Abstract
PurposeAlthough 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).MethodsThe 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.ResultsWe 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.ConclusionSIRT1 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.
Highlights
Oncological advances have improved survival rates of female cancer patients, after completing therapies many patients experience a reduced quality of life due to treatment side effects such as reduced fertility [1, 2]
sirtuin 1 (SIRT1), being a regulator of both follicle dormancy and ovarian stress response to CPM, is a good candidate as a sentinel molecule directly involved in the damage of ovarian reserve by alkylating agents. By virtue of these considerations, the principal aim of the present study is to evaluate the potential beneficial effects of EX-527, a specific inhibitor of SIRT1, on the action exerted by CPM on dormancy, activation and growth of primordial follicles (PFs), along with the underlying molecular mechanisms
Leibovitz’s L-15, α-MEM GlutaMax, Insulin-Transferrin-Selenium (ITS) 100 × and Live/Dead Fixable Far Red Stain were purchased from Invitrogen (Milan, Italy). l-Ascorbic acid, l-glutamine, bovine serum albumin (BSA), penicillin–streptomycin (PEN/STREP), amphotericin B, Hoechst 33,342, fructose, α-thioglycerol, eosin-Y, radio-immunoprecipitation assay (RIPA) buffer, polyvinylidene difluoride (PVDF) membrane, Tris-buffered and saline Tween 20 were purchased from Sigma Aldrich (Milan, Italy)
Summary
Oncological advances have improved survival rates of female cancer patients, after completing therapies many patients experience a reduced quality of life due to treatment side effects such as reduced fertility [1, 2] In addition to their effects on ovarian follicle growth, chemo- and Alkylating agents are the most toxic to the ovaries, and cyclophosphamide (CPM), which is routinely used against solid and haematological cancers, is associated with the highest rate of ovarian toxicity [5]. They involve accelerated follicle activation due to direct targeting of the mechanisms regulating follicle dormancy (burnout) [12] including the lack of suppressive factors such as anti-Müllerian hormone (AMH) as a consequence of developing follicle degeneration [13]. Relevant research in the animal model has highlighted the activation of molecular stress responses to DNA damage (DDR) and oxidative stress (OSR) induced by CPM [6, 8, 10, 17–22]
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