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As an essential mediator in the Gonadotropin-releasing hormone (GnRH) signaling pathway, GnRH receptor (GnRHR) coupled to GnRH, plays an important role in activating the downstream pathway after stimulating a series of cascades to regulate reproduction. To detect the existence of GnRHR and potential GnRH signaling pathway, we cloned and characterized GnRHR in the Chinese mitten crab, Eriocheir sinensis (named EsGnRHR). The full-length EsGnRHR cDNA is 2038 bp in length, including an open reading frame (ORF) of 1566 bp, a 57 bp 5'-untranslated region (5'-UTR) and a 415 bp 3'-UTR. Prediction of transmembrane domains in protein sequence revealed that the EsGnRHR protein contained seven hydrophobic transmembrane regions (TMs). Reverse transcription PCR revealed that EsGnRHR was mainly expressed in the thoracic nerve group and ovary, and weakly distributed in the testis and brain. In situ hybridization further demonstrated that EsGnRHR mRNA was localized at the protocerebrum and deutocerebrum. In the ovary and testis, the hybridization signal was dominantly at the earlier developmental stages. The signal was mainly localized in the cytoplasm cell in the ovary, and in the epithelium cell in the testis. During the different stages of gonadal development, EsGnRHR displayed increasing trends in both female and male when analyzed by quantitative real-time PCR, suggesting that EsGnRHR was involved in controlling gonadal development. Our study provides important information for further research on the molecular mechanisms underlying crab development., (Copyright © 2018 Elsevier B.V. All rights reserved.)

DNA cis-acting elements involved in gene regulation may actively contribute to adaptation processes because they are submitted to lower evolutionary constraints than coding DNA. In this regard, comparisons of the mechanisms underlying basal and regulated Gnrhr expression have revealed some features that promote stable and consistent Gnrhr expression in pituitary gonadotroph cells in different species. The presence of two divergent SF1 (NR5A1) response elements in all analysed mammalian Gnrhr promoters probably comprises one of the features that ensures reliable expression in the pituitary. By contrast, in other tissues, such as the hippocampus and testis, our analyses revealed dissimilar levels of Gnrhr expression among species. Indeed, Gnrhr was consistently expressed after birth in the rat but not the mouse hippocampus. Similar discrepancies were observed in foetal and adult testes. The ability of the rat promoter to drive reporter gene expression in the hippocampus and testis of transgenic mice just as it naturally directs the expression of the endogenous Gnrhr in rats strongly suggests that regulatory DNA sequences contained species-specific instructions prevailing over other controls. The major conclusion emerging from these studies is that Gnrhr promoter sequences are mainly responsible for directing transcriptional programmes and play a predominant role over the species-specific cell environment., (© 2015 British Society for Neuroendocrinology.)

Several evidences supported the existence of melatonin effects on reproductive system in fish. In order to investigate whether melatonin is involved in the modulation of GnRH systems in the European sea bass, we have injected melatonin (0.5 µg/g body mass) in male specimens. The brain mRNA transcript levels of the three GnRH forms and the five GnRH receptors present in this species were determined by real time quantitative PCR. Our findings revealed day-night variations in the brain expression of GnRH-1, GnRH-3 and several GnRH receptors (dlGnRHR-II-1c, -2a), which exhibited higher transcript levels at mid-light compared to mid-dark phase of the photocycle. Moreover, an inhibitory effect of melatonin on the nocturnal expression of GnRH-1, GnRH-3, and GnRH receptors subtypes 1c, 2a and 2b was also demonstrated. Interestingly, the inhibitory effect of melatonin affected the expression of hypophysiotrophic GnRH forms and GnRH receptors that exhibit day-night fluctuations, suggesting that exogenous melatonin reinforce physiological mechanisms already established. These interactions between melatoninergic and GnRH systems could be mediating photoperiod effects on reproductive and other rhythmic physiological events in the European sea bass.

The neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) is present in high concentrations within the hypothalamus, suggesting that it may be a hypophysiotropic factor, whereas pituitary expression suggests a paracrine function. PACAP stimulates gonadotropin secretion and enhances GnRH responsiveness. PACAP increases gonadotropin α-subunit (αGSU), lengthens LHβ, but reduces FSHβ mRNA levels in adult pituitary cell cultures in part by increasing follistatin. PACAP stimulates LH secretion in rats; however, acceptance of PACAP as a regulator of reproduction has been limited by a paucity of in vivo studies. We created a transgenic mouse model of pituitary PACAP overexpression using the αGSU subunit promoter. Real-time PCR was used to evaluate PACAP, follistatin, GnRH receptor, and the gonadotropin subunit mRNA in male transgenic and wild-type mice of various ages. Transgenic mice had greater than 1000-fold higher levels of pituitary PACAP mRNA; and immunocytochemistry, Western blot, and ELISA analyses confirmed high peptide levels. FSH, LH, and testosterone levels were significantly suppressed, and the timing of puberty was substantially delayed in PACAP transgenic mice in which gonadotropin subunit and GnRH receptor mRNA levels were reduced and pituitary follistatin expression was increased. Microarray analyses revealed 1229 of 45102 probes were significantly (P < 0.01) different in pituitaries from PACAP transgenic mice, of which 83 genes were at least 2-fold different. Genes involved in small molecule biochemistry, cancer, and reproductive system diseases were the top associated networks. The GnRH signaling pathway was the top canonical pathway affected by pituitary PACAP excess. These experiments provide the first evidence that PACAP affects gonadotropin expression and sexual maturation in vivo.

Neuromodulators including gonadotropin-releasing hormone (GnRH) and sex steroids help integrate an animal's internal physiological state with incoming external cues, and can have profound effects on the processing of behaviorally relevant information, particularly from the olfactory system. While GnRH and steroid receptors are present in olfactory processing regions across vertebrates, little is known about whether their expression levels change with internal physiological state or external social cues. We used qRT-PCR to measure mRNA levels of two GnRH receptors (GnRH-R1, GnRH-R2), five sex steroid receptors (estrogen receptors: ERalpha, ERbetaa, ERbetab; androgen receptors: ARalpha, ARbeta), and aromatase in the olfactory bulb of the highly social African cichlid fish Astatotilapia burtoni. We asked whether these receptor levels changed with reproductive condition in females, or with social status, which regulates reproductive capacity in males. Our results reveal that mRNA levels of multiple sex steroid, GnRH receptor subtypes, and aromatase in the olfactory bulb vary with sex, social status in males, and reproductive condition in females, which highlights the potential importance of changing receptor levels in fine-tuning the olfactory system during the reproductive cycle. Further, steroid receptor mRNA levels were positively correlated with circulating steroid levels in males, but negatively correlated in females, suggesting different regulatory control between sexes. These results provide support for the hypothesis that the first-order olfactory relay station is a substrate for both GnRH and sex steroid modulation, and suggest that changes in receptor levels could be an important mechanism for regulating reproductive, social, and seasonal plasticity in olfactory perception observed across vertebrates., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Five types of gonadotropin-releasing hormone receptor (GnRH-R) genes, designated as msGnRH-R1, R2, R3, R4, and R5, are expressed in the brain and pituitary of masu salmon (Oncorhynchus masou). In the present study, seasonal changes in the expression of these five genes were examined in the pituitary to elucidate their roles in GnRH action during growth and sexual maturation. In addition, the seasonal variation of these genes in response to GnRH was examined in a GnRH analog (GnRHa) implantation experiment. Pituitary samples were collected 1 week after the implantation every month from immaturity through spawning. The absolute amount of GnRH-R mRNA in single pituitaries was determined by real-time PCR assays. Among the five genes, R4 was predominantly expressed in the pituitaries. In the immature fish, the amount of GnRH-R mRNA varied with seasons and subtypes. In the pre-spawning period, R1 and R4 mRNAs in both sexes and R2 and R3 mRNAs in the females increased 4- to 20-fold and then decreased in the spawning season. The effects of GnRHa treatment were significantly different in both sexes. In the females, GnRHa tended to elevate the expression of all the subtypes of GnRH-R genes in various stages during the experimental period, whereas it had almost no apparent effects in the males. These results indicate that the expression of the five GnRH-R genes is seasonally variable and may be related to the responses of the pituitary hormone genes to GnRH, and the regulation of GnRH-R genes by GnRH is different in both sexes.

Because the GnRH receptor plays a paramount role within the reproductive axis, the understanding of the molecular apparatus that governs the tissue-specific expression and regulation of this gene must lead to a better knowledge of the physiology and the physiopathology of the gonadotrope function. To elucidate these mechanisms, we have used two complementary in vivo and in vitro approaches. Firstly, we have isolated the pituitary promoter of the rat GnRH receptor gene and investigated its activity using transient transfection into two gonadotrope-derived cell lines, the alphaT3-1 and the LbetaT2 cell lines. We have thus defined a primary set of transcription factors involved in the tissue-specific expression of the GnRH receptor gene. These include the steroidogenic factor-1 (SF-1) which plays a decisive role while functionally interacting with proteins related to the GATA and LIM homeodomain families of transcription factors. In addition, we highlighted the critical implication of SF-1 and its functional interaction with a CREB-related factor in the stimulatory action of PACAP (Pituitary Adenylate Cyclase Activating Polypeptide) on promoter activity. These results have led us to analyze the activity of this promoter by transgenesis in the mouse using human placental alkaline phosphatase as a reporter gene. In agreement with the in vitro data, the pituitary promoter was found to confer gonadotrope-specific activity in the pituitary. It was also able to direct transgene expression in several areas of the central nervous system known to express the endogenous GnRH receptor, in particular in the hippocampo-septal complex. Some of these tissue do not express SF-1, suggesting that, in vivo, its role would not be as decisive as suggested by the in vitro experiments. Surprisingly, during pituitary ontogenesis, the transgene is expressed as early as E 13.5 whereas SF-1 is not yet present in the pituitary. Thus, in vivo, SF-1 would not be necessary for the activation of the GnRH receptor gene during the early developmental stages in the pituitary. These results are consistent with data obtained following general or pituitary-specific knockout of the gene encoding SF-1, suggesting that the GnRH receptor is expressed despite the absence of this factor. Identifying the factors responsible for the activation of the GnRH receptor gene at these early developmental stages should make it possible to refine the role of SF-1, not only in gene regulation but more generally, in the physiology and the physiopathology of the gonadotrope function.

Consistent with its neuroendocrine role, gonadotropin-releasing hormone (GnRH) is located principally within the hypothalamus, although extra-hypothalamic expression has been reported. The present study characterized the expression of GnRH and GnRH receptor (GnRH-R) in sheep spinal cord using real-time PCR and immunocytochemistry. Both GnRH and GnRH-R mRNA were detected in sheep spinal cord. Expression of GnRH peptide was localized to discrete locations in the spinal cord, including lamina X (the area surrounding the central canal) and motoneurons in the ventral horn. Although there is no known functional role for GnRH in spinal cord, a role as a potential neurotransmitter/neuromodulator is supported by the expression of both GnRH and GnRH-R in this tissue.

To provide information about the kidney GHRH receptor (GHRH-R), we assessed its tissue and cellular localization, defined its pattern of expression in developing and aging rats, and studied the effects of GHRH on the regulation of GHRH-R mRNA levels and receptor internalization. In situ hybridization and ribonuclease protection assay demonstrated that GHRH-R mRNA is restricted to the Henle's loop (HL). GHRH-R mRNA levels were low in the medulla from 3- and 12-d-old male rats, increased significantly in that from 30- to 70-d-old rats, and decreased in that from 12- and 18-month-old animals. Compared with the GHRH-R mRNA profile obtained in the pituitary, these data support the concept of a tissue-specific regulation of GHRH-R. In HL cell cultures from 70-d-old rats, a 4-h incubation with 1-100 nM rat GHRH-(1-29)NH(2) reduced GHRH-R mRNA levels significantly. As anti-GHRH-R- (392-404) immunoreactivity was demonstrated in HL cells, internalization of [N(alpha)-5-carboxyfluoresceinyl-D-Ala(2),Ala(8), Ala(15),Lys(22)]hGHRH-(1-29)NH(2) in a time- and temperature-dependent manner and inhibition of this process by phenyl arsine oxide indicate that desensitization to GHRH involves both GHRH-R internalization and down-regulation of GHRH-R mRNA levels. Localization of a functional GHRH-R in HL and its regulation during development and aging suggest roles associated with cellular proliferation, differentiation, and/or water/electrolyte transport.

A cytotoxic analogue of LH-RH, AN-207, consisting of 2-pyrrolinodoxorubicin (AN-201) linked to carrier [D-Lys6]LH-RH, was developed for targeted therapy of cancers expressing LH-RH-receptors. To determine its possible side-effects on the pituitary gland, we investigated the gene expression of pituitary LH-RH-receptors and LH secretion in ovariectomized female and normal male rats after treatment with the maximum tolerated dose of AN-207. The effect of AN-207 on the gene expression of the pituitary GH-RH-receptors and GH secretion was also assessed in male rats. Five hours after a single i.v. injection of AN-207 at 175 nmol/kg, there was a 39-51% decrease in mRNA expression for the pituitary LH-RH-receptors in male and female rats. The carrier, at an equimolar dose, caused a similar reduction (37-39%), whereas the cytotoxic radical AN-201, at an equitoxic dose (110 nmol/kg), produced only a 12-24% decrease (NS) in the mRNA expression of LH-RH-receptors. AN-207 and the carrier analogue induced a comparable 90-100-fold increase in serum LH concentrations in male rats, and the same 12-fold elevation in OVX rats at 5 h. Seven days after treatment with AN-207, the mRNA levels for the LH-RH receptors and the serum LH concentration were back to normal in both sexes. AN-207, the carrier, and AN-201 had no significant effect on the expression of mRNA for GH-RH-receptors in the pituitary. In vitro, a continuous perfusion of pituitary cells with 10 nM AN-207 did not affect the hormone-releasing function of the targeted LH cells or the nontargeted GH cells. Our results demonstrate that cytotoxic LH-RH analogue AN-207, at the maximum tolerated dose causes only a transient decrease in the gene expression of the pituitary LH-RH receptors, and the levels of mRNA for LH-RH receptor fully recover within 7 days. Moreover, the carrier hormone moiety, and not the cytotoxic radical in AN-207 is responsible for this transient suppression. Our findings suggest that the therapy with cytotoxic LH-RH analogues will not inflict permanent damage to pituitary function.

The presence of progesterone response element (PRE) in the 5'-flanking region of the human GnRH receptor (GnRHR) suggests the possible regulation of this gene by progesterone (P). In the present study, we examined the effects of P in transcriptional regulation of human GnRHR gene expression at the pituitary and placenta levels since the GnRHR has been detected in both tissues. By the use of transient transfection assays, a differential regulation of human GnRHR promoter activity by P was observed. P treatment resulted in a decrease in promoter activity in the pituitary alphaT3-1 cells, suggesting a P-mediated inhibitory action. Interestingly, P is found to have a stimulatory role at the placental expression of this gene. Addition of RU486 to, or inhibition of endogenous P production by, the placental JEG-3 cells leads to a decrease in promoter activity, which is reversed by the replacement of P. Further studies have identified a putative PRE, namely human GR-PRE (located between -535 and -521, related to translation start site), that may be responsible for the P action since the mutation of these motifs reversed the P-mediated effects. The binding of PR to this element is confirmed by antibody supershift assays. The physiological effects of P are mediated through two PR isoforms, namely PR-A and PR-B. In the present study, overexpression of human PR-A resulted in a decrease in human promoter activity in both pituitary and placental cells. Interestingly, overexpression of PR-B exhibits a cell-dependent transcriptional activity, whereby it functions as a transcription activator in the placenta but as a transcription repressor in the pituitary. In summary, our results demonstrated a differential usage of PR-A and PR-B in transcriptional regulation of human GnRHR gene expression by P at the pituitary and placenta levels.

We have cloned the full-length complementary DNA (cDNA) for a GnRH receptor from Xenopus laevis pituitary cDNA and determined its gene structure. The cDNA encodes a 368-amino acid protein that has a 46% amino acid identity to the human GnRH receptor. The X laevis GnRH receptor has all of the amino acids identified in the mammalian GnRH receptors as sites of interaction with the GnRH ligand. However, this receptor cDNA shares the same distinguishing structural features of the GnRH receptor that have been characterized from other nonmammalian vertebrates. These include the pair of aspartate residues in the transmembrane domains II and VII compared with the aspartate/asparagine arrangement in mammalian receptors, the amino acid PEY motif in extracellular loop III (SEP in mammals), and the presence of a carboxyl-terminal tail. Previous studies have reported that mammalian GnRH was equipotent to other naturally occurring GnRH subtypes in stimulating LH release from the amphibian pituitary. However, in this study we show that the X. laevis GnRH receptor has ligand selectivity for the naturally occurring GnRHs similar to other nonmammalian GnRH receptors. The order of potency of the GnRHs in stimulating inositol phosphate production in COS-1 cells transiently transfected with the X. laevis GnRH receptor cDNA was chicken GnRH II>salmon GnRH>mammalian GnRH. Transcripts of this GnRH receptor are expressed in the pituitary and midbrain of X. laevis.

There is evidence that GnRH and its binding sites are expressed in numerous extrapituitary tissues, including the primate ovary. However, the factors that regulate ovarian GnRH and its receptor (GnRH-R) remain poorly characterized. Since gonadal steroids are key regulators of ovarian functions, the present study investigated the role of 17beta-estradiol (E2) in regulating GnRH and GnRH-R messenger RNA (mRNA) from human granulosa-luteal cells (hGLCs). RT-PCR was used to isolate the ovarian GnRH-R transcript equivalent to the full-length coding region in the pituitary from hGLCs. Sequence analysis revealed that the ovarian GnRH-R mRNA is identical to its pituitary counterpart. Basal expression studies indicated that GnRH and GnRH-R mRNA levels significantly increased with time in vitro, reaching levels of 160% and 170% on day 8 and 10 of culture, respectively (P < 0.05). Treatment with various concentrations of estradiol (1-100 nM) for 24 h resulted in a dose-dependent decrease (P < 0.05) in GnRH and GnRH-R mRNA levels. Time course studies indicated that short-term treatment (6 h) with E2 (1 nM) had no significant effect on GnRH mRNA levels, while long-term treatment (48 h) with E2 resulted in a 40% decrease (P < 0.001) in GnRH mRNA levels. In contrast, GnRH-R mRNA levels exhibited a biphasic pattern, such that a short-term treatment (6 h) with E2 increased GnRH-R mRNA levels by 20% (P < 0.05), whereas long-term treatment (48 h) resulted in a 60% decrease (P < 0.001) in GnRH-R expression in hGLCs. Cotreatment of estradiol and tamoxifen blocked the E2 induced-regulation of GnRH and its receptor mRNAs, indicating that the E2 effect was mediated through its receptor. In summary, our studies demonstrate that the ovary possesses an intrinsic GnRH axis that is regulated during luteinization in vitro, and that E2 is capable of regulating GnRH and its receptor in the human ovary.

Although the ability of estradiol to enhance pituitary sensitivity to GnRH is established, the underlying mechanism(s) remain undefined. Herein, we find that approximately 9,100 bp of 5' flanking region from the ovine GnRH receptor (oGnRHR) gene is devoid of transcriptional activity in gonadotrope-derived cell lines and is not responsive to either estradiol or GnRH. In stark contrast, this same 9,100 bp promoter fragment directed tissue-specific expression of luciferase in multiple lines of transgenic mice. To test for hormonal regulation of the 9,100-bp promoter, ovariectomized transgenic females were treated with a GnRH antiserum alone or in combination with estradiol. Treatment with antiserum alone reduced pituitary expression of luciferase by 80%. Pituitary expression of luciferase in animals receiving both antiserum and estradiol was approximately 50-fold higher than animals receiving antiserum alone. The estradiol response of the -9,100-bp promoter was equally demonstrable in males. In addition, a GnRH analog (D-Ala-6-GnRH) that does not cross-react with the GnRH antiserum restored pituitary expression of luciferase in males passively immunized against GnRH to levels not different from castrate controls. Finally, treatment with both estradiol and D-Ala-6-GnRH increased pituitary expression of luciferase to a level greater than the sum of the individual treatments suggesting synergistic activation of the transgene by these two hormones. Thus, despite the complete absence of transcriptional activity and hormonal responsiveness in vitro, 9,100 bp of proximal promoter from the oGnRHR gene is capable of directing tissue-specific expression and is robustly responsive to both GnRH and estradiol in transgenic mice. To begin to refine the functional boundaries of the critical cis-acting elements, we next constructed transgenic mice harboring a transgene consisting of 2,700 bp of 5' flanking region from the oGnRHR gene fused to luciferase. As with the -9,100 bp promoter, expression of luciferase in the -2,700 lines was primarily confined to the pituitary gland, brain and testes. Furthermore, the passive immunization-hormonal replacement paradigms described above revealed both GnRH and estradiol responsiveness of the -2,700-bp promoter. Thus, 2,700 bp of proximal promoter from the oGnRHR gene is sufficient for tissue-specific expression as well as GnRH and estradiol responsiveness. Given the inability to recapitulate estradiol regulation of GnRHR gene expression in vitro, transgenic mice may represent one of the few viable avenues for ultimately defining the molecular mechanisms underlying estradiol regulation of GnRHR gene expression.

Mammalian GnRH receptor (GnRHR) is unique among G protein-coupled seven-transmembrane segment receptors due to the absence of an intracellular C-terminal tail frequently important for internalization and/or desensitization of other G protein-coupled receptors. The recent cloning of nonmammalian (i.e. catfish, goldfish, frog, and chicken) GnRHRs shows that these contain an intracellular C terminus. Addition of the 51-amino acid intracellular C terminus from catfish GnRHR (cfGnRHR) to rat GnRHR (rGnRHR) did not affect rGnRHR binding affinity but elevated receptor expression by about 5-fold. Truncation of the added C terminus impaired the elevated receptor-binding sites by 3- to 8-fold, depending on the truncation site. In addition, introducing the C terminus to rGnRHR altered the pattern of receptor regulation from biphasic down-regulation and recovery to monophasic down-regulation. The extent of down-regulation was also enhanced. The alteration in receptor regulation due to the addition of a C terminus was reversed by truncation of the added C terminus. Furthermore, addition of the cfGnRHR C terminus to rGnRHR significantly augmented the inositol phospholipid (IP) response of transfected cells to Buserelin, but this did not result from the elevation of receptor-binding sites. Addition of the C terminus did not affect Buserelin-stimulated cAMP and PRL release. GH3 cells transfected with wild-type cfGnRHR did not show measurable Buserelin binding or significant stimulation of IP, cAMP, or PRL in response to Buserelin (10[-13]-10[-9] M). GH3 cells transfected with C terminus-truncated cfGnRHR showed no IP response to Buserelin (10[-13]-10[-7] M). These results suggest that addition of the cfGnRHR intracellular C terminus to rGnRHR has a significant impact on rGnRHR expression and regulation and efficiency of differential receptor coupling to G proteins.

The present study is designed to evaluate the relationship between gonadotropin-releasing hormone (GnRH) and GnRH receptor (GnRHR) gene expression during the steroid-induced LH surge. One week after ovariectomy (OVX), a capsule containing 17beta-estradiol (E) or vehicle (V) was implanted into OVX rats, and 2 days later a single injection of progesterone (P) or V was administered s.c. at 10:00 h. Poly(A)-rich RNA samples were isolated from the micropunches of the preoptic area (POA) and the posterior mediobasal hypothalamus (pMBH) from both sides of individual brain slices. Using competitive reverse transcription-polymerase chain reaction (RT-PCR) procedures, three parameters (POA GnRH, pMBH GnRHR) and pituitary GnRHR mRNA levels were simultaneously determined in each individual animal. POA GnRH mRNA and pituitary GnRHR mRNA levels were decreased by treatment with E, but increased by a combination of E and P. In contrast, pMBH GnRHR mRNA levels were clearly augmented by treatment with E, and decreased by the combination of E and P. Temporal changes in such parameters were determined in OVX+E+V- and OVX+E+P-treated rats. P augmented POA GnRH mRNA levels at the time of the LH surge (17:00 h) and the increased GnRH mRNA levels were remained until 22:00 h, while E alone failed to alter POA GnRH mRNA levels. In the pMBH micropunch samples, P substantially decreased E-induced increase in GnRHR mRNA levels at 17:00 h and further lowered those until 22:00 h. Antisense oligonucleotides of GnRHR mRNA administered into the lateral ventricle of OVX+E-treated rats blocked the E-induced increase in pMBH GnRHR mRNA levels. The antisense oligonucleotides also prevented the LH surge as well as the increase in pituitary GnRHR mRNA levels in the OVX+E+P-treated group. However, administration of this antisense oligonucleotides failed to alter POA GnRH mRNA levels. In conclusion, the present study demonstrated that there is an inverse relationship between POA GnRH mRNA levels and pMBH GnRHR mRNA levels in response to E and/or P, and that the blockade of the E-induced increase in pMBH GnRHR mRNA levels effectively nullified the P-induced LH surge. These results indicate that pMBH GnRHR gene expression is involved in synchronizing the GnRH neuronal activity, which is crucial for the generation of the LH surge.

The hypothalamic hormone gonadotropin-releasing hormone (GnRH) is released in a pulsatile fashion, with its frequency varying throughout the reproductive cycle. Varying pulse frequencies and amplitudes differentially regulate the biosynthesis and secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by pituitary gonadotropes. The mechanism by which this occurs remains a major question in reproductive physiology. Previous studies have been limited by lack of available cell lines that express the LH and FSH subunit genes and respond to GnRH. We have overcome this limitation by transfecting the rat pituitary GH3 cell line with rat GnRH receptor (GnRHR) cDNA driven by a heterologous promoter. These cells, when cotransfected with regulatory regions of the common alpha, LH beta, or FSH beta subunit gene fused to a luciferase reporter gene, respond to GnRH with an increase in luciferase activity. Using this model, we demonstrate that different cell surface densities of the GnRHR result in the differential regulation of LH and FSH subunit gene expression by GnRH. This suggests that the differential regulation of gonadotropin subunit gene expression by GnRH observed in vivo in rats may, in turn, be mediated by varying gonadotrope cell surface GnRHR concentrations. This provides a physiologic mechanism by which a single ligand can act through a single receptor to regulate differentially the production of two hormones in the same cell.

PRL has been shown to induce a number of genes after the stimulation of quiescent Nb2 T-cells, including c-fos, c-myc, ornithine decarboxylase, interferon regulatory factor-1, and others. One of these genes, LHRH, has not previously been reported to respond in this manner, although we and others have reported its presence in rat and human T- and B-cells. Furthermore, recent evidence suggests that LHRH functions as an immunoregulator in a cytokine-like manner. Using the rat immature T-cell line Nb2, we present data showing for the first time that 1) the LHRH gene is regulated by PRL at various times during the cell cycle; 2) an alternatively spliced LHRH messenger RNA exists in Nb2 cells and may produce a new truncated GnRH-associated peptide (alternatively called PIF for PRL-inhibiting factor); 3) the LHRH receptor is expressed in lymphocytes in a manner similar to the LHRH gene after PRL addition, and its complementary DNA sequence is identical to that of the pituitary receptor; 5) the SH gene, found on the opposite strand of the LHRH gene, is expressed in lymphocytes at the same time and in the same manner as the LHRH gene; 6) the LHRH messenger RNA has a very short half-life in these cells; and 7) the lymphocyte LHRH transcription start site is essentially the same as the hypothalamic site. These data strengthen the relationship between PRL and LHRH expression in the immune system and further support our contention that LHRH is an important immunoregulator, on par with other known cytokines.

The binding of gonadotropin-releasing hormone (GnRH) to its receptor in the anterior pituitary gland is the key molecular interaction regulating the reproductive process of mammals. Here, we report the isolation of a cDNA representing this receptor from rat anterior pituitary and the regulation of expression of its mRNA. The rat GnRH receptor cDNA was composed of 2909 nucleotides and encoded a protein containing 327 amino acids having a seven transmembrane topology. Northern blot analysis on RNA from rat pituitary, ovary and testis showed four different transcripts (5.0, 4.5, 2.5 and 1.3 kb) of which the 5.0 kb form was most abundant. The levels of expression of the transcripts were found to be highest in the pituitary followed by the ovary and the testis (about 40% and 5% compared to pituitary, respectively). Using the more sensitive reverse transcriptase/PCR technique, we also detected GnRH receptor mRNA in the adrenal and the hypothalamus. Measurement of pituitary GnRH receptor mRNA levels (the 5.0 kb form) during the estrous cycle showed the lowest levels at estrus (1.0-fold), a 2.2 +/- 0.57 (mean +/- SEM) -fold increase at diestrus I, a 3.5 +/- 0.41-fold increase at diestrus II, a 2.6 +/- 0.34-fold increase on the morning of proestrus, and a 1.9 +/- 0.25-fold on the afternoon of proestrus. Removal of the ovaries led to a 2.7 +/- 0.29-fold increase in GnRH receptor mRNA levels in the pituitary gland; treatment of ovariectomized rats with estrogen resulted in a significant decrease in GnRH receptor mRNA levels. Our studies demonstrate ovarian regulation of GnRH receptor mRNA expression in the anterior pituitary gland.

Receptors for gonadotropin releasing hormone (GnRH), located in the cell membranes of adeno-hypophysial gonadotropes, mediate the action of GnRH to stimulate the secretion of the gonadotropic hormones (LH and FSH). In the present studies, we have isolated a GnRH receptor cDNA from bovine pituitary, determined its primary structure, and studied the regulation of its gene expression. The cDNA is composed of 1326 nucleotides and encodes a protein containing 328 amino acids. The GnRH receptor of cattle, like that in humans and mice, is a seven transmembrane receptor and has structural characteristics homologous with the family of G protein-coupled receptors. It exhibits 91% identity at the amino acid level with the human and 86% identity with mouse and rat receptors. Northern blot analysis of the RNA from bovine pituitary, probed with 32P-labeled bovine GnRH receptor cDNA, revealed the presence of four different transcripts (5.0, 3.5, 2.5 and 1.5 kb) in the pituitary of which the 5.0 kb form was most abundant. Using the reverse transcriptase/PCR technique, we detected expression of GnRH receptor mRNA in the pituitary but not in any other extrapituitary tissues such as the hypothalamus, hippocampus, testis, corpus luteum, ovary (containing follicles), myoendometrium, adrenal, kidney, liver and spleen. Higher levels of GnRH receptor mRNA were found in the pituitaries of steers than in cohort bulls, suggesting regulation of GnRH receptor gene expression by testicular steroids.