Objective  To analyze the proteomic differences between mouse secondary oocytes, also known as metaphase Ⅱ oocytes (MⅡ), and the first polar bodies (PB1) using high-resolution single-cell proteomics, to identify key proteins regulating embryonic development, and to provide a molecular basis for optimizing in vitro oocyte maturation systems.

Methods  Paired samples of MⅡ (n = 5) and PB1 (n = 5) were analyzed using high-resolution single-cell mass spectrometry (timsTOF HT). Quantitative proteomics and bioinformatics approaches were employed to conduct differential protein screening and functional enrichment.

Results  Using the timsTOF HT platform, we achieved the detection of over 3000 proteins per single cell and identified 277 proteins specifically enriched in MⅡ. Gene Set Enrichment Analysis (GSEA) revealed that these MⅡ-specific proteins were significantly enriched in gene regulation and DNA damage repair pathways associated with mitochondrial energy metabolism. Cross-species GSEA comparison between human and mouse homologs demonstrated elevated expression of heat shock proteins, including Hsp90b1, Hspa5, etc., in the mTORC1 pathway in MⅡ (P < 0.05). In addition, key factors regulating cumulus complex development, such as Calr, Aldoa, etc., were significantly upregulated.

Conclusion  MⅡ strategically retains proteins essential for embryonic development through asymmetric division. The timsTOF HT platform demonstrated superior sensitivity in analyzing and identifying these proteins. According to the protein analysis results, the distribution of mTORC1 pathway proteins indicates that they play a key role in embryonic metabolism regulation. In particular, heat shock proteins facilitate protein folding and maintain endoplasmic reticulum homeostasis, thereby ensuring oocyte maturation and the embryonic developmental potential.