The Cloning And Mrna Expression Of GnIH And Its Receptors In Rabbit

GnIH(gonadotropin-inhibitory hormone), a RFamide novel neuropeptide (Arg-Phe-NH2in the C terminus) of12amino acids, was first discovered in the hypothalamus of birds in2000by Tsutsui. Cell bodies containing the isolated novel neuropeptide were located in the paraventricular nucleus (PVN). The isolated novel neuropeptide was shown to decrease gonadotropin release from cultured anterior pituitary in a dose-dependent manner. Therefore Tsutsui designated this novel hypothalamic neuropeptide as GnIH. Subsequently, three GnIH homologs were also isolated in mammals. They have the Arg-Phe-NH2in the C terminus, called RF ralated peptide with the similar effects to GnIH. It was reported to have many important physiological roles mediated by its receptors, such as inhibiting gonadotropin synthesis and release from the pituitary. GnIH and its related peptides can also stimulate feeding behavior. However, the present studies about GnIH mainly focused on the human, mouse, rat and so on, there is little research of GnIH and NPFFR in rabbit.Therefore we investigated the gene cloning and mRNA distribution of GnIH and its receptors in rabbit by RT-PCR. The results are as follows:1. The gene cloning of partial regments of GnIH and its two receptors was studied by RT-PCR. The nucleotide sequences of GnIH and its receptors were submitted to the GenBank database by us, and the accession number were HM744733、 JF826513and JF907578, respectively. The amino acids of the GnIH are MPHAAATLPLRFGR (RFRP-1), SPQPVANLPLRFGR (RFRP-2) and IPNLPQRFGR (RFRP-3)2. The nucleotide sequences and amino acids of the GnIH and its receptors were analyzed by the bioinformatics software. Nucleotide sequence of rabbit GnIH was high homologous compared with other animal species such as sheep, cattle, rhesus monkey, human (88%、83%、83%and82%nucleotide sequence homology, respectively), and had low homology with pigs, hamster, and mouse (79%、77%and75%respectively). The homology of the predicted amino acids was basically similar with that of nucleotide sequence. Compared with other animal species, the nucleotide sequence and amino acids of rabbit NPFFR-1and NPFFR-2both were high homologous. The transmembrance analysis of both receptors revealed that they had typical transmembrane features. 3. The expression of GnIH and its receptors mRNA in rabbit was studied by RT-PCR. The results showed that, GnIH had the highest level of expression in the wall of eyeball, and the lower in hypothalamus. It was also detected in liver, kidney, adrenal, pituitary, midbrain, amygdala, spinal cord, olfactory bulb, pons, cerebellum, medulla oblongata, hippocampus, heart, spleen, pancreas and other organizations. Its receptors were widely distributed in the central nervous system and peripheral organs. NPFFR-1was distributed in the hypothalamus, midbrain, cerebellum, pyriform lobe, amygdala, medulla oblongata, olfactory bulb, spinal cord, hippocampus, pituitary, pons, ovary, testis, epididymis, uterus, bladder, wall of eyeball, esophagus, breast, pancreas, thymus and duodenum. NPFFR-2had different levels of expression in the hypothalamus, midbrain, ovary, testis, wall of eyeball, adrenal gland, pyriform lobe, amygdala, medulla oblongata, olfactory bulb, spinal cord, hippocampus, pituitary, pons, and so on. All these results were similar to that of other animals.