Abstract
Once ovulated, the oocyte has to be fertilized in a short time window or it will undergo post-ovulation aging (POA), whose underlying mechanisms are still not elucidated. Here, we optimized single-cell proteomics methods and performed single-cell transcriptomic, proteomic, and phosphoproteomic analysis of fresh, POA, and melatonin-treated POA oocytes. POA oocytes showed downregulation of most differentially expressed proteins, with little correlation with mRNA expression, and the protein changes can be rescued by melatonin treatment. MG132 treatment rescued the decreased fertilization and polyspermy rates and upregulated fragmentation and parthenogenesis rates of POA oocytes. MG132-treated oocytes displayed health status at proteome, phosphoproteome, and fertilization ability similar to fresh oocytes, suggesting that protein stabilization might be the underlying mechanism for melatonin to rescue POA. The important roles of proteasome-mediated protein degradation during oocyte POA revealed by single-cell multi-omics analyses offer new perspectives for increasing oocyte quality during POA and improving assisted reproduction technologies.