Melatonin regulates the antioxidant capacity of sheep granulosa cells through a novel uORF-Nrf2aa mediated Nrf2/KEAP1 pathway

Ovarian dysfunction stands as a prevalent contributor to female infertility, with its etiology intertwined with genetic, autoimmune, and environmental factors. Within the ovarian follicles, granulosa cells (GCs) represent the predominant cell population. Alterations in GCs, notably oxidative stress (OS) and the consequential surge in reactive oxygen species (ROS), play pivotal roles in the orchestration of ovarian function. Nrf2aa, a newly identified upstream open reading frame (uORF), is situated within the 5' untranslated region (5'UTR) of sheep Nrf2 mRNA and is regulated by melatonin, a crucial intrafollicular antioxidant. In this study, we have noted that Nrf2aa has the capacity to encode a peptide and exerts a negative regulatory effect on the translation efficiency (TE) of the Nrf2 CDs region. Further in vitro experiments, we observed that interfering with Nrf2aa can enhance the cellular functionality of GCs under 3-np-induced oxidative stress, while overexpressing Nrf2aa has the opposite effect. Furthermore, overexpression of Nrf2aa counteracts the rescuing effect of melatonin on the cellular functions of GCs under oxidative stress conditions, including estrogen secretion, proliferation, apoptosis, and many more. Finally, we confirmed that Nrf2aa, by regulating the expression of key proteins in the Nrf2/KEAP1 signaling pathway, further modulates the antioxidant levels in GCs.