The Study Of Expression Of OPN In Male Developmental Reproductive Tract Of Hu Sheep

Osteopontin(OPN) is a highly acidic glycoprotein, rich in aspartic acid, glutamic acid and serine and was initially identified in the mineralized matrix of bovine bone. Subsequently some studies reported that OPN was expressed in many tissues and fluids, including gastrointestinal, testis, milk, uterine and so on and involved in various physical and pathological processes. Recent studies have found that OPN was widespreadly existed in male reproductive tract, but there were some differences between homogeneity and heterogeneity, and only 55 KDa isoform was positively correlated with bovin fertility. Hu sheep was with ith the capacity of high fertility and recent researches were mainly focus on the female Hu sheep, however there was few reports about mle Hu sheep fertility. Therefore, the experiment took the male developmental reproductive tract(testis, caput, corpus, cauda and ductus deferens) and cauda epididymis sperm of sexual maturity Hu sheep as research objects and studied the expression level of OPN mRNA and protein for providing the theory basis about its connection with male Hu sheep fertility.The study was divided into three parts, the first part focused on the localization of OPN protein in the male develpmental reproductive tract of Hu sheep; the second part was to study the relative exression level of OPN mRNA in that process; the third part detected the molecular weight of OPN protein and analyzed the expressive differences. The main results were as follows:1. In the experiment of OPN protein localization in development, we found that OPN was mainly located in the late development of germ cells in 8-month-old Hu sheep(sexual maturity). In the epididymis and ductus deferens, the apical region of principal cells cytoplasm mainly expressed OPN and there was no positive signal in basical cells. IF indicated that OPN was strongly expressed in the posterior of acrosome, midpiece and principal piece, while weakly stained in the sperm acrosome acrosomal cap and end piece. Not all sperm in a given sample showed a consistent staining for OPN. In the 4-month-old and 10-day-old Hu sheep, OPN was expressed in the testicular interstitium, not in germ cells. In addtion, OPN was mainly expressed in the lamina propria and smooth muscle in the caput and corpus of 4-month-old and epithelial cells in the cauda and ductus deferens expressed OPN. In the 10-day-old Hu sheep epididymis, OPN was mainly expressed in the smooth muscle, while in epithelial cells in the ductus deferens. Moreover, the exfoliative epithelium cells in the sheep ductus deferens lumen of 10-day-old Hu also showed the positive signal of OPN.2. Q-PCR detected that the OPN mRNA expression level in the testis, epididymis and ductus deferens of 8-month-old was signifcantly higher than 4-month-old and 10-day-old, between which it had no siginifant difference. In addition, there also existed significant difference in these tissues with the same age stage.3. The study first detected a prominent 34 KDa isoform in the testis, epididymis and ductus deferens of each age stage by western blot(WB). In addition, small quantities of the 30 KDa isoform were also present in these tissues. Moreover, gray scale analysis found that the relative expresion level of OPN protein in these tissues had no significant difference.