Comparative analysis of the pituitary and ovarian GnRH systems in the leopard gecko: signaling crosstalk between multiple receptor subtypes in ovarian follicles.

GnRH regulates reproductive functions through interaction with its pituitary receptor in vertebrates. The present study demonstrated that the leopard gecko possessed two and three genes for GnRH ligands and receptors, respectively, though one of the three receptor subtypes had long been thought not to exist in reptiles. Each receptor subtype showed a distinct pharmacology. All types of ligands and receptors showed different expression patterns, and were widely expressed both inside and outside the brain. This report also shows a comparison of the pituitary and ovarian GnRH systems in the leopard gecko during and after the egg-laying season. All three receptor subtypes were expressed in both the whole pituitary and ovary; however, only one receptor subtype could be detected in the anterior pituitary gland. In situ hybridization showed spatial expression patterns of ovarian receptors, and suggested co-expression of multiple receptor subtypes in granulosa cells of larger follicles. Co-transfection of receptor subtypes showed a distinct pharmacology in COS-7 cells compared with those of single transfections. These results suggest that distinct signaling mechanisms are involved in the pituitary and ovarian GnRH systems. Seasonal and developmental variations in receptor expression in the anterior pituitary gland and ovarian follicles may contribute to the seasonal breeding of this animal.

A system for receptor functional analysis based on c-fos mRNA expression: analysis of GnRH receptors as a test system.

This report describes the establishment of a system for assessing receptor activation by RT-PCR-based detection of c-fos mRNA induction. In this system, COS-7 cells were transiently transfected with GnRH receptor expression plasmid, and ligand-induced c-fos expression was quantified by the RT-competitive PCR method. The results were compared with those of a conventional inositol phosphate (IP) assay. Changes in c-fos expression levels were observed in a dose- and ligand-dependent manner. Similar tendencies were observed in ligand selectivity between c-fos expression and IP production. The novel system developed and established in the present study is sensitive by using RT-PCR and convenient because it requires only basic methods of cell culture and molecular biology. It also has the merit that it does not need any specific measuring devices or radioactive substances. Given the ability of c-fos to respond to diverse stimuli, the present system may be applicable for various receptors for bioactive substances in addition to GnRH receptor, and useful for various purposes including screening ligands for orphan receptors.

Comparative genomics of the endocrine systems in humans and chimpanzees with special reference to GNRH2 and UCN2 and their receptors.

To identify the genetic basis of the differences between chimpanzees and humans, it is indispensable to analyze a whole gene set constituting a particular regulatory system as well as to compare the whole genome or chromosomes randomly. We compared genes encoding hormones of the endocrine system, one of the most fundamental regulatory systems in organisms. The present study covered a total of 111 genes generating 115 precursors and 172 peptides. Decisive differences were observed in GNRH2 and UCN2 and their corresponding receptor genes. It is often postulated that mechanisms underlying the basic functions of life are common and would not be readily altered. The present study demonstrated that, on the contrary, substantial differences have been generated in genes composing the endocrine system, even between humans and our closest living relative.

Molecular and evolutionary characterization of the GnRH-II gene in the chicken: distinctive genomic organization, expression pattern, and precursor sequence.

Of all the structural variants of GnRH (gonadotropin-releasing hormone), GnRH-II has been found to be universally present in and uniquely conserved among jawed vertebrates without any sequence substitutions. Our previous study found that the GnRH-II precursor sequences have become divergent in the lineage of eutherian mammals, based on a comparison between reptilian and mammalian GnRH-II. To elucidate the molecular evolution of GnRH-II throughout amniotes, we have performed the first identification of the avian GnRH-II cDNA/gene from the chicken, the species used for the initial discovery of GnRH-II peptide. Gene arrangement around the GnRH-II in the chicken was similar to that in mammals; however, a gene MRPS26 was partly overlapped with the downstream part of the GnRH-II in the chicken. It was identified that the GnRH-II/MRPS26 locus generated at least five distinct types of transcripts with different expression patterns and three of them may produce functional GnRH-II decapeptide. Sequence comparison revealed that the prepro-GnRH-II polypeptide of the chicken was substantially different from those of other species regarding the length and similarity. The present results strongly indicated that considerable variations were generated in the precursor sequence of the evolutionarily conserved GnRH-II during amniote evolution. It was also suggested that the sequence divergence seen in the chicken may have occurred independently of that in the mammalian lineage.

Chicken RFamide-related peptide (GnIH) and two distinct receptor subtypes: identification, molecular characterization, and evolutionary considerations.

RFamide-related peptides (RFRPs) regulate the release of various pituitary hormones in vertebrates. It is completely unknown how the functions of RFRPs vary among animal classes and whether vertebrate RFRPs are orthologous to each other and belong to the same peptide family. This report concerns identification of avian RFRP (gonadotropin-inhibitory hormone, GnIH) from the chicken. Chromosome-wide synteny conservation demonstrated the orthologous relationships among vertebrate RFRPs. The consensus motif for RFRP was modified to Pro-Xaa-Arg-Phe-NH2. We also describe the first identification of two distinct types of receptors for non-mammalian RFRP (RFRPR and NPFFR) from the chicken. Amino acid comparison revealed substantial differences in both termini of receptors among classes of vertebrates. The 5'-flanking regions of chicken RFRPR and NPFFR suggested their expressions in the pituitary gland, and this was confirmed by the RT-PCR analysis. Localizations of both chicken RFRP and its receptors were distinct from those of mammals. These results indicated that avian RFRP, unlike the mammalian one, directly acts on the pituitary gland via receptors to regulate gonadotropin release. It was also suggested that functional differences, especially between avian and mammalian RFRPs, are substantially due to divergences in the structures and expression sites of their receptors.

Identification of the reptilian prolactin and its receptor cDNAs in the leopard gecko, Eublepharis macularius.

In spite of their physiological significance, there is no available information about the nucleotide sequences of prolactin (PRL) and its receptor in reptilian species. In order to fill this gap, PRL and its receptor cDNAs were identified in a reptilian species, the leopard gecko Eublepharis macularius. The deduced leopard gecko PRL polypeptide showed high identities with the corresponding polypeptides of other reptiles. The leopard gecko PRL receptor (PRLR) was estimated to have tandem repeated regions in its extracellular domain, which had been originally found in avian PRLR. Molecular phylogenetic analysis suggests that these tandem repeated regions were generated by the duplication of the extracellular region in the latest common ancestor among reptiles and birds. In addition, tissue distributions of PRL and PRLR in the leopard gecko were examined by the reverse transcription-polymerase chain reaction (RT-PCR). PRLR mRNA was detected in all tissues examined and highly expressed in the whole brain, pituitary, intestine, kidney, ovary, oviduct and testis. Whereas, PRL mRNA was expressed in the whole brain, pituitary, ovary and testis. The co-expressions of PRL and its receptor in some extrapituitary organs suggest that PRL acts as an autocrine/paracrine factor in such organs of the leopard gecko.

Molecular cloning and characterization of a gonadotropin-releasing hormone receptor in the guinea pig, Cavia porcellus.

Guinea pig gonadotropin-releasing hormone (gpGnRH) is predicted to have a unique structure among all known forms of GnRH molecule [Endocrinology 138 (1997) 4123] and it is of great interest to determine whether the unique structure of gpGnRH is manifested in the characteristics of the guinea pig GnRH receptor. In the present study, we isolated a full-length cDNA for a GnRH receptor from the pituitary gland of the guinea pig. The putative guinea pig GnRH receptor protein has an amino acid identity of 79-87% with mammalian type I GnRH receptors. The amino acid residues which have been demonstrated to be important for ligand binding and signal transduction were conserved in the guinea pig GnRH receptor. However, there are several specific amino acid substitutions among mammalian type I GnRH receptors. Moreover, though the guinea pig has generally been classified as a rodent, the putative GnRH receptor protein did not have some rodent-specific characteristics. Total IP assays demonstrated that the cloned guinea pig GnRH receptor is a functional GnRH receptor and that it shows different preference of ligand sensitivities from the rat GnRH receptor.

Identification and characterization of the reptilian GnRH-II gene in the leopard gecko, Eublepharis macularius, and its evolutionary considerations.

To elucidate the molecular phylogeny and evolution of a particular peptide, one must analyze not the limited primary amino acid sequences of the low molecular weight mature polypeptide, but rather the sequences of the corresponding precursors from various species. Of all the structural variants of gonadotropin-releasing hormone (GnRH), GnRH-II (chicken GnRH-II, or cGnRH-II) is remarkably conserved without any sequence substitutions among vertebrates, but its precursor sequences vary considerably. We have identified and characterized the full-length complementary DNA (cDNA) encoding the GnRH-II precursor and determined its genomic structure, consisting of four exons and three introns, in a reptilian species, the leopard gecko Eublepharis macularius. This is the first report about the GnRH-II precursor cDNA/gene from reptiles. The deduced leopard gecko prepro-GnRH-II polypeptide had the highest identities with the corresponding polypeptides of amphibians. The GnRH-II precursor mRNA was detected in more than half of the tissues and organs examined. This widespread expression is consistent with the previous findings in several species, though the roles of GnRH outside the hypothalamus-pituitary-gonadal axis remain largely unknown. Molecular phylogenetic analysis combined with sequence comparison showed that the leopard gecko is more similar to fishes and amphibians than to eutherian mammals with respect to the GnRH-II precursor sequence. These results strongly suggest that the divergence of the GnRH-II precursor sequences seen in eutherian mammals may have occurred along with amniote evolution.