[show abstract][hide abstract] ABSTRACT: Objective: Gonadotropin-inhibitory hormone (GnIH)-3 is a neuropeptide that plays a major role in the regulation of reproduction and feeding in mammals. Materials and Methods: We measured endocrine and behavioural parameters of reproduction in sheep, and sexual behaviour in sheep, mice and cynomolgus monkeys. In addition, GnIH gene expression (in situ hybridization) was examined in ewes, and effects of GnIH-3 on food intake and energy expenditure were measured in various species. GnIH-3 was infused (i.v.) into ewes after an i.m. injection of estradiol benzoate to determine whether the peptide blocks the surge in luteinizing hormone (LH) secretion. Results: GnIH gene expression was reduced in the preovulatory period in ewes. Infusion (i.v.) of GnIH-3 blocked the estrogen-induced LH surge (in ewes). Intracerebroventricular infusion had no effect on female or male sexual behaviour in each of the three species, but increased food intake. There were no effects on energy expenditure in sheep or rats. GnIH increased fos protein (immunohistochemistry) was seen in orexigenic neurons (in sheep and rats), but also in anorexigenic neurons (in sheep). Conclusions: GnIH-3 reduces reproductive hormone levels and increases food intake in mammals without reducing energy expenditure. There is minimal effect on reproductive behaviour. The dual effect on reproduction and feeding suggests that GnIH-3 provides a molecular switch between these two functions. Blockade of the positive feedback effect of estrogen with parenteral infusion indicates that this peptide may have utility as a blocker of reproductive function in mammals.
[show abstract][hide abstract] ABSTRACT: There is strong evidence that kisspeptin acts to regulate GnRH secretion, but whether there is also a component of action on the gonadotropes is not clear. Using quantitative RT-PCR, we found that G protein-coupled receptor-54 mRNA is expressed in ovine pituitary cell fractions enriched for gonadotropes as well as in somatotropes and lactotropes. To test whether kisspeptin acts directly on the pituitary gonadotropes, we first examined LH release from primary ovine pituitary cell cultures treated with kisspeptin. We found that kisspeptin treatment increased the concentration of LH in culture media by 80%, compared with control, but only in pituitary cultures from ewes during the follicular phase of the estrous cycle. After this, we determined whether kisspeptin acts on the pituitary gland in vivo. Using GnRH-replaced ovariectomized hypothalamo-pituitary-disconnected ewes, we were not able to achieve any effect of kisspeptin on LH under steady-state conditions or during the period of an estrogen-induced LH surge. Finally, we collected hypophysial portal blood samples from ovariectomized ewes and measured kisspeptin levels. Low but detectable amounts of kisspeptin were found in portal plasma, but levels were similar in ovariectomized ewes that were untreated or given estrogen to elicit an LH surge. Thus, although we observed an effect of kisspeptin on LH release in vitro in some situations, similar findings were not obtained in vivo. Moreover, the low concentrations of kisspeptin in hypophysial portal blood and the lack of any change during the period of an estrogen-induced GnRH/LH surge suggest that action on the pituitary gland is not of major consequence in terms of LH release.
[show abstract][hide abstract] ABSTRACT: Mammalian gonadotropin-releasing hormone (GnRH I: pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) stimulates pituitary gonadotropin secretion, which in turn stimulates the gonads. Whereas a hypothalamic form of GnRH of variable structure (designated type I) had been shown to regulate reproduction through a cognate type I receptor, it has recently become evident that most vertebrates have one or two other forms of GnRH. One of these, designated type II GnRH (GnRH II: pGlu-His-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2), is conserved from fish to man and is widely distributed in the brain, suggesting important neuromodulatory functions such as regulating K+ channels and stimulating sexual arousal. We now report the cloning of a type II GnRH receptor from marmoset cDNA. The receptor has only 41% identity with the type I receptor and, unlike the type I receptor, has a carboxyl-terminal tail. The receptor is highly selective for GnRH II. As with the type I receptor, it couples to G(alpha)q/11 and also activates extracellular signal-regulated kinase (ERK1/2) but differs in activating p38 mitogen activated protein (MAP) kinase. The type II receptor is more widely distributed than the type I receptor and is expressed throughout the brain, including areas associated with sexual arousal, and in diverse non-neural and reproductive tissues, suggesting a variety of functions. Surprisingly, the type II receptor is expressed in the majority of gonadotropes. The presence of two GnRH receptors in gonadotropes, together with the differences in their signaling, suggests different roles in gonadotrope functioning.
Proceedings of the National Academy of Sciences 09/2001; 98(17):9636-41. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: The availability of large expressed sequence tag (EST) databases has led to a revolution in the way new genes are identified. Mining of these databases using known protein sequences as queries is a powerful technique for discovering orthologous and paralogous genes. The scientist is often confronted, however, by an enormous amount of search output owing to the inherent redundancy of EST data. In addition, high search sensitivity often cannot be achieved using only a single member of a protein superfamily as a query. In this paper a technique for addressing both of these issues is described. Assembled EST databases are queried with every member of a protein superfamily, the results are integrated and false positives are pruned from the set. The result is a set of assemblies enriched in members of the protein superfamily under consideration. The technique is applied to the G protein-coupled receptor (GPCR) superfamily in the construction of a GPCR Resource. A novel full-length human GPCR identified from the GPCR Resource is presented, illustrating the utility of the method.
Briefings in Bioinformatics 03/2000; 1(1):93-9. · 5.30 Impact Factor
[show abstract][hide abstract] ABSTRACT: Gonadotropin releasing hormone (GnRH) regulates the reproductive system through a specific G-protein-coupled receptor (GPCR) in pituitary gonadotropes. The existence of two (or more) forms of GnRH in most vertebrates suggested the existence of GnRH receptor subtypes (I and II). Using sequence information for extracellular loop 3 of a putative Type II GnRH receptor from a reptile species, we have looked for a Type II GnRH receptor gene in the human genome EST (expressed sequence tag) database. A homolog was identified which has 45% and 41% amino acid identity with exons 2 and 3 of the known human GnRH pituitary receptor (designated Type I) and much lower homology with all other GPCRs. A total of 27 contiguous ESTs was found and comprised a continuous sequence of 1642 nucleotides. The EST sequences were confirmed in the cloned human gene and in PCR products of cDNA from several tissues. All EST transcripts detected were in the antisense orientation with respect to the novel GnRH receptor sequence and were highly expressed in a wide range of human brain and peripheral tissues. PCR of cDNA from a wide range of tissues revealed that intronic sequence equivalent to intron 2 of the Type I GnRH receptor was retained. The failure to splice out putative intron sequences in transcripts which spanned exon-intron boundaries is expected in antisense transcripts, as candidate donor and acceptor sites were only present in the gene when transcribed in the orientation encoding the GnRH receptor homolog. No transcripts extended 5' to the sequence corresponding to intron 2 of the Type I GnRH as the antisense transcripts terminated in poly A due to the presence of a polyadenylation signal sequence in the putative intron 2 when transcribed in the antisense orientation. These findings suggest that a Type II GnRH receptor gene has arisen during vertebrate evolution and is also present in the human. However, the receptor may have become vestigial in the human, possibly due to the abundant and universal tissue transcription of the opposite DNA strand to produce antisense RNA.
Journal of Endocrinology 08/1999; 162(1):117-26. · 4.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: The majority of vertebrates have two or three forms of gonadotropin-releasing hormone (GnRH), which appear to have arisen by successive gene duplication events. This suggests the possibility of concordant gene duplications of the GnRH receptor to produce two or more cognate receptors. Since the extracellular loop 3 (EC3) domain of mammalian GnRH receptors plays a role in distinguishing the different forms of GnRH, we have contemplated that the sequence of this domain will differ significantly in the putative cognate receptors. Degenerate oliognucleotides encoding the sequences of the transmembrane domains preceding and following EC3 were used for PCR amplification of genomic DNA from zebrafish (Brachydanio rerio), goldfish (Carassius auratus), African clawed frog (Xenopus laevis), chicken (Gallus domesticus), and lizard (Agama atra). Isolation and sequencing of specific clones revealed that they fell into three groups. Two of these were most similar to the mammalian pituitary GnRH receptor and were therefore designated Type IA and Type IB. The third form (designated Type II) was most different from the others and was identified in Xenopus, lizard, and human DNA. These findings support the concept of the existence of three distinct GnRH receptors, which have evolved in conjunction with three distinct GnRH ligand classes present in many vertebrates.
General and Comparative Endocrinology 01/1999; 112(3):296-302. · 2.82 Impact Factor
[show abstract][hide abstract] ABSTRACT: The number of pituitary gonadotropin-releasing hormone receptors (GnRH-R) varies across the estrous cycle. We report that there is variable expression of the differently-sized GnRH-R transcripts in cyclic ewes and in an experimental model. During the follicular phase of the cycle, and compared to the luteal phase, there was increased expression of the 1.5, 2.3 and 3.7 kilobase (kb) transcripts with no change in the levels of the 5.6 or the 1.2 kb transcripts. Steady state levels of mRNA for luteinising hormone beta and common alpha subunit were also increased in the follicular phase of the cycle. In hypothalamo-pituitary disconnected ovariectomised ewes given pulsatile GnRH replacement, injection of estrogen increased the 1.5, 2.3 and 3.7 kb, while the levels of the 5.6 and 1.2 kb transcripts were not altered. We conclude that the differential regulation of GnRH-R mRNA occurs through a direct effect of E on the pituitary.
Molecular and Cellular Endocrinology 12/1998; 146(1-2):141-9. · 4.04 Impact Factor
[show abstract][hide abstract] ABSTRACT: The effects of neonatal immunization against GnRH were studied in sheep after they had reached adulthood (3-4 yr) and the antibody titers had fallen to undetectable levels. The immunized animals had small gonads, and the females did not have large follicles (>3 mm) or corpora lutea in their ovaries. Compared with controls, the immunized animals had low or nondetectable levels of LH and FSH in peripheral plasma, and the immunized animals generally failed to respond to a single i.v. GnRH challenge. After ovariectomy, the control ewes, but not the immunized ewes, showed an elevation in plasma LH and FSH levels. The sampling of hypophysial portal blood, with a newly described method, showed that the secretion of GnRH was reduced in the immunized animals, but the amount of GnRH in the median eminence was similar in the control and immunized ewes. The pituitary content of LH and FSH was reduced in the immunized ewes as was messenger RNA for the gonadotropin subunits and the GnRH receptor. These data indicate that neonatal immunization does not affect the synthesis of GnRH in adulthood but reduces the secretion of GnRH, causing long-term sterility in these animals.
[show abstract][hide abstract] ABSTRACT: Photoaffinity cross-linking with [azidobenzoyl-d-Lys6]GnRH leads to irreversible activation of the gonadotropin-releasing hormone (GnRH) receptor. In order to localize the cross-linking site, the disulfide bridge structure was initially probed by mutagenesis. A consistent pattern of changes in the ability of GnRH to stimulate signal transduction after Ser substitutions of extracellularly located Cys residues indicated that Cys14 in the N-terminal domain is connected to Cys200 in the second extracellular loop, while Cys196 in this loop is connected to the highly conserved Cys114 at the extracellular end of transmembrane helix 3. Protein chemical analysis of radioactive fragments of cross-linked GnRH receptor following deglycosylation and enzymatic digest with endoproteinase Glu-C and trypsin before and after introduction or elimination of potential protease cleavage sites indicated that 125I[azidobenzoyl-d-Lys6]GnRH cross-links to a segment comprising residues 12-18 of the N-terminal domain. The existence of the Cys114-Cys196 bridge was directly confirmed as a labeled fragment, including that Cys114 was resolvable only under reducing conditions. The observation that the cross-linked N-terminal enzymatic fragments had identical apparent size under non-reducing conditions shows that the cross-linking reaction disconnected the disulfide bridge between Cys14 and Cys200 and indicates that Cys14 is probably the residue involved in cross-linking of the ligand. It is concluded that covalent tethering of GnRH through a photoreactive side chain located at position 6 in the middle of the peptide leads to continued activation of the receptor presumably through covalent binding to Cys14 in the N-terminal domain of the receptor.