Melatonin prevents post-ovulatory oocyte aging in the mouse and extends the window for optimal fertilization in vitro Short title: Melatonin prevents oocyte aging in vitro

The quality of metaphase II oocytes deteriorates rapidly following ovulation as the result of an aging process associated with impaired fertilizing potential, disrupted developmental competence and an increased likelihood of embryonic resorption. As oxidative stress has been shown to accelerate the onset of apoptosis in oocytes and influence their capacity for fertilization, this study aimed to characterize the significance of such stress in the post-ovulatory aging of mouse oocytes in vitro. In the course of these studies, we investigated the ability of the potent antioxidant, melatonin, to arrest the aging process when used to supplement oocyte culture medium. This study demonstrated that oxidative stress may occur in oocytes after as little as 8 h in culture and coincides with the appearance of early apoptotic markers such as phosphatidylserine externalization, and is followed 16 h later by caspase activation (P < 0.05) and morphological evidence of oocyte senescence (P < 0.001). Importantly, supplementation of oocyte culture medium with 1 mM melatonin was able to significantly relieve the timedependent appearance of oxidative stress in oocytes (P < 0.05) and, as a result, significantly delay the onset of apoptosis (P < 0.05). Furthermore, melatonin supplementation extended the optimal window for fertilization of oocytes aged for 8 h and 16 h in vitro (P < 0.05), and significantly improved the quality of the resulting embryos (P < 0.01). We conclude that melatonin may be a useful tool in a clinical setting to prevent the time-dependent deterioration of oocyte quality following prolonged culture in vitro. Summary: Addition of the antioxidant melatonin to oocyte culture media delays the onset of post-ovulatory oocyte apoptosis; it not only extends the window for optimum fertilization but also improves the quality of resulting blastocysts.