Regulation Of Reproductive Endocrine Function By Melanocortin-4 Receptor(MC4R) In Scatophagus Argus

Melanocortin-4 receptor(MC4R), one of the members in G protein-coupled receptor superfamily(GPCRs), exists widely in all areas of the central nervous system, including the cerebral cortex, thalamus, hypothalamus, brain stem and the spinal cord. Previous studies suggested that MC4 R is involved in modulating reproductive function in mammals. MC4 R is also expressed in brain、pituitary and gonads in fish, and nonfunctional Y-linked MC4 R copies in larger male swordtails act as dominant negative mutations and delay the onset of puberty. Results suggested that MC4 R might also be associated with the reproductive regulation in fish. These studies will elucidate the potential reproductive endocrine function of MC4 R and the site(s) of action in fish, and contribute to a better understanding of Mc4 r physiology in vertibrates.In this study, spotted scat was selected as our study objects. The ORF sequences of mc4 r were cloned. Expression of mc4 r in 11 tissues(brain, pituitary, testis, ovary, liver, kidney, spleen, intestines, heart, gill and muscle) in males and females were determined by semi-quantitative reverse transcription PCR(Sq RT-PCR). The binding activity and c AMP generation of SAMC4 R with Mc4 r broad-spectrum agonist(NDP-MSH, a-MSH) and MC4 R selective agonist(THIQ) were determined by radioimmunoassay(RIA). Expressions of mc4 r, as well as that of genes related to reproduction treated with MC4 R ligands in spotted scat were detected by Real-time quantitative PCR(Real-time PCR). The main results were as following: 1. Molecular cloning and tissues distribution of SAmc4 r.The ORF of mc4 r was 984 bp which encoded 589 amino acids. Amino acid alignment analyses showed that mc4 r in S.argus is the member of rhodopsin family, which contain all the typical functional domains of G protein-coupled receptor. Phylogenetic analyses indicated that spotted scat was most closely related to Perciformes, and its MC4 R shared 97% sequence identity with large yellow croaker.In addition,mc4 r m RNA was widely expressed in spotted scat, with the highest levels of expression in brain, pituitary, and ovary in male and female. 2. Ligand binding properties of SAMC4 R.Ligand binding properties of SAMC4 R showed that it bound NDP-MSH with the highest affinity followed by a-MSH, THIQ. a-MSH and NDP-MSH were identified as potent agonists to stimulate c AMP generation followed by THIQ in SAMC4 R.3. Effect of MC4 R agonist on mc4 r and genes related to reproduction in S.argus.For in virto incubation experiment, MC4 R broad-spectrum agonist NDP-MSH increased significantly sb Gn RH in hypothalamus,as well as the fsh and lh in pituitary at 3 h. Expression of fsh returned to its normal levels after 6 hours, while that of sb Gn RH and lh were significant higher in the treated group compared to the control group. Result treated with the selective agonist THIQ was similar to that with NDP-MSH.For the in vivo experiment, THIQ increased significantly mc4 r and sb Gn RH in hypothalamus,as well as the fsh and lh in pituitary after abdominal injection 12 h and 24 h. 4. Effect of MC4 R antagonist on mc4 r and genes related to reproduction in S.argus.For in virto incubation experiment, MC4 R broad-spectrum antagonist SHU9119 inhibited significantly sb Gn RH in hypothalamus,as well as the fsh and lh in pituitary at 3 h. Expression of sb Gn RH、fsh and lh returned to its normal levels after 6 hours, while the selective antagonist Ipsen5 i of sb Gn RH and lh were significant lower in the treated group compared to the control group.For the in vivo experiment, Ipsen5 i inhibited significantly mc4 r and sb Gn RH in hypothalamus,as well as the fsh and lh in pituitary after abdominal injection at 12 h. Expression of fsh and lh returned to its normal levels after 24 hours, while that of sb Gn RH and mc4 r were significant lower in the treated group compared to the control group.In a word, MC4 R mainly regulates sb Gn RH in the hypothalamus, thereby modulating the synthesis and secretion of LH in the pituitary. In addtion, Mc4 r directly regulates LH levels of pituitary in S.argus.