Molecular characterization of two sea bass g-protein-coupled receptior 54 (GPR54): cDNA cloning and expression analysis

Resumen del trabajo presentado en la 1st World Conference on Kisspeptin Signaling in the Brain, celebrada en Córdoba (España), del 8 al 10 de octubre de 2008From late 2003 onwards, a large number of experimental studies, which have been mainly carried out in mammals, have evidenced that the G protein-coupled receptor GPR54 and its ligands (kisspeptins, encoded by the KiSS-1 gene) play a critical role in the control of reproduction, activation of gonadotropic axis and regulation of puberty in vertebrates. Nevertheless, the potential role of the KiSS-1 system and its receptor in other biological processes is not discarded. While KiSS-1 cDNA sequences have been described in few vertebrate non-mammalian species, the GPR54 gene has been already cloned and characterized from several fish species, including tilapia (Oreochromis niloticus), grey mullet (Mugil cephalus), zebrafish (Danio rerio), fathead minnow (Pimephales promelas) and fish cobia (Rachycentron canadum) among others. Here, we report a study based on the cloning and molecular characterization of two GPR54 genes in sea bass (Dicentrarchus labrax), an important marine teleost aquacultured species. A comparative tissue expression analysis of these two genes is described according to developmental stage (prepubertal and pubertal fish) and sex. The sequence of the tilapia GPR54 receptor was used to query the putative gene sequence in fish species by TBLASTN searches. To amplify fragments of sea bass GPR54, degenerate PCR primers were designed based on conserved amino acids from fish genes. PCR amplifications were performed using a genome walking approach and the complete cDNAs were obtained from a sea bass brain cDNA library. Nucleotide sequences of the cloned DNA fragments were confirmed by sequencing, evidencing that two GPR54 genes exist in sea bass. Searches in the medaka and zebrafish databases evidenced that both genes were also present in other vertebrate non-mammalian species, thus suggesting that a duplication of the GPR54 gene occurred in fish. Tissue expression analysis of both GPR54 genes in sea bass revealed that both are mainly expressed in the brain, pituitary, testis and ovary in prepubertal and pubertal male and female sea bass. Temporal tissue expression of the two sea bass GPR54 genes in these tissues during the reproductive season (January-April) was independent of developmental stage or sex. Finally, expression of both genes was also confirmed in other tissues, including the spleen, kidney, liver, intestine, gill heart, eye, skin and muscle. Further studies on the activation of sea bass receptors and their ligands controlling the KiSS-1 system and gonadotropic axis in this fish species are now required.Funded by GV06/268 to A.G. A.F. supported by a Ramón y Cajal contract from MEC of Spain