Distribution, cloning and sequencing of GnRH, its receptor, and effects of gastric acid secretion of GnRH analogue in gastric parietal cells of rats

Our objective was to study the distribution of gonadotropin-releasing hormone (GnRH) and its receptor, cloning and sequencing of GnRH and its receptor gene in cultured gastric parietal cells of rats. The distribution of GnRH and its receptor mRNA were investigated through immunocytochemical ABC methods and in situ hybridization methods in cultured gastric parietal cells of rats. After isolation of the total RNA from the parietal cells, RT-PCR was conducted to obtain GnRH and its receptor cDNA. Then, the products of PCR was purified, digested by the restriction enzyme of Hind III and EcoR I, and DNA fragments of interests were cloned into pUC19 vector. The products of PCR were analyzed by sequencing with Sanger's method after identified by PCR and digestion of restriction enzyme. Gastric parietal cells showed GnRH and its receptor immunoreactivity; positive material was located in cytoplasm other than in nuclei. GnRH and its receptor mRNA hybridized signals were also detected in cytoplasm with negative nuclei. The specific amplified band of GnRH and its receptor sequences were detected through Agarose gel electrophoresis, and GnRH gene sequence is identical to that of GnRH which has been reported in rat hypothalamus and GnRH receptor sequence is identical to that of the pituitary of rat. GnRH analogue (Alarelin) could inhibit the gastric acid secretion both by direct actions on parietal cells and by inhibiting vagous function. Our data suggest that GnRH could be produced by gastric parietal cells of rats and may modulate physiological function of gastric parietal cells of rats through autocrinal and paracrinal way.     

Effects of GABA(A) receptor modulation on the expression of GnRH gene and GnRH receptor (GnRH-R) gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland of follicular-phase ewes

The effect of prolonged, intermittent infusion of GABA(A) receptor agonist (muscimol) or GABA(A) receptor antagonist (bicuculline) into the third cerebral ventricle on the expression of GnRH gene and GnRH-R gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland was examined in follicular-phase ewes by real-time PCR. The activation or inhibition of GABA(A) receptors in the hypothalamus decreased or increased the expression of GnRH and GnRH-R genes and LH secretion, respectively. The present results indicate that the GABAergic system in the hypothalamus of follicular-phase ewes may suppress, via hypothalamic GABA(A) receptors, the expression of GnRH and GnRH-R genes in this structure. The decrease or increase of GnRH-R mRNA in the anterior pituitary gland and LH secretion in the muscimol- or bicuculline-treated ewes, respectively, is probably a consequence of parallel changes in the release of GnRH from the hypothalamus activating GnRH-R gene expression. It is suggested that GABA acting through the GABA(A) receptor mechanism on the expression of GnRH gene and GnRH-R gene in the hypothalamus may be involved in two processes: the biosynthesis of GnRH and the release of this neurohormone in the hypothalamus.     

Immunoassay of gonadotrophin-releasing hormone in brain tissue of Booroola Merino ewes

The presence of a fecundity gene (F) in Booroola Merino ewes increases the ovulation rate. To test how F gene expression affects the gonadotrophin-releasing hormone (GnRH) concentration in hypothalamic or extrahypothalamic regions of the brain, GnRH was measured by radioimmunoassay in acetic acid extracts of various brain tissues from Booroola ewes which were homozygous (FF), heterozygous (F+) or non-carriers (++) of the F gene. The GnRH concentration in brain tissues from FF, F+ and ++ animals which had been ovariectomized 5 months previously was also evaluated. No significant F gene-specific differences were noted in any of the brain areas tested, in intact or ovariectomized animals. However, in ovariectomized ewes, the concentrations of GnRH increased about 2-fold in the median eminence of the hypothalamus, remained unchanged in the medial basal hypothalamus and dropped to less than 10% of the values in intact ++ animals in the preoptic area. These studies suggest that the changed pituitary sensitivity and increased gonadotrophin release in Booroolas carrying the F gene(s) is not attributable to increased hypothalamic GnRH concentrations in these animals.     

Steroid Hormone Regulation and Tissue-Specific Expression of the Human GnRH Gene in Cell Culture and Transgenic Animals

In order to study the molecular mechanisms involved in the control of GnRH gene expression, the human GnRH gene was cloned and characterized. The gene was expressed in cells obtained from CNS tumors in transgenic mice generated utilizing 1131 bp of 5' flanking GnRH DNA fused to the simian virus 40 large T antigen. We have shown a stimulatory estrogen response element in the human GnRH gene by transient transfection studies. DNase I footprinting and an avidinbiotin DNA binding assay demonstrated that the human GnRH gene bound ER. The GN cell line was found to have nuclear ERs utilizing an ¹²⁵I estradiol binding study and by in situ hybridization histochemistry. In order to study GnRH expression in vivo, either 5000 or 484 bp of GnRH flanking DNA was fused to the luciferase (Luc) reporter gene, and transgenic mice generated. Expression in the transgenic animals was found in the hypothalamus of animals bearing the -500Luc transgene, but not in animals bearing the -484Luc transgene. The transgenic mice expressing the -5000Luc gene were gonadectomized resulting in a 20-30% increase in hypothalamic Luc expression in the males and a 65% increase in females, while mice who were gonadectomized and replaced with testosterone (males) or E₂ (females) showed a 50% decrease in Luc expression over control levels. Thus, these studies present in vitro evidence of E₂ modulation of GnRH gene expression and an in vivo model in which sensitive studies of GnRH regulation and expression can be performed.     

Regulation of gonadotropin-releasing hormone (GnRH) gene expression in hypothalamic neuronal cells

Summary 1. Gonadotropin-releasing hormone (GnRH) is the hypothalamic releasing factor that controls pituitary gonadotropin subunit gene expression and indirectly gametogenesis and steroidogenesis from the gonad, which results in reproductive competence.2. GnRH is synthesized in only about 1000 neurons in the hypothalamus and released in an episodic fashion down the median eminence to regulate gonadotropin biosynthesis.3. Although much is known about the secretory dynamics of GnRH release, little is known about the pretranslational control of GnRH biosynthesis due to lack of appropriate model systems. The recent availability of immortalized neuronal cell lines that produce GnRH allows investigators for the first time to begin to dissect the factors that directly regulate GnRH gene expression.4. This article reviews the current state of knowledge concerning the mechanisms that direct tissue-specific and peptide hormone control of GnRH biosynthesis.     

Homologous regulation of the gonadotropin-releasing hormone receptor gene is partially mediated by protein kinase C activation of an activator protein-1 element

Homologous regulation of GnRH receptor (GnRHR) gene expression is an established mechanism for controlling the sensitivity of gonadotropes to GnRH. We have found that expression of the GnRHR gene in the gonadotrope-derived alpha T3-1 cell line is mediated by a tripartite enhancer that includes a consensus activator protein-1 (AP-1) element, a binding site for SF-1 (steroidogenic factor-1), and an element we have termed GRAS (GnRHR-activating sequence). Further, in transgenic mice, approximately 1900 b.p. of the murine GnRHR gene promoter are sufficient for tissue-specific expression and GnRH responsiveness. The present studies were designed to further delineate the molecular mechanisms underlying GnRH regulation of GnRHR gene expression. Vectors containing 600 bp of the murine GnRHR gene promoter linked to luciferase (LUC) were transiently transfected into alpha T3-1 cells and exposed to treatments for 4 or 6 h. A GnRH-induced, dose-dependent increase in LUC expression of the -600 promoter was observed with maximal induction of LUC noted at 100 nM GnRH. We next tested the ability of GnRH to stimulate expression of vectors containing mutations in each of the components of the tripartite enhancer. GnRH responsiveness was lost in vectors containing mutations in AP-1. Gel mobility shift data revealed binding of fos/jun family members to the AP-1 element of the murine GnRHR promoter. Treatment with GnRH or phorbol-12-myristate-13-acetate (PMA) (100 nM), but not forskolin (10 microM), increased LUC expression, which was blocked by the protein kinase C (PKC) inhibitor, GF109203X (100 nM), and PKC down-regulation (10 nM PMA for 20 h). In addition, a specific MEK1/MEK2 inhibitor, PD98059 (60 microM), reduced the GnRH and PMA responses whereas the L-type voltage-gated calcium channel agonist, +/- BayK 8644 (5 microM), and antagonist, nimodipine (250 nM), had no effect on GnRH responsiveness. Furthermore, treatment of alpha T3-1 cells with 100 nM GnRH stimulated phosphorylation of both p42 and p44 forms of extracellular signal-regulated kinase (ERK), which was completely blocked with 60 microM PD98059. We suggest that GnRH regulation of the GnRHR gene is partially mediated by an ERK-dependent activation of a canonical AP-1 site located in the proximal promoter of the GnRHR gene.     

The Otx2 homeoprotein regulates expression from the gonadotropin-releasing hormone proximal promoter

The GnRH gene is expressed exclusively in a highly restricted population of approximately 800 neurons in the mediobasal hypothalamus in the mouse. The Otx2 homeoprotein has been shown to colocalize with GnRH in embryonic mouse brain. We have identified a highly conserved bicoid-related Otx target sequence within the proximal promoter region of the GnRH gene from several species. This element from the rat GnRH promoter binds baculovirus-expressed Otx2 protein and Otx2 protein in nuclear extracts of a hypothalamic GnRH-expressing neuronal cell line, GT1-7. Transient transfection assays indicate that the GnRH promoter Otx/bicoid site is required for specific expression of the GnRH gene in GT1-7 cells and that it can confer specificity to a neutral Rous sarcoma virus (RSV) promoter in GT1-7 cells but not in NIH3T3 cells. Overexpression of mouse Otx2 in GT1-7 cells induces expression of a GnRH promoter plasmid, an effect that is dependent upon the Otx binding site. Thus, the GnRH proximal promoter is regulated by the Otx2 homeoprotein. Finally, we have now demonstrated the presence of Otx2 protein in the GnRH neurons of the adult mouse hypothalamus. These data suggest that Otx2 is important in the development of the GnRH neuron and/or in the maintenance of GnRH expression in the adult mouse hypothalamus.     

A role for foxd3 and sox10 in the differentiation of gonadotropin-releasing hormone (GnRH) cells in the zebrafish Danio rerio

Gonadotropin-releasing hormone (GnRH) is found in a wide range of vertebrate tissues, including the nervous system. In general, GnRH has two functions: endocrine, acting as a releasing hormone; and neuromodulatory, affecting neural activity in the peripheral and central nervous system. The best understood population of GnRH cells is that of the hypothalamus, which is essential for reproduction. Less well understood are the populations of GnRH cells found in the terminal nerve and midbrain, which appear to be neuromodulatory in function. The GnRH-containing cells of the midbrain are proposed to arise from the mesencephalic region of the neural tube. Previously, we showed that neuromodulatory GnRH cells of the terminal nerve arise from cranial neural crest. To test the hypothesis that neuromodulatory GnRH cells of the midbrain also arise from neural crest, we used gene knockdown experiments in zebrafish to disrupt neural crest development. We demonstrate that decrement of the function of foxd3 and/or sox10, two genes important for the development and specification of neural crest, resulted in a reduction and/or loss of GnRH cells of the midbrain, as well as a reduction in the number of terminal nerve GnRH cells. Therefore, our data support a neural crest origin for midbrain GnRH cells. Additionally, we demonstrate that knockdown of kallmann gene function resulted in the loss of endocrine GnRH cells of the hypothalamus, but not of neuromodulatory GnRH cells of the midbrain and terminal nerve, thus providing additional evidence for separate pathways controlling the development of neuromodulatory and endocrine GnRH cells.     

GnRH deficiency: new insights from genetics

The acquisition of a sexually dimorphic phenotype is a critical event in mammalian development. Hypogonadotropic hypogonadism (HH) results from impaired secretion of GnRH. The patients display with delayed puberty, micropenis and cryptorchidism in the male reflecting gonadotropin insufficiency, and amenorrhea in the female. Kallmann's syndrome (KS) is defined by the association of HH and anosmia or hyposmia (absent smelling sense). Segregation analysis in familial cases has demonstrated diverse inheritance patterns, suggesting the existence of several genes regulating GnRH secretion. The X-linked form of the disease was associated with a genetic defect in the KALI gene located on the Xp22.3 region. KAL1 gene encodes an extracellular matrix glycoprotein anosmin-1, which facilitates neuronal growth and migration. Abnormalities in the migratory processes of the GnRH neurons with the olfactory neurons explain the association of HH with anosmia. Recently, mutations in the FGF recepteur 1 (FGFR1) gene were found in KS with autosomal dominant mode of inheritance. The role of FGFR1 in the function of reproduction requires further investigation. Besides HH with anosmia, there are isolated HH (IHH). No human GnRH mutations have been reported although hypogonadal mice due to a GnRH gene deletion exist. In patients with idiopathic HH and without anosmia an increasing number of GnRH receptor (GnRHR) mutations have been described which represent about 50% of familial cases. The clinical features are highly variable and there is a good relationship between genotype and phenotype. A complete loss of function is associated with the most severe phenotype with resistance to pulsatile GnRH treatment, absence of puberty and cryptorchidism in the male. In contrast, milder loss of function mutations causes incomplete failure of pubertal development. The preponderant role of GnRH in the secretion of LH by the gonadotrophs explains the difference of the phenotype between male and female with partial GnRH resistance. Affected females can have spontaneous telarche and normal breast development while affected males exhibit no pubertal development but normal testis volume, a feature described as "fertile-eunuch". High-dose pulsatile GnRH has been used to induce ovulation. Another gene, called GPR54, responsible for idiopathic HH has been recently described by segregation analysis in two different consanguineous families. The GPR54 gene is an orphan receptor, and its putative ligand is the product of the KISS-1 gene, called metastine. Their roles in the function of reproduction are still unknown.     

Normal sequence of the gonadotropin-releasing hormone gene in patients with idiopathic hypogonadotropic hypogonadism.

Idiopathic hypogonadotropic hypogonadism (IHH) results from absent or greatly diminished secretion of GnRH. Defects in the GnRH gene have been identified in an animal model of IHH and have been hypothesized as a possible basis for GnRH deficiency in humans. In this study, we used the polymerase chain reaction to clone and sequence the coding regions, promoter, and 3' untranslated tract of the GnRH genes from both alleles of four unrelated patients with IHH. One of the patients studied is a member of a kindred in which X-linked inheritance has been excluded by father-to-son transmission of the disease. No DNA sequence mutations were found. We conclude that most cases of IHH in humans do not involve mutations in the GnRH gene and are presumably caused by mutations at one or more other genetic loci that are required for normal function of GnRH-producing neurons.