N-acetylserotonin promotes synchronized nuclear and cytoplasmic maturation of porcine oocytes through granulosa cell-associated melatonin signaling during in vitro maturation

Nuclear-cytoplasmic asynchrony remains a key factor limiting the efficiency of porcine oocyte in vitro maturation (IVM). N-acetylserotonin (NAS), an intermediate in melatonin biosynthesis, has antioxidant and mitochondria-related biological activities, but its role in porcine oocyte maturation remains unclear. This study investigated the effects of NAS on nuclear-cytoplasmic synchrony during IVM and evaluated its relationship with melatonin signaling in cumulus-oocyte complexes (COCs). COCs were matured in vitro with different concentrations of NAS, and oocyte maturation quality was assessed by cumulus expansion, first polar body extrusion, spindles arrangement, blastocyst rate, cortical granule migration, redox status, mitochondrial function, cytoskeletal organization, transzonal projections (TZPs), melatonin synthesis, and melatonin receptor signaling. The results showed that 10⁻⁹ M NAS significantly increased cumulus expansion, cortical granule migration and blastocyst rate, but did not significantly affect first polar body extrusion and spindles normal rate. NAS improved oocyte cytoplasmic quality by reducing ROS levels, increasing GSH content and mitochondrial distribution, and upregulating SIRT3 , ND1 , and COX3 mRNA levels. NAS reduced mitochondrial membrane potential while ATP content remained unchanged. NAS also enhanced cytoskeletal organization without significantly altering the level of apoptosis. In COCs, NAS maintained stronger TZP signals at 30 h of IVM. ASMT, MT1, and MT2 were detected in COCs. NAS promoted melatonin synthesis during IVM and increased MT1 and MT2 mRNA expression in granulosa cells, whereas receptor expression in oocytes was not significantly changed. The melatonin receptor antagonist luzindole abolished the NAS-induced increase in cumulus expansion and caused a decreasing trend in first polar body extrusion. These findings indicate that NAS improves porcine oocyte cytoplasmic maturation mainly through melatonin-related signaling within COCs, thereby supporting nuclear-cytoplasmic synchronization during IVM.