Mechanism Of Oxidative Stress On The Expression Of Puma In Ovarian Granulosa Cells Of Mice

The environmental stress in the pig production often causes the body and the organization to elevate oxidize the free radicals,oxidative stress is a common mechanism by which multiple stress factors impair reproductive performance.On the other hand,the reproductive activity of animals is accompanied by an increase in oxidative stress levels,and oxidative stress is considered to be the cost of animal breeding.In the process of female animal breeding,the ovary is one of the most important organs,the quality of ovaries will determine the animal's reproductive capacity and production performance,ovarian development process,the vast majority of follicles have degenerated and disappeared,the process known as follicular atresia,and recent studies have shown that follicular atresia is the main cause of granulocyte apoptosis.Puma is a new member of BH3-only subfamilies of the Bcl-2 family.and it can be rapidly induced and has potent pro-apoptotic effects and is a key mediator of P53-dependent and non-dependent apoptotic pathways.Puma is associated with follicular atresia,tumorigenesis and tissue damage,and is a very important target for the regulation of follicular atresia,tumorigenesis and tissue damage.Puma is regulated by multiple transcription factor,such as P53,P73,NF-?B,FoxO family and c-Myc.Previous studies of our lab showed that FoxO1 could mediate the expression of Puma to induce granulosa cells apoptosis after adding 200 ?M H2O2 in mouse ovarian granulosa cells.However,the mechanism of oxidative stress induced Puma expression in mouse ovarian granulosa cells was not deeply studied.A large number of studies have confirmed that P53 and NF-?B are the two most important transcription factors that regulate the expression of Puma.In this research,this two transcription factor inhibitors were used to investigate whether P53 and NF-?B were also involved in oxidative stress induced Puma expression on mouse ovarian follicular granulosa cells.In this study,15?M PFT-a and 20?M PDTC were used to detect the effect of oxidative stress induced up-regulation of Puma gene expression.Immunofluorescence staining and chromatin immunoprecipitation were used to verify the effect of 200?M H2O2 on the P53 nucleation and the binding to the Puma promoter.In addition,the combination of PFT-a and H2O2 can inhibit oxidative stress-induced P53 nucleation and the increase in binding to the Puma promoter.TUNEL assay was used to study the effect of PFT-a on oxidative stress-induced granulosa cells apoptosis,and the apoptosis-related genes were detected by fluorescence quantitative PCR.TUNEL also examined the effect of SP600125 on oxidative stress-induced granulosa cells apoptosis.The effect of SP600125 on oxidative stress-induced P53 nucleation was also detected by immunofluorescence staining.The results of qRT-PCR showed that PFT-a could inhibit the up-regulation of oxidative stress-induced Puma gene mRNA expression,PDTC could not inhibit the up-regulation of Puma gene mRNA expression.Immunofluorescence staining and chromatin immunoprecipitation showed that 200?M H2O2 treatment could promote P53 nucleation and the increase of binding with Puma promoter,And the combination of PFT-? and H2O2 could inhibit P53 nucleation and bind to Puma promoter.TUNEL assay showed that PFT-a could inhibit oxidative stress-induced granulosa cells apoptosis,and PFT-a could decrease the expression of Bim and Caspase-3 mRNA induced by oxidative stress,but the difference was not significant.Finally,immunofluorescence staining showed that SP600125 could inhibit oxidative stress-induced P53 nucleation,and SP600125 treatment could inhibit oxidative stress-induced granulosa cells apoptosis.This study elucidates the mechanism of oxidative stress on the expression of Puma in ovarian granulosa cells,Oxidative stress treatment can activate the activity of JNK,activation of JNK phosphorylation of P53 and P53 nucleation;P53 and Puma promoter increased,causing Puma gene mRNA expression increased,and thus induce apoptosis of granulosa cells.Revealing the molecular mechanism of mouse ovarian granulosa cell apoptosis not only can provide a valuable reference for people to develop new technologies and measures to improve sow animal fecundity,but also can provide reference for the prevention and treatment of human reproductive diseases and to extend the growth period of women.