Investigation of NAD+ metabolism alterations during follicular atresia in porcine ovaries and their regulatory mechanisms on granulosa cell apoptosis.

Follicular atresia plays a significant role in the depletion of the ovarian reserve and the decline of overall ovarian function. Understanding its molecular mechanisms is essential to preventing ovarian aging and maintaining female reproductive health. Although NAD+ depletion is known to induce apoptosis in various cell types, the dynamic changes in NAD+ metabolism during follicular atresia remain unclear. Moreover, its specific impact on granulosa cells has not been fully elucidated. In this study, we examined the metabolic alterations of NAD+ during follicular atresia. We also explored the molecular mechanisms underlying granulosa cell apoptosis to provide a theoretical basis for developing novel therapeutic strategies to preserve female fertility. Using a well-established porcine model of follicular atresia, we observed a downregulation of key enzymes involved in NAD+ biosynthesis during follicular regression, accompanied by an increased progesterone to estradiol (P4/E2) ratio in the follicular fluid. In granulosa cells, reduced NAD+ levels and decreased NAD+/NADH ratios activated caspase-3 thereby triggering apoptosis. In a vitro model of granulosa cell apoptosis, we further demonstrated that NAD+ precursors can bypass the rate-limiting enzyme NAMPT but NMNAT1 remaines essential for effective NAD salvage. Deficiency of NMNAT1 compromised nuclear SIRT1 activity, leading to excessive p53 acetylation and the induction of apoptosis Additionally, pharmacological inhibition of NAMPT impaired mitochondrial SIRT3 activity, which enhanced p53-mediated apoptotic pathways. In conclusion, the NAD+ salvage pathway is crucial for regulating follicular atresia, NMNAT1 acts as a key regulator in granulosa cells within this pathway. It supports cell survival through SIRT1-mediated deacetylation of p53. These findings identify NMNAT1 as a potential therapeutic target to delay ovarian aging and preserve females fertility.