Energy metabolism is central to oogenesis, yet our understanding of metabolic regulation in early-stage oocytes remains limited, largely due to the difficulty of isolating sufficient numbers of small oocytes for protein and metabolic analyses. We developed a method to isolate dormant and growing oocytes using the OCT4-GFP mice combined with flow cytometry, yielding a pure population of thousands of small oocytes within a short timeframe. Purity of the isolated oocytes was confirmed by GFP signals via microscopy and by proteomic detection of oocyte- and somatic cell-specific markers. Before conducting label-free proteomics, protein concentrations were normalised across groups by adjusting the protein injection amount to 100 ng per sample. We then used proteomics data to characterise metabolic pathways, including oxidative phosphorylation (OXPHOS), glycolysis, the TCA cycle, and fatty acid β-oxidation in dormant, growing, and fully grown oocytes.

All electron transport chain complexes were progressively downregulated during oocyte growth, with complex IV showing the smallest decline, suggesting a general suppression of OXPHOS. Proteins regulating TCA cycle showed a differential pattern, with some downregulated, some upregulated, and others unchanged. Proteins involved in mitochondrial β-oxidation declined during oocyte growth, whereas peroxisomal β-oxidation proteins were upregulated. Unexpectedly, almost all glycolytic enzymes were upregulated in fully grown oocytes, and pathway enrichment analysis confirmed a relative decrease in OXPHOS and an enrichment of the proteins in the glycolytic pathway during oocyte growth.

Together, these findings establish a robust new method for isolating small oocytes and reveal a coordinated change in the oocyte metabolic proteome, from OXPHOS towards glycolysis. Given that glycolysis is dispensable for oocyte function, this shift suggests maternal glycolytic enzymes may contribute to the upregulation of glycolysis in preimplantation development.

Keywords: oocyte growth, oocyte isolation, energy metabolism, OXPHOS, glycolysis, proteomics analysis