Protective effects of a SIRT1 inhibitor on primordial follicle activation and growth induced by cyclophosphamide: insights from a bovine in vitro folliculogenesis system

Purpose: Although oncological advances have improved survival rates of female cancer patients, they often suffer a reduced fertility due to treatment side effects. In the present study, we evaluated the potential fertoprotective effects of the specific inhibitor of SIRT1, EX-527, on the gonadotoxic action exerted by cyclophosphamide (CPM) on loss of primordial follicles (PFs). Methods: The effects of the CPM metabolite phosphoramide mustard (PM) on follicle activation, growth and viability and the protective action of EX-527 against PM effects were evaluated on bovine ovarian cortical strips in vitro cultured for 1 or 6 days. To understand whether PFs exposed to PM plus EX-527 were able to activate and grow to the secondary stage after suspension of the treatment, strips cultured for 3 days in PM plus EX-527 for 3 days were transferred to plain medium until day 6. Follicle growth and health were evaluated through histology and viability assay at a confocal microscope. In order to investigate the molecular pathways underlying the ovarian response to PM in the presence of EX-527, we analysed the protein level of SIRT1, HuR, PARP1 and SOD2 after 1 day of in vitro culture. Results: We found that (1) PM, the main CPM active metabolite, promotes PF activation; (2) the ovarian stress response induced by PM includes a SIRT1-dependent pathway; and (3) EX-527 reduces PF activation and growth induced by PM. Conclusion: SIRT1 can represent a candidate molecule to be targeted to protect ovarian follicles from alkylating agents and EX-527 could represent a potential fertoprotective agent for cancer patients.