Stage-dependent DNA damage and mitochondrial dysfunction under simulated microgravity constrain oocyte maturation and are mitigated by melatonin.

During spaceflight, the female reproductive system undergoes substantial adaptation to microgravity and faces an increased risk of reproductive impairment. Mammalian oocytes remain arrested for extended periods at the first meiotic prophase, a stage particularly vulnerable to DNA damage, yet whether simulated microgravity (SMG) directly induces oocyte genomic damage and how such damage, together with the associated meiotic arrest, can be mitigated remain unclear. Melatonin (MLT), a pineal hormone with broad physiological roles, has shown benefits in improving oocyte quality in vivo and in vitro. Using SMG as a stress model, we demonstrate that during the prophase-arrested stage, SMG exposure induces DNA double-strand breaks with activation of the ATM-CHK2 DNA damage response, without detectable elevation of global cellular ROS or mitochondrial superoxide. These changes were accompanied by abnormal mitochondrial distribution and increased early apoptosis. In vitro MLT supplementation during SMG exposure alleviated DNA damage through a DNA-PKcs-associated NHEJ repair response, improved mitochondrial distribution, and this protective effect was largely independent of canonical MT1 or MT2 receptor signaling. During meiotic maturation, MLT improved SMG-induced spindle assembly defects, promoted MTOC coalescence, suppressed mitochondrial unfolded protein response overactivation, reduced SMG-induced mitochondrial hyperpolarization, and reduced early apoptosis. Consequently, oocytes exposed to MLT exhibited increased first polar body extrusion, improved spindle integrity, and enhanced oocyte-intrinsic developmental competence, as reflected by increased blastocyst formation after parthenogenetic activation. Together, these findings show that SMG induces DNA damage in prophase-arrested oocytes and identify MLT as a stage- and dose-sensitive modulator of DNA repair and mitochondrial homeostasis, offering a potential strategy to protect female reproductive health during spaceflight.