Putrescine promotes maturation of oocytes from reproductively old mice via mitochondrial autophagy

ABSTRACT Research question: Does putrescine improve oocytes from reproductively old mice by promoting mitochondrial autophagy༟ Design: Germinal vesicle (GV) stage cumulus-oocyte complexes (COCs) were obtained from 9-month-old female C57B6L/N mice and divided into three groups: NC (control group), PUT (putrescine group), and DFMO (difluoromethylornithine, inhibitor group). These GV-COCs underwent mouse in vitro maturation (IVM) culture to observe the extrusion of the first polar body in each group. Using JC-1 and DCFH-DA fluorescent probes and a confocal microscope to measure the mitochondrial membrane potential integrity and ROS levels in MII stage oocytes. The expression and cellular localization of the p53 protein were examined by immunofluorescence. RT-qPCR was used to detect the activation of mitochondrial autophagy pathways. The potential mechanisms through which putrescine improves oocytes from reproductively old mice were explored by single-cell transcriptomic analysis. Autophagosomes, autolysosomes, and mitochondria in different groups were directly observed using transmission electron microscopy. Results: The study found that adding exogenous putrescine can promote the in vitro maturation of oocytes from reproductively old mice. Putrescine reduces oxidative stress in oocytes from reproductively old mice by promoting the autophagy of damaged mitochondria, decreasing the levels of reactive oxygen species, and increasing mitochondrial membrane potential. Putrescine affects the expression and subcellular localization of the p53 protein, and increases the expression of TFEB, which may be the potential mechanism behind its promotion of autophagy. Conclusion: This study clarified the target and regulatory pathway of putrescine in oocytes, suggesting that putrescine is an effective small molecule compound with significant potential for non-invasively improving the fertility of elderly women.