Establishment of porcine ovarian follicles treated with t-BHP for an in vitro aging model

Ovarian aging is closely associated with low female fertility. Excessive oxidative stress can induce ovarian senescence and follicular atresia, thereby reducing reproductive performance. In this study, intact antral follicles of pigs were treated with tert-butyl hydroperoxide (t-BHP) to simulate aging stimulation conditions. The results showed that 200 μM t-BHP induced the senescence-related phenotype of antral follicles, which was time-dependent and changed significantly after 6 h of treatment. t-BHP can induce an increase in reactive oxygen species (ROS) and senescence mediated by Caspase-3, P53, Foxo1, and SOD. Senescence-associated β-galactosidase (SA-β-Gal) staining revealed an increase in the number of positive cells. Enzyme-linked immunosorbent assays (ELISA) showed that the level of reactive oxygen species increased, and that the ratio of progesterone (P 4 ) to estradiol (E 2 ) increased. Finally, the findings of this study showed that the relative expression levels of Caspase-3, P53, Foxo1 mRNA, and protein increased, while the relative expression levels of SOD decreased. In conclusion, treatment with 200 μM of t-BHP effectively induced follicular senescence at 6 h. Therefore, the in vitro treatment of follicles with 200 μM t-BHP for 6 h may be a feasible in vitro culture model to simulate ovarian senescence in gilts.