The gene encoding the ovine gonadotropin-releasing hormone (GnRH) receptor: cloning and initial characterization.

We have isolated four lambda clones, which, in their aggregate, contain the entire coding sequence of the ovine gene encoding the gonadotropin-releasing hormone (GnRH) receptor (GnRHR). Like its human and murine counterparts, ovine GnRHR exists as a single-copy gene and is comprised of three exons and two introns. Furthermore, the locations of all exon-intron boundaries are perfectly conserved among the human, ovine and murine genes. The most striking difference among these genes is the location of the transcription start points (tsp) and, thus, the length of 5' untranslated region (UTR). This variation in size of the 5' UTR between the murine, human and ovine genes raises the possibility that different mechanisms have evolved for cell-specific expression of this gene. Isolation of the ovine GnRHR and its associated 5' flanking region is the essential first step in defining the molecular mechanisms underlying cell-specific and hormonal regulation of its expression in ruminants.

Effects of follicle-stimulating hormone administration on oestradiol-induced cystic ovaries in guinea pigs.

A consistent defect in follicle-stimulating hormone (FSH) secretion is seen in humans with Polycystic Ovarian Syndrome (PCOS); therefore, we evaluated whether Metrodin (a highly purified urinary FSH) administration concurrent with cyst induction or following cyst induction inhibits estrogen-induced cyst development and augments ovarian follicular growth in an established guinea pig model. All animals in these studies received subcutaneous implants containing oestradiol-17 beta (E2)-filled Silastic capsules for a 48-hour period. Guinea pigs in study #1 were administered four 0.25 mL injections of FSH or placebo at twelve-hour intervals simultaneously with the E2 treatment; guinea pigs assigned to study #2 were administered four 0.25 mL injections of FSH or placebo at twelve-hour intervals following the induction of the cystic condition by E2. Exogenous FSH appears to negate cyst formation when superimposed upon the cyst-inducing agent (E2). Further, treatment with FSH augmented the number of mid-sized follicles in both paradigms. This study is the first to establish evidence of an anti-cystic effect of FSH in an animal model.

Cell-specific expression of the mouse gonadotropin-releasing hormone (GnRH) receptor gene is conferred by elements residing within 500 bp of proximal 5' flanking region.

Gonadotropin-releasing hormone (GnRH) is a decapeptide produced by the hypothalamus. Upon binding to specific high-affinity receptors on gonadotrope cells of the anterior pituitary gland, GnRH stimulates the synthesis and secretion of LH. In light of the critical role of GnRH in reproduction much effort has been directed toward understanding the regulation of this hormone and its cognate receptor. The recent availability of genomic clones for the GnRH receptor has facilitated research to address the molecular mechanisms underlying regulation of GnRH receptor gene expression. We have expanded the analysis of the promoter for the mouse GnRH receptor gene and report that in addition to transcriptional start sites located within 100 bp of the translation start codon there is a more distal transcriptional start site approximately 200 bp 5' of the initiation codon. The initiation of transcription from this more distal site was sufficient to confer cell-specific expression on luciferase. Further, transient expression assays of constructs containing progressive 5' deletions in the GnRH receptor gene promoter reveal the presence of one or morecis-acting elements located between -500 and -400 (relative to ATG) necessary for transcriptional activity in the gonadotrope-derived αT3 cell line. Finally, αT3 but not COS-7 cell nuclear extract contained protein(s) that bind to at least two separate motifs contained within the -500 to -400 region. We suggest that activation of GnRH receptor gene expression in the αT3 cell line requires the binding of at least two transcriptional regulatory proteins to basal enhancer elements located within a 100 bp region between -500 to -400 relative to the translation start codon in the mouse GnRH receptor gene.

Regulation of gonadotropin-releasing hormone (GnRH) receptor messenger ribonucleic acid and GnRH receptors during the early preovulatory period in the ewe.

Estradiol increases the number of GnRH receptors in the ewe. Although results from studies conducted in vitro indicate that progesterone may have a negative influence on the number of ovine GnRH receptors, this effect of progesterone has not been documented in vivo. To explore the regulation of GnRH receptors at the level of gene expression, a partial complementary DNA (cDNA) encoding ovine GnRH receptor was isolated using reverse transcription and polymerase chain reaction methodology. This partial cDNA (701 basepairs) was used to isolate a full-length cDNA encoding GnRH receptor from an ovine pituitary cDNA library. Northern blot analysis of RNA from ovine pituitary glands using the partial cDNA as a molecular probe revealed four messenger RNA (mRNA) transcripts at 5.6, 3.8, 2.1, and 1.3 kilobases. In some samples, a fifth transcript at 0.8 kilobases was also evident. GnRH receptor mRNA was not detected in ovine brain, heart, kidney, adrenal, or liver tissues. To examine the regulation of GnRH receptor mRNA and GnRH receptors during the early preovulatory period, relationships among steady state concentrations of GnRH receptor mRNA, numbers of GnRH receptors, and circulating concentrations of progesterone and estradiol during luteolysis were characterized. We hypothesized that during luteolysis, decreased concentrations of progesterone would be associated with increased concentrations of GnRH receptor mRNA and increased numbers of GnRH receptors. On day 11 or 12 of the estrous cycle, luteolysis was induced in 14 ewes by treatment with prostaglandin F2 alpha (PGF2 alpha). Four ewes were treated with saline (saline controls). Anterior pituitary tissue was collected 4 h (n = 4), 12 h (n = 5), and 24 h (n = 5) after treatment with PGF2 alpha or 24 h after treatment with saline and from four untreated ewes on day 11 or 12 of the estrous cycle (untreated controls). Twelve hours after treatment with PGF2 alpha, circulating concentrations of progesterone had decreased (P < 0.05) to 46% of the control values; however, concentrations of estradiol were not different from those in control ewes. Concentrations of GnRH receptor mRNA increased 2-fold during luteolysis and were higher than control values 12 h after PGF2 alpha treatment (P < 0.05). This increase in GnRH receptor mRNA was not accompanied by an increase in the number of GnRH receptors. Twenty-four hours after treatment with PGF2 alpha, concentrations of progesterone in PGF2 alpha-treated ewes had decreased (P < 0.05) to 15% of control values, whereas concentrations of estradiol had increased (P < 0.05) to 321% of control values.(ABSTRACT TRUNCATED AT 400 WORDS)