The Regulation Of P53Function By SUMOylation In Mouse Ovarian Granulosa Cells

Follicular atresia is a process of spontaneous degradation of follicles thus hindering its growth and development in mammalian ovary. Present study shows that follicular atresia is caused by apoptosis of granulose cells for which a number of apoptosis-related genes has already been identified, including p53gene. The half-life of the p53protein in normal cells is increased when they are exposed to various kinds of external stimuli. These stimuli influences the post-translational events and hence the stability of the proteins. Studies show that SUMO enhances the stability of p53protein but its activity is not clear so far. Besides this, little is known about the role of p53in apoptosis of mouse granulosa cells. The main objective of this study is to explore the role of p53in mouse granulosa cells and the effects of SUMOylation. The experimental design for this study and the respective results are briefly discussed below.In-vitro cultured of mouse ovarian granulosa cells were carried out for transformation. The granulosa cells were transfected/co-transfected with sumo-1and ubc9to reveal the modification pattern of SUMOylation, determine the SUMOylation site and its effects on the stability of p53protein. In addition, for the detection of biological activity of p53b i.e. SUMOylation including proliferation and apoptosis of granulosa cells and related genes were also quantitatively analyzed. The data initially revealed the SUMO modification of p53results in the proliferation and apoptosis of granulosa cells.The experimental results showed that (1) p53b protein can be modified by SUMOylation. p53b and potential SUMOylation site mutant expression vectors were co-transfected with the SUMO-1eukaryotic expression vector respectively to prove that its SUMOylation site is1ys375.(2) To discover whether modification is responsible for increase in the stability of p53b in a dose-dependent manner, SUMO-1doses were increased while keeping the p53b in a fixed proportion for the co-transfection of granulosa cells.(3) In addition, mutation in p53b SUMOylation site significantly reduced its ability to promote apoptosis of granulosa cells, but have no significant impact on its proliferation.(4) p53b has an inverse relation with CHK1gene i.e. overexpression of the p53b significantly reduce the expression of CHK1but is in direct relation with PCNA and Bax as the over expression of p53b causes dramatic increase in the expression of PCNA and Bax resulting in the destruction of SUMO binding site of p53b. Meanwhile it has no effect on the expression of CHK1and PCNA but the Bax expression is significantly reduced which reveals that regulation of granulosa cells proliferation by p53may be achieved through negative regulation of CHK1but the SUMO-1have no noticeable effects. Meanwhile SUMO-1modification of p53stimulates the apoptosis in granulosa cells.In short, findings in this study show that SUMO modification of p53b in mouse ovarian granulosa cells is involved in the regulation of p53b function. These results will help in better understanding of follicular granulosa cells development, follicular atresia and provide a theoretical/practical basis for ovarian cancer mechanism studies. Besides this it also provides a strong base for theoretical/practical studies related to human reproductive health, livestock genetics, breeding and reproduction.