The Effect Of FSH On The Expression Of Skp2,p27^kip1 In Cultured Immature Boar Sertoli Cells

Sertoli cell plays an important role in sperm production.It is necessary for a constant ratio in Sertoli cells and germ cells to promote germ cell differentiation.Furthormore, the number of SC determines the size of adult animal testes and sperm's quantity. The process of SC proliferation is regulated by many factors, in all of which FSH is one of the most important hormones. S-phase kinase-associated protein 2(Skp2) is a key factor to regulating cell proliferation, which could bind ubiquitination of p27kip1 on the 26S proteasome degradation and promote cell process.The objective of the study is to identify whether FSH could regulate the expression of Skp2, p27kip1, and how FSH regulates the process. In the study,2 to 3 week-old piglets was chosen as material, and the expression of Skp2 and p27kip1 were detected by adding Inhibitors of various signaling pathways, using Western blot to drtect Skp2, p27kip1 protein's expression, and real time quantitative PCR detection of Skp2 mRNA expression.The results were as followed:(1) FSH promoted the expression of Skp2 protein from 15-90min, and the expression fo Skp2 protein was the highest in 30 min, then the expression of Skp2 began to decreased. FSH also promotes Skp2 mRNA expression after adding FSH 15min, and Skp2 mRNA expression was significantly increased compared with the control group increased by 11.56 times (P<0.05), while in 30min, Skp2 mRNA expression was the highest, which 17.76-fold (P<0.05)to control group, then the expression level decreased.(2)Adding cAMP enhancers Forskolin (10uM) as same as FSH were significantly promoted Skp2 protein's expression, inhibited the degradation of p27kip1 protein; the cAMP inhibitor Rp-cAMP (20 uM) alone were no significant effect for Skp2, p27kip1 proteins expression, but inhibited FSH on Skp2, p27kip1 proteins.Meanwhile, Ca2+ channel blocker (Verapamil) alone was not significantly affected for Skp2, p27kip1 protein expression, but it can inhibited FSH induced the expression of Skp2, p27kip1 protein. The effect of Verapamil alone for Skp2 mRNA expression when compared with control group was not significant (P> 0.05). (3) ERK inhibitor U0126 alone was not significantly affected,on the protein expression of Skp2, p27k'pl,but inhibited FSH-induced the expression of Skp2, p27kipl. the effect of U0126 alone on the expression of Skp2 mRNA had no significant effect (P> 0.05), but U0126 inhibited FSH induced the expression of Skp2 mRNA compared with FSH alone decreased 53.0%(P<0.05).In summary, this study can be the following conclusions:(1) FSH (50ng/ml) promote the expression of Skp2 in Sertoli cell in time-dependent manner and the expression fo Skp2 protein was the highest in 30 min.(2)FSH induced Skp2 mRNA and protein expression through cAMP generation and promoting the flow of Ca2+and the activation of ERK 1/2 cascade.(3)The level of Skp2 and the level of p27kipl is negative correlation, in Sertoli cells.