| Neuropeptide B(NPB)is a neuropeptide which extracted from bovine hypothalamus in 2002.Because its first tryptophane residue was brominated,it was named Neuropeptide B.NPB has two isomorph,NPB23 and NPB29,but only human NPB29 was found in other mammals.Neuropeptide W(NPW),which was first isolated from the porcinehypothalamus in 2002 by Shimomura et al,exists in two isoforms,consisting of 23(NPW23)or 30(NPW30)amino acids.Because both the first and last amino acids of the NPW30 are Trp,so named the peptide as neuropeptide W.NPB and NPWwere the endogenous ligands for G protein-coupled receptors GPR7(NPBWR1)and GPR8(NPBWR2).Previous reports have confirmed that NPB and NPBWR1 were widely distributed in mammalian animals and participated in regulation of various physiological functions such as feeding,neuroendocrine,reproductive,inflammatory pain and emotion.Since the discovery of NPB,there were a lot of study on its genetic structure,distribution and physiological function in humans and rats,but has not yet seen the relevant research in the pig.Therefore,the we took the pig as the research object in this study,we used the electron cloning,RT-PCR,Q-PCR cloned NPB and its receptor of pig and studyed their distribution in the pig by electron cloning and RT-PCR.Furthermore,we found that the NPBWR1 and NPBWR2 were expressed in pig ovarian granulosa cells and leydig cells,and NPB and NPW affected secretion of sex homons from these cells in vitro.So we speculated that NPB and NPW play a role in regulation of pig reproduction.1.Gene cloning and expression of pig NPB and NPBWR1We got pig NPB and NPBWR1 sequences by electronic cloning and studied the distribution of their mRNAs in pig by QPCR.The results are as follows:(1)We cloned NPB and NPBWR1 full-length sequence in pigs,whose fragment sizes were 372 bp and 993 bp,prespectively.Both nucleotide and amino acid of NPB and NPBWR1 in pigs had high homology with that of other species.(2)NPB and NPBWR1 mRNAs were widely expressed in central nervous system and peripheral tissues and organs in pig.2.The expression and distribution of NPBWR1 and NPBWR2 in pig leydig and ovarian granulosa cells(1)The expressions of NPBWR1 and NPBWR2 mRNAs were detected in pig leydig and ovarian granulosa cells.(2)The distribution of NPBWR1 in these was confirmed by the imunohistochemistry and immunofluorescence.3.The effects of NPB and NPW on homone secretion of pig leydig and ovarian granulosa cells in vitroDifferent concentrations of NPB or NPW(10-12,10-10,10-8 and 10-6 M)promoted the secretion of testosterone in leydig cells in concentration-dependent manner.Different doses of NPB or NPW could promote the secretion of progesterone in ovarian granulosa cells in dose-dependent manner.Low concentrations of NPB or NPW(10-8 and 10-10 M)promoted estradiol secretion,but high concentrations of NPB or NPW(10-6 M)inhibited its secretion.Both NPB or NPW affected significantly the proliferation of leydig cells. |
