 Gon-Msi1/2-null cells. Our analyses revealed that the Gon-Msi1/2-null pools had a higher % of cells expressing Fshb, as well as an expected significant drop in Msi2-expressing gonadotropes and no change in Lhb-expressing cells. We have recently identified Fshb as an MSI target in silico, and qRT-PCR of female pituitary lysate immunoprecipitated with anti-MSI1 shows a 7-fold enrichment in Fshb mRNA. We identified differentially expressed genes comparing the control and Gon-Msi1/2-null gonadotrope clusters. Using Gene Ontology analyses, the Gon-Msi1/2-null gonadotrope cluster appears to have aberrant expression of mRNAs involved in protein folding and cellular responses to nutrients. Our high-output sequencing has allowed us to achieve 25,000 reads/cell and will provide greater resolution of the role of Musashi in control of gonadotrope function. Taken together, our data indicate that Musashi influences the molecular landscape and subsequent physiology of the female gonadotrope. We have identified potential gonadotrope-specific MSI targets, including pathways that may underlie the dysregulated gonadotropin production and secretion seen in our Gon-Msi1/2-null females. Future studies will compare pubertal and adult females, as well as females from different estrous cycle stages.

Effects of central orexin A on gonadotropins and progesterone secretion in ewes during the luteal phase of the estrous cycle and during anestrus

Prior studies, mainly on rodents demonstrated that orexin A (OXA) plays an important role in regulating the hypothalamo-pituitary-gonadal (HPG) axis. However, the results of those works have indicated that the central impact of OXA on the HPG axis may vary depending on the species or sex of the animal, the phase of the estrous cycle, or the experimental model system (in vivo vs. in vitro). The aim of the present study was to further investigate the role of OXA in the regulation of reproduction in seasonally breeding, intact, non-steroid-primed ewes during the luteal phase of the estrous cycle and during anestrus. We examined the effect of fourth-ventricle injections of OXA (0.3 μg/kg body weight) on plasma luteinizing hormone (LH), follicle-stimulating hormone (FSH) and progesterone concentrations and on the level of gonadotropin-releasing hormone receptor (GnRH-R) protein in the anterior pituitary (AP). Radioimmunoassays revealed that OXA treatments inhibited (P < 0.001) LH and FSH secretion during both examined periods and progesterone (P < 0.001) during the luteal phase. The influence of OXA on LH and FSH release may be partially attributable to the additional response of the pituitary to OXA, as, under the influence of OXA, the concentration of GnRH-R protein in the AP was downregulated during the luteal phase (P < 0.01) and during anestrus (P < 0.05). We conclude that OXA may be a negative regulator of gonadotropin secretion in intact, non-steroid-primed ewes in periods when the HPG axis is suppressed by negative feedback through steroid sex hormones.

Leptin Regulation of Gonadotrope Gonadotropin-Releasing Hormone Receptors As a Metabolic Checkpoint and Gateway to Reproductive Competence.

The adipokine leptin signals the body’s nutritional status to the brain, and particularly, the hypothalamus. However, leptin receptors (LEPRs) can be found all throughout the body and brain, including the pituitary. It is known that leptin is permissive for reproduction, and mice that cannot produce leptin (Lep/Lep) are infertile. Many studies have pinpointed leptin’s regulation of reproduction to the hypothalamus. However, LEPRs exist at all levels of the hypothalamic–pituitary–gonadal axis. We have previously shown that deleting the signaling portion of the LEPR specifically in gonadotropes impairs fertility in female mice. Our recent studies have targeted this regulation to the control of gonadotropin releasing hormone receptor (GnRHR) expression. The hypotheses presented here are twofold: (1) cyclic regulation of pituitary GnRHR levels sets up a target metabolic checkpoint for control of the reproductive axis and (2) multiple checkpoints are required for the metabolic signaling that regulates the reproductive axis. Here, we emphasize and explore the relationship between the hypothalamus and the pituitary with regard to the regulation of GnRHR. The original data we present strengthen these hypotheses and build on our previous studies. We show that we can cause infertility in 70% of female mice by deleting all isoforms of LEPR specifically in gonadotropes. Our findings implicate activin subunit (InhBa) mRNA as a potential leptin target in gonadotropes. We further show gonadotrope-specific upregulation of GnRHR protein (but not mRNA levels) following leptin stimulation. In order to try and understand this post-transcriptional regulation, we tested candidate miRNAs (identified with in silico analysis) that may be binding the Gnrhr mRNA. We show significant upregulation of one of these miRNAs in our gonadotrope-Lepr-null females. The evidence provided here, combined with our previous work, lay the foundation for metabolically regulated post-transcriptional control of the gonadotrope. We discuss possible mechanisms, including miRNA regulation and the involvement of the RNA binding protein, Musashi. We also demonstrate how this regulation may be vital for the dynamic remodeling of gonadotropes in the cycling female. Finally, we propose that the leptin receptivity of both the hypothalamus and the pituitary are vital for the body’s ability to delay or slow reproduction during periods of low nutrition.

The expression of several reproductive hormone receptors can be modified by perfluorooctane sulfonate (PFOS) in adult male rats

This study was undertaken to evaluate the possible role of several reproductive hormone receptors on the disruption of the hypothalamic-pituitary-testis (HPT) axis activity induced by perfluorooctane sulfonate (PFOS). The studied receptors are the gonadotropin-releasing hormone receptor (GnRHr), luteinizing hormone receptor (LHr), follicle-stimulating hormone receptor (FSHr), and the androgen receptor (Ar). Adult male rats were orally treated with 1.0; 3.0 and 6.0 mg of PFOS kg−1 d−1 for 28 days. In general terms, PFOS can modify the relative gene and protein expressions of these receptors in several tissues of the reproductive axis. At the testicular level, apart from the expected inhibition of both gene and protein expressions of FSHr and Ar, PFOS also stimulates the GnRHr protein and the LHr gene expression. The receptors of the main hormones involved in the HPT axis may have an important role in the disruption exerted by PFOS on this axis.

Equine chorionic gonadotropin influence on sheep oocyte in vitro maturation, apoptosis, and follicle-stimulating hormone receptor and luteinizing hormone receptor expression.

We assessed the effects of equine chorionic gonadotropin (eCG) on oocyte in vitro maturation (IVM), apoptosis, and follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), and gonadotropin-releasing hormone receptor (GnRHR) expression and mRNA levels. Cumulus-oocyte complexes (COCs) were recovered from sheep ovaries and pooled in groups, before being cultured in IVM media containing varying eCG concentrations. Maturation and apoptosis rates were then calculated. Expression of FSHR, LHR, and GnRHR mRNA in oocytes was measured using quantitative reverse transcription polymerase chain reaction. Protein levels were ascertained by western blotting. Matured oocytes displayed and released an intact first polar body. Sheep oocyte maturation rates gradually increased as eCG concentration was raised from 0 to 20 µg/mL. Apoptosis rates of eCG-treated oocytes were lower than those of the control group, and were lowest using 20 µg/mL eCG. FSHR, LHR, and GnRHR mRNA expression increased (P < 0.01, P < 0.05, and P < 0.05, respectively, compared to 0 µg/mL eCG) with eCG concentration, being highest following exposure to 20 µg/mL. FSHR and GnRHR protein levels were significantly higher in oocytes administered 20 µg/mL eCG compared with those matured in the absence of eCG. eCG dose positively correlated with FSHR, LHR, and GnRHR mRNA and protein expression. In conclusion, eCG enhances maturation and decreases apoptosis of oocytes undergoing IVM, and heightens FSHR, LHR, and GnRHR expression. Such increased expression may facilitate oocyte IVM. These findings contribute to our understanding of the mechanisms of underlying hormonal control of sheep oocyte IVM, advancing ovine reproductive methods.

Molecular cloning, sequencing, and distribution of feline GnRH receptor (GnRHR) and resequencing of canine GnRHR

GnRH receptors play vital roles in mammalian reproduction via regulation of gonadotropin secretion, which is essential for gametogenesis and production of gonadal steroids. GnRH receptors for more than 20 mammalian species have been sequenced, including human, mouse, and dog. This study reports the molecular cloning and sequencing of GnRH receptor (GnRHR) cDNA from the pituitary gland of the domestic cat, an important species in biomedical research. Feline GnRHR cDNA is composed of 981 nucleotides and encodes a 327 amino acid protein. Unlike the majority of mammalian species sequenced so far, but similar to canine GnRHR, feline GnRHR protein lacks asparagine in position three of the extracellular domain of the protein. At the amino acid level, feline GnRHR exhibits 95.1% identity with canine, 93.8% with human, and 88.9% with mouse GnRHR. Comparative sequence analysis of GnRHRs for multiple mammalian species led to resequencing of canine GnRHR, which differed from that previously published by a single base change that translates to a different amino acid in position 193. This single base change was confirmed in dogs of multiple breeds. Reverse transcriptase PCR analysis of GnRHR messenger RNA in different tissues from four normal cats indicated the presence of amplicons of varying lengths, including full-length as well as shortened GnRHR amplicons, pointing to the existence of truncated GnRHR transcripts in the domestic cat. This study is the first insight into molecular composition and expression of feline GnRHR and promotes better understanding of receptor organization, and distribution in various tissues of this species.

Selective deletion of leptin receptors in gonadotropes reveals activin and GnRH-binding sites as leptin targets in support of fertility.

The adipokine, leptin (LEP), is a hormonal gateway, signaling energy stores to appetite-regulatory neurons, permitting reproduction when stores are sufficient. Dual-labeling for LEP receptors (LEPRs) and gonadotropins or GH revealed a 2-fold increase in LEPR during proestrus, some of which was seen in LH gonadotropes. We therefore investigated LEPR functions in gonadotropes with Cre-LoxP technology, deleting the signaling domain of the LEPR (Lepr-exon 17) with Cre-recombinase driven by the rat LH-β promoter (Lhβ-cre). Selectivity of the deletion was validated by organ genotyping and lack of LEPR and responses to LEP by mutant gonadotropes. The mutation had no impact on growth, body weight, the timing of puberty, or pregnancy. Mutant females took 36% longer to produce their first litter and had 50% fewer pups/litter. When the broad impact of the loss of gonadotrope LEPR on all pituitary hormones was studied, mutant diestrous females had reduced serum levels of LH (40%), FSH (70%), and GH (54%) and mRNA levels of Fshβ (59%) and inhibin/activin β A and β B (25%). Mutant males had reduced serum levels of GH (74%), TSH (31%), and prolactin (69%) and mRNA levels of Gh (31%), Ghrhr (30%), Fshβ (22%), and glycoprotein α-subunit (Cga) (22%). Serum levels of LEP and ACTH and mRNA levels of Gnrhr were unchanged. However, binding to GnRH receptors was reduced in LEPR-null LH or FSH gonadotropes by 82% or 89%, respectively, in females (P < .0001) and 27% or 53%, respectively, in males (P < .03). This correlated with reductions in GnRH receptor protein immunolabeling, suggesting that LEP's actions may be posttranscriptional. Collectively, these studies highlight the importance of LEP to gonadotropes with GnRH-binding sites and activin as potential targets. LEP may modulate population growth, adjusting the number of offspring to the availability of food supplies.

Aberrant gonadotropin-releasing hormone receptor (GnRHR) expression and its regulation of CYP11B2 expression and aldosterone production in adrenal aldosterone-producing adenoma (APA)

Aberrant expression of gonadotropin-releasing hormone receptor (GnRHR) has been reported in human adrenal tissues including aldosterone-producing adenoma (APA). However, the details of its expression and functional role in adrenals are still not clear. In this study, quantitative RT-PCR analysis revealed the mean level of GnRHR mRNA was significantly higher in APAs than in human normal adrenal (NA) (P=0.004). GnRHR protein expression was detected in human NA and neoplastic adrenal tissues. In H295R cells transfected with GnRHR, treatment with GnRH resulted in a concentration-dependent increase in CYP11B2 reporter activity. Chronic activation of GnRHR with GnRH (100nM), in a cell line with doxycycline-inducible GnRHR (H295R-TR/GnRHR), increased CYP11B2 expression and aldosterone production. These agonistic effects were inhibited by blockers for the calcium signaling pathway, KN93 and calmidazolium. These results suggest GnRH, through heterotopic expression of its receptor, may be a potential regulator of CYP11B2 expression levels in some cases of APA.

Characterization of the Gonadotropin Releasing Hormone Receptor (GnRHR) Expression and Activity in the Female Mouse Ovary

GnRH agonists (GnRHa) are increasingly used for fertility preservation in women undergoing gonadotoxic chemotherapy. However, the protective mechanisms of action for these compounds have not yet been elucidated. In this study, we aimed to determine whether GnRHa have a direct effect on ovarian granulosa cells. GnRH receptor (GnRHR) expression was determined in mouse somatic and gonadal tissues including granulosa/cumulus cells and oocytes using quantitative RT-PCR and immunohistochemistry. Granulosa cells were isolated from mouse ovaries primed with pregnant mare serum gonadotropin. Response to GnRHa in cultured granulosa cells was assessed by determining the increase of intracellular cAMP and by assessing phosphorylation of downstream mediators of GnRH signaling: ERK and p38. To measure intracellular cAMP in our system, the cells were transfected with a cAMP-responsive luciferase reporter plasmid and stimulated with GnRHa. For all experiments, pituitary tissue and/or the αT3-1 mouse pituitary cell line were used as controls. GnRHR mRNA and protein were detected in mouse ovaries, granulosa/cumulus cells, and oocytes. After GnRHa stimulation at various time intervals, we were unable to detect a cAMP increase or activation of the ERK or p38 signaling pathway in cultured primary mouse granulosa cells, whereas activation was detected in the control αT3-1 mouse pituitary cells. In this study, we have not detected activation of the canonical GnRH signaling pathways in mouse ovarian somatic cells. Our findings suggest that the mechanism of action of GnRHa in the ovary is either below the detection level of our experimental design or is different from that in the pituitary.

Alarelin active immunization influences expression levels of GnRHR, FSHR and LHR proteins in the ovary and enhances follicular development in ewes

We investigated the effects of gonadotropin releasing hormone (GnRH) agonist on expressions of GnRH receptor (GnRHR), follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) proteins in the ovaries and follicular development in the ewes. Forty-two pre-pubertal ewes were assigned to experimental groups 1 to 5 (EG-I to EG-V) and control group (CG). Ewes in EG-I, EG-II and EG-III were subcutaneously injected with 200, 300 or 400 μg alarelin antigens twice (on days 0 and 14), respectively. Ewes in EG-IV and EG-V were subcutaneously injected with 200 μg and 300 μg alarelin antigen four times (on days 0, 7, 14 and 21). Ewes in CG were subcutaneously injected with a solvent twice (on days 0 and 14). Serum concentrations of GnRH antibody in the EGs increased and were higher than (P<0.05) that of CG from day 14 to day 60. GnRH antibody concentrations in EG-IV and EG-V were higher than that in EG-I, EG-II and EG-III from days 35 to 45. Expressions of GnRHR protein in EG-IV and EG-V were lower than that in CG (P<0. 01). Expressions of FSHR and LHR proteins in EGs increased. Levels of FSHR and LHR proteins in EG-IV and EG-V (P<0.05) were higher than CG. Ovarian weights in EGs increased. Values of follicle vertical diameter, follicle transverse diameter, follicle wall thickness, follicle externatheca thickness and follicle internatheca thickness in EG-III and EG-V were greater than other groups. Primordial follicles and primary follicles developed quickly in alarelin-immunized animals. Secondary follicles and mature follicles became more abundant. Mitochondria, mitochondrial cristaes and cortical granules increased. Serum FSH concentrations of EGs remained higher than that in CG from days 28 to 70 (P<0.05). Alarelin immunization stimulated GnRH antibody production, suppressed expression of GnRHR protein, enhanced expressions of FSHR and LHR proteins in ovaries, promoted FSH secretion and thereby accelerated the development of ovaries and follicles in ewes.

Targeting Gonadotropin-releasing Hormone Receptor Inhibits the Early Step of Ovarian Cancer Metastasis by Modulating Tumor-mesothelial Adhesion

Ovarian cancer has a clear predilection to metastasize to the peritoneum, which represents one of the most important prognostic factors of poor clinical outcome. Gonadotropin-releasing hormone (GnRH) receptor is significantly overexpressed during the malignant progression of human ovarian cancer. Here, using lentiviral-based small interfering RNA (siRNA) technology to downregulate GnRH receptor in metastatic ovarian cancer cells, we show that GnRH receptor is an important mediator of ovarian cancer peritoneal metastasis. GnRH receptor downregulation dramatically attenuated their adhesion to the peritoneal mesothelium. By inhibiting the expression of GnRH receptor, we showed decreased expression of α2β1 and α5β1 integrin and adhesion to specific extracellular matrix (ECM) proteins. This was also associated with a reduction of P-cadherin. Furthermore, adhesion of ovarian cancer cells to different ECMs and the mesothelium were abrogated in response to β1 integrin and P-cadherin reduction, confirming that the effects were β1 integrin- and P-cadherin-specific. Using a mouse model of human ovarian cancer metastasis, we found that the inhibition of GnRH receptor, β1 integrin, and P-cadherin significantly attenuated tumor growth, ascites formation, and the number of metastatic implants. These results define a new role for GnRH receptor in early metastasis and offer the possibility of novel therapeutic targets.

G protein-coupled receptor expression in the adult and fetal adrenal glands

Hormonal regulation of adrenal function occurs primarily through G protein-coupled receptors (GPCR), which may play different roles in fetal vs. adult adrenal glands. In this study, we compared the transcript levels of GPCR between fetal and adult adrenal and found that gonadotropin-releasing hormone receptor (GnRHR), latrophilin 3 receptor, G protein-coupled receptor 37, angiotensin II receptor type 2, latrophilin 2 receptor and melanocortin receptor were expressed at significantly higher levels in fetal adrenal. High GnRHR protein expression was also detected in fetal adrenal using immunohistochemical analysis. To define potential ligand sources for fetal adrenal GnRHR, we demonstrated that GnRH1 mRNA was expressed at high levels in the placenta, while fetal adrenal had high expression of GnRH2. In summary, certain GPCR particularly GnRHR were highly expressed in fetal adrenal and the expression of GnRH mRNA in the placenta and the fetal adrenal raises the possibility of endocrine and/or paracrine/autocrine influences on fetal adrenal function. However, the exact function of GnRHR in fetal adrenal remains to be determined.

Immunolocalization of Gonadotropin-Releasing Hormone (GnRH)-I, GnRH-II, and Type I GnRH Receptor during Follicular Development in the Human Ovary

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. 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. Immunohistochemistry, immunofluorescence, immunoblot assay, and real-time RT-PCR were performed. 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. 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.

Molecular cloning and characterization of a gonadotropin-releasing hormone receptor in the guinea pig, Cavia porcellus

Guinea pig gonadotropin-releasing hormone (gpGnRH) is predicted to have a unique structure among all known forms of GnRH molecule [Endocrinology 138 (1997) 4123] and it is of great interest to determine whether the unique structure of gpGnRH is manifested in the characteristics of the guinea pig GnRH receptor. In the present study, we isolated a full-length cDNA for a GnRH receptor from the pituitary gland of the guinea pig. The putative guinea pig GnRH receptor protein has an amino acid identity of 79–87% with mammalian type I GnRH receptors. The amino acid residues which have been demonstrated to be important for ligand binding and signal transduction were conserved in the guinea pig GnRH receptor. However, there are several specific amino acid substitutions among mammalian type I GnRH receptors. Moreover, though the guinea pig has generally been classified as a rodent, the putative GnRH receptor protein did not have some rodent-specific characteristics. Total IP assays demonstrated that the cloned guinea pig GnRH receptor is a functional GnRH receptor and that it shows different preference of ligand sensitivities from the rat GnRH receptor.

Human chorionic gonadotropin down-regulates the expression of gonadotropin-releasing hormone receptor gene in GT1-7 neurons.

Previous studies have shown that human CG (hCG) can down-regulate the expression of GnRH gene in GT1-7 neurons and that these neurons contain GnRH receptors and a self-stimulatory mechanism in the synthesis and release of GnRH. These findings have led us to hypothesize that hCG may down-regulate GnRH receptors to disrupt the self-stimulatory mechanism. To test this hypothesis, we cultured GT1-7 neurons in the presence or absence of an optimal concentration of highly purified hCG (100 ng/ml) and then measured steady-state levels of GnRH receptor messenger RNA (mRNA) transcripts and protein. Northern blotting demonstrated that GT1-7 neurons contain a major 5.5-kb and minor 2.4-kb and 1.6-kb GnRH receptor mRNA transcripts. Ligand blotting showed that GT1-7 neurons also contain 53-kDa and 43-kDa GnRH receptor proteins. Culturing with hCG resulted in a significant decrease in steady-state levels of GnRH receptor mRNA transcripts by 12 h and GnRH receptor proteins by 6 and 12 h, followed by a return to the controls by 24 h. The treatment with hCG had no obvious effect on the transcription rate of GnRH receptor gene. The hCG treatment, however, significantly decreased the half-life of GnRH receptor mRNA transcripts from 27 h to 16 h. In summary, we conclude that treatment with hCG can down-regulate the expression of GnRH receptor gene by decreasing the stability of transcripts in GT1-7 neurons. By down-regulating GnRH receptors, hCG may disrupt the self-stimulatory mechanism in GnRH synthesis.

Human chorionic gonadotropin down-regulates the expression of gonadotropin-releasing hormone receptor gene in GT1-7 neurons

Previous studies have shown that human CG (hCG) can down-regulate the expression of GnRH gene in GT1-7 neurons and that these neurons contain GnRH receptors and a self-stimulatory mechanism in the synthesis and release of GnRH. These findings have led us to hypothesize that hCG may down-regulate GnRH receptors to disrupt the self-stimulatory mechanism. To test this hypothesis, we cultured GT1-7 neurons in the presence or absence of an optimal concentration of highly purified hCG (100 ng/ml) and then measured steady-state levels of GnRH receptor messenger RNA (mRNA) transcripts and protein. Northern blotting demonstrated that GT1-7 neurons contain a major 5.5-kb and minor 2.4-kb and 1.6-kb GnRH receptor mRNA transcripts. Ligand blotting showed that GT1-7 neurons also contain 53-kDa and 43-kDa GnRH receptor proteins. Culturing with hCG resulted in a significant decrease in steady-state levels of GnRH receptor mRNA transcripts by 12 h and GnRH receptor proteins by 6 and 12 h, followed by a return to the controls by 24 h. The treatment with hCG had no obvious effect on the transcription rate of GnRH receptor gene. The hCG treatment, however, significantly decreased the half-life of GnRH receptor mRNA transcripts from 27 h to 16 h. In summary, we conclude that treatment with hCG can down-regulate the expression of GnRH receptor gene by decreasing the stability of transcripts in GT1-7 neurons. By down-regulating GnRH receptors, hCG may disrupt the self-stimulatory mechanism in GnRH synthesis.

Gonadotropin-Releasing Hormone and Its Receptors in Rat Brain

Since the chemical identification of gonadotropin-releasing hormone (GnRH) in 1971, significant progress has been made in understanding the mechanisms by which GnRH action is mediated in the anterior pituitary. In contrast, relatively little information is available which identifies the intracerebral sites and mechanisms of action of GnRH in the brain. Early immunohistochemical studies of GnRH distribution in the central nervous system, together with behavioral and electrophysiological experiments, suggested that GnRH functioned as a neurotransmitter and was, possibly, involved in the expression of reproductive behaviors. The subsequent identification and characterization of GnRH receptors in the brain further strengthened the view that GnRH caused specific effects in select regions of the brain known to be involved in the neuroendocrine regulation of the anterior pituitary and in the generation of reproductive behaviors. After the mouse pituitary GnRH receptor was cloned it became possible to identify brain neurons which contained the GnRH receptor mRNA. Comparison of the locations of the GnRH peptide, the GnRH receptor protein, and the neurons which contain the GnRH receptor mRNA suggests that the GnRH neuronal system itself can potentially provide a direct link between the neuroendocrine regulation of anterior pituitary function and the intracerebral regulation of reproductive behaviors; furthermore, it is possible that the GnRH neuronal system takes an active part in intracerebral feedback loop systems between the mediobasal hypothalamus and the septum-diagonal band which regulate GnRH release from the median eminence.

Localization of putative gonadotrophin releasing hormone receptor protein in the anterior pituitary.

Specific binding of a fully biologically active 125I-gonadotrophin releasing hormone (GnRH) to isolated anterior pituitary cells is time dependent, saturable and the concentration dependent binding curves exhibit positive cooperativity. Binding to intact or solubilized plasma membranes and an affinity purified GnRH receptor protein reveals in all instances multiple high affinity binding sites. Thus, GnRH receptor protein appears to be an intrinsic constituent of the cell membrane, and perhaps, other membranous organelles. To investigate the latter, the binding of 125I-GnRH to various subcellular fractions was studied and its affinity and time requirements determined. GnRH binding to plasma membranes and secretory granules was to multiple high affinity sites, while that to nuclei and microsomes was to a single high affinity site. Binding was 1.83 +/- 0.07, 0.78 +/- 0.04, 0.31 +/- 0.03 and 0.27 +/- 0.03 fmol micrograms-1 protein for isolated plasma membranes, secretory granules, microsomes and nuclei, respectively, after 30 min incubation with 10(-9) M GnRH. The magnitude of binding to microsomes did not change during the incubation period. It did not show any decrease (p greater than 0.05) in isolated nuclei and plasma membranes, except for the 24 h time period, when a significant drop (p less than 0.001) was seen. Binding to the secretory granule fraction culminated at 15 min and then decreased (p less than 0.001) steadily to a non-detectable level at 24 h. Thus GnRH receptor protein or its portion may be an integral part of some membranous particles in the anterior pituitary cells.(ABSTRACT TRUNCATED AT 250 WORDS)