Gnrhr Expression Research Articles

Regulation of two forms of gonadotropin-releasing hormone receptor gene expression in the protandrous black porgy fish, Acanthopagrus schlegeli

Two GnRH receptors (GnRH-R I and GnRH-R II) were obtained in protandrous black porgy (Acanthopagrus schlegeli). We investigated their tissue distribution, developmental/seasonal changes and regulation of expression using in vivo and in vitro (primary cultures of dispersed pituitary cells) approaches. The relative expressions of GnRH-Rs in the pituitary and gonad were as follows: pituitary: GnRH-R I > GnRH-R II; testicular tissue: GnRH-R I > GnRH-R II; ovarian tissue: GnRH-R I = GnRH-R II. GnRH-R I but not GnRH-R II expression was higher in the pituitary during the spawning period as compared to the prespawning. The expression profiles of both forms of GnRH-R were variable in the gonads according to the gonadal stage and season. In vivo, hCG stimulated GnRH-R I and GnRH-R II expression in testis and ovary. The LHRH analog also up-regulated both receptors in testis and but increased only GnRH-R II in the ovary. Sex steroids (estradiol, E2 and testosterone, T) increased the expression of both receptors in the testis and ovary. In the pituitary, sex steroids (E2 and T) increased the expression of GnRH-R I, but not GnRH-II, both in vivo and in vitro. The LHRH analog also specifically up-regulated the expression of GnRH-R I, but not GnRH-R II, by pituitary cells in vitro. All these data suggest that GnRH-R I rather than GnRH-R II may play a major physiological role in the pituitary. In contrast, both GnRH-R I and GnRH-R II may participate in the regulation of gonadal functions, including a possible role during sex change.

Transcriptional Regulation of the Porcine Gonadotropin Releasing Hormone II Receptor Gene.

Gonadotropin-releasing hormone (GnRH) II has been implicated in the interaction between female energy balance and reproductive function, in addition to an anti-proliferative/apoptotic role in cancer cell lines. Specifically, administration of GnRH II to underfed female mice or musk shrews rescued mating behavior. Moreover, endometrial and ovarian cancer cell proliferation is reduced by GnRH II, likely via interaction with the GnRH I receptor. Although the GnRH I receptor has been well characterized, the molecular mechanisms underlying regulation of the GnRH II receptor (GnRHRII) remain to be elucidated. To determine the importance of the GnRH II ligand-receptor system, the pig represents an ideal model since it produces GnRH II and 2 isoforms of GnRHRII. Our laboratory isolated 3029 bp of 5' flanking sequence for the porcine GnRHR II gene. Transient transfections with vectors containing the GnRHR II promoter fused to the cDNA encoding luciferase (3029pGL3) indicated that this promoter is active in cell lines derived from both reproductive and non-reproductive tissues. The objective of this study was to determine the transcription factors and corresponding binding sites that confer cell-specific activity of the porcine GnRHRII gene promoter in the swine testes (ST) cell line. Progressive 5' deletions of the GnRHR II promoter indicated that the region from -707/-488 bp upstream of the translational start site contained elements critical to promoter activity in ST cells. Electrophoretic mobility shift assays (EMSAs) were performed by incubating radiolabeled oligonucleotide probes spanning the -707/-488 bp promoter region with ST nuclear extracts. Specificity of binding was determined by addition of 50-fold molar excess of either homologous or heterologous competitors. Analysis of the region between -591 and -570 bp of proximal promoter indicated a specific binding complex, which was abrogated upon the addition of an unlabeled competitor containing consensus binding sites for members of the Sp family of transcription factors. Further, addition of antibodies directed against the Sp1 and Sp3 transcription factors, but not Sp2 or Sp4, resulted in a supershift of the specific DNA:protein complex. Transient transfection of ST cells with vectors containing a mutation of the Sp1/Sp3 site located at -581/-576 bp within the context of the 3029 bp promoter resulted in a 61% reduction in promoter activity relative to the unaltered promoter (P < 0.05). To determine the specific contribution of the Sp1 and Sp3 transcription factors to GnRHR II promoter activity in ST cells, Sp1 or Sp3 overexpression vectors were co-transfected with the 3029pGL3 reporter vector. Overexpression of Sp1 increased promoter activity 2-fold compared to control (P < 0.05), whereas increasing levels of Sp3 was unable to activate the promoter. Furthermore, analysis of the region between -606/-580 bp displayed specific binding complexes that were competed by either Sp1 or Egr1 consensus oligonucleotides. Additional EMSAs revealed that the specific complex binding to the -606/-580 radiolabeled probe shifted upon the addition of Egr1 or Sp1 antibodies. Mutation of the overlapping Sp1/Egr1 site residing between -597/-587 bp within 3029pGL3 and subsequent transfection of the ST cell line resulted in a 56% decrease in promoter activity compared to promoterless control (P < 0.05). Thus, Sp1 and Sp1/Egr1 binding sites are critical to expression of the porcine GnRHR II gene in ST cells. (poster)

Pulse Frequency-Dependent Gonadotropin Gene Expression by Adenylate Cyclase-Activating Polypeptide 1 in Perifused Mouse Pituitary Gonadotroph LbetaT2 Cells1

We examined how pulsatile stimulation with adenylate cyclase-activating polypeptide 1 (ADCYAP1) affected gonadotrophs. In static culture, gonadotropin-releasing hormone (GnRH) stimulated transcription of all the gonadotropin subunits. In contrast, ADCYAP1 increased common alpha-glycoprotein subunit gene (Cga) promoter activity but failed to increase luteinizing hormone beta (Lhb) and follicle-stimulating hormone beta (Fshb) promoters. Messenger RNAs for Lhb and Fshb were slightly but significantly increased by ADCYAP1 stimulation. The results of cotreatment of the cells with GnRH and ADCYAP1 was not different from the effects of GnRH alone on Lhb and Fshb transcriptional activities as well as on mRNA expressions. To determine the effect of pulsatile ADCYAP1 stimulation on gonadotropin subunit gene expression, perifused LbetaT2 cells were stimulated either at high frequency (5-min ADCYAP1 pulse every 30 min) or at low frequency (5-min ADCYAP1 pulse every 120 min). High-frequency ADCYAP1 pulses preferentially increased Lhb gene expression 2.29-fold +/- 0.15-fold, and low frequency pulses resulted in a 1.55-fold +/- 0.16-fold increase. Fshb gene expression was increased 1.87-fold +/- 0.3-fold by high-frequency ADCYAP1 pulses and 4.3-fold +/- 0.29-fold by low-frequency pulses. These results were similar to the frequency-specific effects of pulsatile GnRH. Follistatin (Fst) gene expression was specifically increased by high-frequency GnRH pulses. High-frequency ADCYAP1 pulses increased Fst to a larger extent (4.7-fold +/- 0.57-fold) than did low-frequency pulse (2.72-fold +/- 1.09-fold). ADCYAP1 receptor gene (Adcyap1r) expression was increased significantly following pulsatile GnRH regardless of pulse frequency. Low-frequency ADCYAP1 pulses, however, increased Adcyap1r expression (16.49-fold +/- 8.41-fold) to a larger extent than high frequency pulses did. In addition, high-frequency ADCYAP1 pulses specifically increased Gnrhr (GnRH receptor) expression by 4.38-fold +/- 0.81-fold; however, low-frequency pulses did not result in an increase. These results suggest that ADCYAP1, like GnRH, specifically regulates Lhb and Fshb subunit gene in a pulse frequency-specific manner. This regulation may involve alteration in numbers of GnRH and ADCYAP1 receptors as well as FST expression.

Genetic Labeling: New Approaches to Creating a Gonadotroph “ID”

Genetic Labeling: New Approaches to Creating a Gonadotroph “ID”

Welcoming β-Catenin to the Gonadotropin-Releasing Hormone Transcriptional Network in Gonadotropes

GnRH binds its G-coupled protein receptor, GnRHR, on pituitary gonadotropes and stimulates transcription of Cga, Lhb, and Fshb. These three genes encode two heterodimeric glycoprotein hormones, LH and FSH, that act as gonadotropins by regulating gametogenesis and steroidogenesis in both the testes and ovary. GnRH also regulates transcription of Gnrhr. Thus, regulated expression of Cga, Lhb, Fshb, and Gnrhr provides a genomic signature unique to functional gonadotropes. Steadily increasing evidence now indicates that GnRH regulates transcription of its four signature genes indirectly through a hierarchical transcriptional network that includes distinct subclasses of DNA-binding proteins that comprise the immediate early gene (IEG) family. These IEGs, in turn, confer hormonal responsiveness to the four signature genes. Although the IEGs confer responsiveness to GnRH, they cannot act alone. Instead, additional DNA-binding proteins, including the orphan nuclear receptor steroidogenic factor 1, act permissively to allow the four signature genes to respond to GnRH-induced changes in IEG levels. Emerging new findings now indicate that beta-catenin, a transcriptional coactivator and member of the canonical WNT signaling pathway, also plays an essential role in transducing the GnRH signal by interacting with multiple DNA-binding proteins in gonadotropes. Herein we propose that these interactions with beta-catenin define a multicomponent transcriptional network required for regulated expression of the four signature genes of the gonadotrope, Cga, Lhb, Fshb, and Gnrhr.

Weaning and the Developmental Changes in Follicle-Stimulating Hormone, Pituitary Adenylate Cyclase-Activating Polypeptide, and Inhibin B in the Male Rat1

Pituitary Fshb concentrations increase markedly and selectively beginning on Postnatal Day 20 in the male rat. To evaluate the factors potentially responsible for this rise in FSH, we adjusted the time of weaning, which is generally also on Day 20. Male rat pups were provided nutrients by suckling only and were weaned to laboratory chow earlier (Day 17) or later (Day 23) than normally performed in animal facilities (Day 20). Between ages 17 and 29 days, significant increases were seen in serum LH (1.4-fold) and FSH (2.4-fold) levels; pituitary expression of Lhb (5.4-fold), Fshb (21.3-fold), and inhibin beta B (Inhbb, 2.26-fold) mRNAs; and testicular expression of Inhbb (10-fold) mRNA. Concurrently, significant decreases occurred in serum inhibin B levels (1.8-fold); pituitary adenylate cyclase-activating polypeptide (Adcyap1, 4.2-fold), total follistatin (Fst, 3.5-fold), and Fst isoform 288 (5.6-fold) mRNAs; and testicular expression of inhibin beta A (8.2-fold) mRNA. Early weaning significantly increased serum FSH but not LH and increased pituitary expression of Fshb and GnRH receptor (Gnrhr) mRNAs but not Lhb. Early weaning also significantly decreased serum inhibin B but increased testicular expression of the Inhbb subunit. Early weaning also caused pituitary expression of Fst and Adcyap1 to decline earlier than in the control group. Immediately after weaning, growth accelerated substantially, and the time of weaning produced significant and differential effects on circulating leptin levels that were not related to indices of FSH production. From these observations, we propose the novel hypothesis that the increase in growth rate subsequent to weaning signals circulating inhibin B levels to fall and pituitary Adcyap1 and consequently Fst expression to decrease, and that these events together facilitate the rise in Fshb and Gnrhr expression by increasing pituitary activin signaling.

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.

Temporal profile of brain and pituitary GnRHs, GnRH-R and gonadotropin mRNA expression and content during early development in European sea bass ( Dicentrarchus labrax L.)

A likely endocrine control mechanism for sexual differentiation in size-graded populations of European sea bass ( Dicentrarchus labrax) is proposed by evaluating the brain expression and pituitary content of two forms of gonadotropin-releasing hormone (GnRH), namely sea bream (sbGnRH) and salmon (sGnRH), the pituitary expression of one subtype of GnRH receptor (dlGnRH-R-2A) and the three gonadotropin (GtH) subunits, namely glycoprotein α (GPα), follicle-stimulating hormone β (FSHβ) and luteinizing hormone β (LHβ), as well as the pituitary and plasma LH levels between 50 and 300 days post-hatching (dph). Four gradings were conducted between 2 and 8 months after hatching, resulting in a population of large and small individuals, having 96.5% females (female-dominant population) and 69.2% males (male-dominant population), respectively, after the last grading. The onset of gonadal differentiation was different in the two sexes, and coincided with a peak of expression of sbGnRH or sGnRH. Furthermore, the expression of these GnRHs was correlated with the expression of dlGnRH-R-2A. Sex-related differences in the brain and pituitary content of sbGnRH were also found at the time of sexual differentiation. Moreover, the observed sexual dimorphism at the transcriptional or synthesis level of these GnRH forms suggests that a different neuro-hormonal regulation is operating according to sex. At the onset of sex differentiation, FSHβ transcriptional activity reached maximal values, which were maintained until the completion of the process. The present study suggests a role for sbGnRH, sGnRH and the dlGnRH-R-2A during gonadal differentiation, possibly through enhancement of FSHβ gene expression. In males, a different endocrine regulation seems to exist also during spermiogenesis and spermiation, when gene transcription, peptide synthesis and release of LH are of greater importance.

Evolved regulation of gonadotropin-releasing hormone receptor cell surface expression.

Dominant negative effects of mutant gonadotropin-releasing hormone (GnRH) receptors (GnRHR; isolated from patients with idiopathic hypogonadotropic hypogonadism) on plasma membrane expression (PME) and function of the wt GnRHR were examined. In addition, we assessed the effect of mutants on wt GnRHR with receptor modifications that, by themselves, diminished PME. Among such mechanisms that restrict PME of GnRHR in primates are: (a) addition of the primate-specific K191 and (b) deletion of the carboxyl tail ("Ctail") found in pre-mammalian species (fish, birds) of GnRHR. We prepared rat (r) and human (h) GnRHR plasmids (88% homologous), each with or without the K191; chimeras were then made with C-tail or each of four truncated fragments (selected to isolate consensus sites for palmitoylation or phosphorylation) of the 51-amino-acid Ser-rich piscine GnRHR C-tail and then expressed in COS-7 cells. The data suggest that the dominant negative effect of the mutants on the hGnRHR requires intrinsic low PME that co-evolved with the dominant-negative effect. The data further reveal that additional modifications must have occurred in primates that are important for both the diminution of the PME and the development of the dominant negative effect of the mutants.

GnRH agonist inhibits human telomerase reverse transcriptase mRNA expression in endometrial cancer cells.

We investigated the relationship between the antiproliferative effect of GnRH agonist and telomerase activity using the endometrial cancer cell line HEC-1A. The subjects were 38 endometrial cancer, and 2 atypical endometrial hyperplasia patients. GnRH-R expression was detected using RT-PCR. HEC-1A cells were incubated with 10(-7)-10(-4) M GnRH agonist (leuprolide acetate), and cell proliferation was determined using MTT assay. The telomerase activity was detected by the TRAP assay and expression of human telomerase reverse transcriptase (hTERT) was assessed by RT-PCR. GnRH-R mRNA was detected at 94.7% (36/38) in endometrial cancer and in both of the atypical endometrial hyperplasia and in HEC-1A cells. Cell proliferation of HEC-1A showed significant inhibition at leuprolide acetate concentrations of 10(-6) M or higher compared with untreated control culture (p<0.05). The telomerase activity showed no marked difference compared with untreated culture. However, hTERT mRNA expression showed a decrease in the leuprolide-treated cells. It is suggested that the mechanism of the antitumor effect of GnRH agonist involved the inhibition of hTERT mRNA expression in the endometrial cancer cells.

The expression, regulation and signal transduction pathways of the mammalian gonadotropin-releasing hormone receptor

Normal mammalian sexual maturation and reproductive functions require the integration and precise coordination of hormones at the hypothalamic, pituitary, and gonadal levels. Hypothalamic gonadotropin-releasing hormone (GnRH) is a key regulator in this system; after binding to its receptor (GnRHR), it stimulates de novo synthesis and release of gonadotropins in anterior pituitary gonadotropes. Since the isolation of the GnRHR cDNA, the expression of GnRHR mRNA has been detected not only in the pituitary, but also in extrapituitary tissues, including the ovary and placenta. It has been shown that change in GnRHR mRNA is one of the mechanisms for regulating the expression of the GnRHR. To help understand the molecular mechanism(s) involved in transcriptional regulation of the GnRHR gene, the 5' flanking region of the GnRHR gene has recently been isolated. Initial characterization studies have identified several DNA regions in the GnRHR 5' flanking region which are responsible for both basal expression and GnRH-mediated homologous regulation of this gene in pituitary cells. The mammalian GnRHR lacks a C-terminus and possesses a relatively short third intracellular loop; both features are important in desensitization of many others G-protein coupled receptors (GPCRs), Homologous desensitization of GnRHR has been shown to be regulated by various serine-threonine protein kinases including protein kinase A (PKA) and protein kinase C (PKC), as well as by G-protein coupled receptor kinases (GRKs). Furthermore, GnRHR was demonstrated to couple with multiple G proteins (Gq/11, Gs, and Gi), and to activate cascades that involved the PKC, PKA, and mitogen-activator protein kinases. These results suggest the diversity of GnRHR-G protein coupling and signal transduction systems. The identification of second form of GnRH (GnRH-II) in mammals adds to the complexity of the GnRH-GnRHR system. This review summaries our recent progress in understanding the regulation of GnRHR gene expression and the GnRHR signal transduction pathways.