Gene regulatory networks orchestrating oocyte fate bifurcation in primordial follicles revealed by single-cell transcriptomics

The first wave of primordial follicle activation occurs around postnatal day 2.5 (P2.5), with the remaining follicles entering a dormant state to ensure a continuous supply of fertilizable oocytes. However, the molecular characterization and underlying mechanisms of this critical fate commitment remain poorly understood. Here, we employ SMART-seq2 to profile transcriptional dynamics in individual perinatal female C57BL/6 mouse germ cells across three developmental stages: cyst stage at embryonic day 17.5 (E17.5), primordial-follicle stage at P2.5, and primary-follicle stage at P6.5. Unsupervised clustering and trajectory inference reveal divergent transcriptional programs within P2.5 primordial oocytes, indicative of a bifurcating process in which pre-determined oocytes commit towards either dormant or activated states. Activated oocytes exhibit upregulation of genes linked to PI3K-Akt/mTORC1 signaling, extracellular matrix (ECM) disassembly, and oocyte maturation, whereas marker genes of dormant oocytes are enriched in oxidative stress response and DNA repair pathways. Transcriptional regulatory network reconstruction based on SCENIC inference identifies key transcription factors (e.g., BHLHE41, TCF3/12) orchestrating the fate bifurcation. Notably, dormant oocytes form a stable transcriptional state distinct from the continuous activation and development trajectory. Our findings provide a comprehensive landscape of gene expression and regulatory networks that orchestrate fate determination in P2.5 primordial-follicle oocytes.