Role of SIRT1 in the adaptive response of mouse ovaries to radiotherapy (X ray radiations)

Study question: Is SIRT1 involved in ovarian adaptive response to gonadotoxic damage induced by radiotherapy in adult, prepubertal and aged mice? Summary answer: SIRT1 orchestrates the adaptive response of mouse ovaries against radiotherapy in adult but not in prepubertal and aged mice. What is known already: Radiotherapy is one of the most effective anticancer therapies and causes damage in both cancer and surrounding healthy tissues via ionization, formation of reactive oxygen species (ROS) and subsequent impairment of DNA double-strand integrity and oxidative stress (OS) establishment. Ovaries are very sensitive to radiation-related damage, which may cause premature ovarian failure and menopause. SIRT1, a histone deacetylase involved in oocyte and ovarian adaptive response to OS and chemotherapy, is activated by changes in the redox state and promotes cell survival/apoptosis. Saffron-derived crocetin is an antioxidant molecule with anti-tumor effects that protects non-malignant tissues against chemotherapy-induced toxicity. Study design, size, duration: Part I) CD1 female mice aged 3-4 weeks (n = 12, Prepubertal mice); 8-10 weeks (n = 12, Adult) or 48-52 weeks (n = 6, Aged) were exposed to 3 Gy total body irradiation. Part II) Ovaries from postnatal day 4 (PND4) CD1 mice were cultured in MEM-alpha with 3 mg/ml BSA supplemented with or without 50 μM crocetin for 24 hrs. Then ovaries were exposed to 2 Gy and cultured for further 24 hrs in medium with or withouth crocetin. Participants/materials, setting, methods: Prepubertal, adult and aged mice were sacrificed at 24 hr after radiotherapy. Ovaries were removed and processed for protein analysis. The relative abundance of SIRT1, HuR, and pFOXO3a was investigated by Western blotting (WB). To investigate potential beneficial effect of a natural antioxidant, crocetin, on damage induced by radiotherapy, PND4 ovaries were processed at 24 hr after the irradiation and the expression of SIRT1, HuR and PGC1-alpha was assessed by WB analysis. Main results and the role of chance: In ovaries from adult mice exposed to radiotherapy, we observed an increase of SIRT1 and SIRT1 mRNA stabilizer, HuR, demonstrating an early involvement of SIRT1 signalling to counteract damage by radiations (One-way ANOVA and Student-Newman-Keuls Multiple comparison p<0.05). By contrast in prepubertal or aged ovaries we observed that SIRT1 has a different expression level according to the class of age and decreases after radiations. FOXO3A is a factor involved in the maintenance of follicle quiescence. Its inactive form pFOXO3a was found to increase in both adult and prepubertal mice after radiations, indicating a wave of primordial follicle activation. PND4 ovaries are endowed of primordial follicle as unique follicle population. In these ovaries SIRT1 and HuR decreased in response to radiations, as observed in prepubertal mice. Medium supplementation with crocetin decreased SIRT1 expression but increased HuR, demonstrating a partial response to damage. Furthermore, since mitochondria have a central role in the regulation of redox state, we investigated the effects of radiations on PGC1-alpha, marker of mitochondria biogenesis and function. Crocetin supplementation prevented the decreased expression of PCG1-alpha induced in PND4 ovaries by radiations. Limitations, reasons for caution: This mouse model only offers information on short-term effects of radiation, and how these ovaries would look a few weeks later is still a question. Wider implications of the findings: This study increases the knowledge on the adaptive response orchestrated by SIRT1 in response to gonadotoxicity induced by radiotherapy and highlights the beneficial effect of saffron-derived crocetin, an antioxidant molecule with anticancer properties. Radiosensitivity is associated with a reduced ability to activate an adaptive response mediated by SIRT1 and HuR.