Single-cell proteomics analysis of human oocytes during GV-to-MI transition

Abstract STUDY QUESTION Which proteins are involved in the transition of human oocytes from the germinal vesicle (GV) to metaphase I (MI) phase? SUMMARY ANSWER A total of 2369 proteins were identified, including 149 with significantly differential expression, 79 with upregulated expression in MI oocytes and 70 with downregulated expression. WHAT IS KNOWN ALREADY During oocyte maturation, maternal proteins and RNA are stored to support early embryo development. However, GV oocytes matured in vitro have a lower chance of developing into blastocysts than MI oocytes. Therefore, identifying key differentially expressed proteins between the GV and MI stages can provide a better understanding of human oocyte development and maturation mechanisms and improve the utilization of oocytes. STUDY DESIGN, SIZE, DURATION In total, 16 oocytes at the GV and MI stages were collected from female patients who underwent ovulation induction due to male factor infertility requiring embryo retrieval for ICSI. Differential proteins were identified in 16 oocytes using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, and the expression of several differential proteins was verified by immunofluorescence (IF). RNA interference was employed to identify the functions of specific proteins during oocyte maturation. PARTICIPANTS/MATERIALS, SETTING, METHODS 16 immature human oocytes discarded during ICSI cycles (eight GV oocytes and eight MI oocytes) were collected from 10 female patients. Two cohorts of oocytes underwent zona pellucida removal, lysis, and enzymatic digestion prior to peptide detection using LC-MS/MS methodology. Peptide detection outcomes were subjected to differential protein screening and functional annotation employing distinct analytical algorithms and datasets. To corroborate the sequencing findings, proteins exhibiting notable differential expression were authenticated via IF. Concerning protein functionality, siRNA was introduced during the GV phase, and oocyte maturation was evaluated through observation of polar body extrusion, alongside assessment of siRNA interference efficacy via IF analysis. MAIN RESULTS AND THE ROLE OF CHANCE A total of 2369 proteins were identified, including 149 with significantly differential expression, 79 with upregulated expression in MI oocytes and 70 with downregulated expression. Gene ontology functional annotation and functional analysis revealed that these differentially expressed proteins are involved mainly in organic matter and cell metabolism, biological regulation, primary metabolism, nitrogen compound metabolism, and other biological processes. Kyoto Encyclopedia of Genes and Genomes analysis revealed that the differentially expressed genes were involved mainly in the following pathways: transport and catabolism, signal transduction, protein folding, and energy and amino acid metabolism. The differentially expressed proteins included actin-related protein 2 (ACTR2), NADH: Ubiquinone Oxidoreductase Core Subunit S1 (NDUFS1), Tubulin Gamma Complex Component 3 (TUBGCP3), Heat Shock Protein Family B (Small) Member 1 (HSPB1), and Eukaryotic Translation Initiation Factor 3 Subunit B, which are involved mainly in mitochondrial function, cell division, and signal transduction. ACTR2, HSPB1, NDUFS1, and TUBGCP3 were selected for IF staining, and the difference in fluorescence intensity between GV and MI oocytes was consistent with the sequencing results. Three pairs of primers were designed for each gene corresponding to the top 10 differentially upregulated and downregulated proteins (with siRNAs successfully designed for eight upregulated and seven downregulated proteins) to study their function, and the results revealed that the protein expression of TUBGCP3 was downregulated after RNA interference. LARGE SCALE DATA See supplementary tables. LIMITATIONS, REASONS FOR CAUTION Although we have identified some differentially expressed proteins during the transition from human oocyte GV to MI stage, their crucial roles in oocyte maturation remain elusive. To elucidate the functions of these proteins in oocyte maturation, we have generated conditional knockout mice targeting selected proteins. WIDER IMPLICATIONS OF THE FINDINGS We conducted single-cell level analysis to identify differentially expressed proteins between the human oocyte GV and MI stages. Our objective is to ascertain the potential of supplementing these proteins in the in vitro maturation culture medium to augment both oocyte maturation rates and quality. STUDY FUNDING/COMPETING INTEREST(S) This research was supported by the National Natural Science Foundation of China (82171599 and 82471657, B.X., 82301871, L.L.); China Postdoctoral Science Foundation (2024M763169, S.B.); and the National Key Research and Development Project of China (2029YFA0802600, B.X.). None of the authors has any conflict of interest to declare. TRIAL REGISTRATION NUMBER N/A.