Functional Study Of Sumolyation P27^kip1 In Mouse Sertoli Cellcycle

The number of Sertoli cells determine the size of the testis and the production of sperm per day, each Sertoli cells support limited germ cells.. p27Kip1(hereafter p27) is the marker of matured sertoli cells which mainly inhibit CDK2 and consquently block cell cycle progression. Protein SUMOylation will affect the substrate function, subcellular location, activity and cellular transport. In this study, we mainly focus on whether p27 could be SUMOylated and the SUMOylation amino acidsite. Since the specific location of p27 on testis and the funcion of p27 on cell cycle control(not Sertoli cells),we also try to illustrate the function of p27 on sertoli cell cycle regulation.The experiment design as follows:1. IHC and WB detect the location and expression level of p27 on day 0pp, day 4pp,day 10 pp and day 42pp;2. Transfect over-expression, wild type or site mutant p27 plasmid into sensitive cell CHO-K1, and then employed WB, we check the SUMOylation pattern of p27 to clarify the SUMOylationsite;3. Isolation and culture day 7pp mouse sertoli cell, and over expressed wild type or site mutant p27 in day 7pp mouse sertoli cell, after that,we detecting the cell cycle of sertoli cell by using flow cytometry.The result showed that1. p27 is expressed in the mouse testis of every period, p27 was posttranslation modified by several forms(phosphorylation,ubiquitylation,SUMOylation), the highest expression of p27 was on day 10 pp, mainly located in the nuclear of germ cell, in adult mouse testis(day 42pp), a strong expression of p27 in all Sertoli cells was found;2. Mouse p27 could be SUMOylated by SUMO-1, only mutant lys189 could abolish p27 SUMOylated, moreover, the stablity of p27 was significant declined after SUMOylation was blocked;3. The G1 rate of sertoli cell increased after over expression of wild type p27 andlysine reidue 190 mutant plasmid(p<0.05), there is no significant affect on cell cycle after lysine reidue 189 and 189/190 mutant plasmid transfection.4. All in all, we identified the SUMOylation site of mouse p27 were Lys189, and the SUMOylation of p27 influence its stability and the cell cycle of sertoli cell. These results will contribute on understanding and exploring the mechanisms of testicular cancer, also it can provide new ideas for increasing reproductive performance of male animals.