The Distribution Of Neuropeptide Y(NPY) And Its Receptors In The Reproductive Tract Of Hens

There is a special tubular glands found in utero-vaginal junction and infundibulum in hen’s repreductive tracts,which could keep chicken sperm alive for a long time and still remain fertilizing capacity,in addition,it also has the ability to store sperm for a long time, therefore, these tubular glands were called sperm storage tubules.Neuropeptidey(NPY) is the different expressed gene in sperm storage tubules. In this experiment,NPY was chosen to disscuss the influence of sperm in reproductive tract.Immunohistochemistry was used to explore the expression sites of NPY and its receptor NPY1 R, NPY5 R in hen’s magnum, infundibulum, uterus, isthmus and utero-vaginal junction. Our results found that the protein of NPY, NPY1 R existed at the epithelium of reproductive tract in hens. The strongest immunoreaction of NPY is at the epithelium of infundibulum, and strong immunoreaction is at the magnum and isthmus fragment. The strong immunoreaction of NPY1 R is at the epithelium of magnum and isthmus.Based on the sequences of NPY and NPY1 R, NPY5 R genes in GeneBank, specific primers were designed to amplify the conserved region sequences. The expression of chicken NPY, NPY1 R, and NPY5 R genes in hen’s magnum, infundibulum, uterus, isthmus, vagina and utero-vaginal junction were analyzed by fluorescence quantitative assay. The results showed that NPY expression in the isthmus and vagina were higher than other regines in hen reproductive tract(P<0.05); The expression of NPY1 R was the highest in vagina(P<0.05); The expression of NPY1 R was the highest in vagina and utero-vaginal junction(P<0.05).According to the mRNA sequence of chicken NPY in GeneBank, we synthesized NPY DNA sequence that suit for codon preference of pichia pastoris and inserted into pPICZαA vector. The recombinant pPICZαA was constructed and then transformed into Pichia pastoris GS115 genome by electroporation. The positive recombinant strains(GS115/NPY) were inducted to express recombinant protein by addition of 1% methanol every 24 h.Then Tricine-SDS-PAGE electrophoresis and Western Blot of His-tag protein analysis were uesd to detected recombinant protein. The results showed that the recombinant NPY protein could be successfully secreted by positive recombinant strains and the molecular weight of NPY protein was approximately 35 kDa.