Integrative Single-Cell Proteomics and Network Analysis Identifies DHX9 as a Key Regulator of Oocyte Maturation and a Therapeutic Target for Oocyte Developmental Arrest

Abstract Oocyte developmental arrest at the germinal vesicle (GV) or metaphase I (MI) stage is a major cause of infertility, yet its molecular mechanisms remain poorly understood. Here, we performed ultradeep single-cell proteomics on 8 human oocytes (4 GV-stage and 4 MI-stage) and 12 mouse oocytes (3 GV, 3 MI, 3 MII, and 3 FGO) to investigate the proteomic landscape of developmental arrest. We identified an average of 5,574 proteins per human oocyte (range: 4,350–6,157) and 3,477 per mouse oocyte (range: 3,315–3,634), revealing aberrant activation of mRNA splicing pathways in arrested oocytes. Integrative network analysis identified the RNA helicase DHX9 as a central hub within a conserved coexpression module. Mechanistically, elevated DHX9 expression maintained transcriptional hyperactivity, preventing the transition to transcriptional quiescence required for maturation. Critically, pharmacological inhibition of DHX9 with ATX968 restored transcriptional silencing and significantly rescued meiotic maturation, achieving 95% GVBD and 80% PBE at 1 μM ATX968, comparable to wild-type controls (98%/82%) and significantly exceeding vehicle-treated arrested oocytes (8% GVBD; p < 0.001). In summary, our study demonstrates that DHX9-driven dysregulation of RNA splicing is a key mechanism underlying oocyte developmental arrest. These findings highlight DHX9 as a promising therapeutic target and provide a potential strategy for improving oocyte competence in clinical infertility treatment.