E2 ubiquitin-conjugating enzyme of mural granulosa cell regulates oocyte maturation via the mTOR-CNP signaling pathway_bulk RNAseq_co-cultured
Bidirectional communication between mural granulosa cells (MGCs) and oocytes is crucial for oocyte maturation during mammalian follicular development. However, the underlying molecular networks remain poorly defined. Here, we report that UBE2T (E2 ubiquitin-conjugating enzyme) is highly expressed in sheep ovarian tissues and preferentially enriched in MGCs. Functionally, we demonstrate that UBE2T in MGCs enhances intra-oocyte cAMP and cGMP levels, thereby preventing spontaneous meiotic resumption. Moreover, UBE2T facilitates oocyte cytoplasmic maturation, as evidenced by enhanced mitochondrial membrane potential and uniform cortical granule distribution, while simultaneously promoting the secretion of C-type natriuretic peptide (CNP) and estradiol. Mechanistically, integrated multi-omics analysis and experimental validation reveal that UBE2T upregulates mTOR protein expression, thereby driving CNP production. These findings reveal a novel function of UBE2T in MGCs that acts via the mTOR-CNP signaling pathway to maintain oocyte meiotic arrest. Furthermore, we demonstrated that UBE2T-mediated signaling in MGCs significantly improves oocyte mitochondrial function and cytoplasmic maturity. Based on these findings, we established an optimized two-step in vitro maturation (IVM) protocol featuring a 4-hour pre-maturation co-culture with MGCs. This system significantly enhanced oocyte quality, as evidenced by a marked increase in mitochondrial DNA (mtDNA) copy number compared to standard IVM. In conclusion, our study not only identifies UBE2T as a key regulator of the MGC-oocyte crosstalk via the mTOR-CNP axis but also provides a practical strategy for improving the efficiency of ovine in vitro embryo production systems by optimizing oocyte cytoplasmic competence.