E2 ubiquitin-conjugating enzyme UBE2T in mural granulosa cells regulates ovine oocyte maturation via the mTOR-CNP signaling pathway.

Mural granulosa cells (MGCs) secrete C-type natriuretic peptide (CNP) to maintain oocyte meiotic arrest, yet the upstream regulatory networks remain unclear. This study aimed to investigate how the ubiquitin-conjugating enzyme E2T (UBE2T) in MGCs modulates CNP signaling to influence oocyte quality and to apply these findings to optimize in vitro maturation (IVM) efficiency. Expression analysis showed that UBE2T was highly expressed in the ovine ovary and preferentially enriched in MGCs (P < 0.05). Functional validation demonstrated that UBE2T in MGCs elevated intra-oocyte cAMP and cGMP levels, preventing spontaneous meiotic resumption (P < 0.05). Concurrently, UBE2T facilitated cytoplasmic maturation, characterized by enhanced mitochondrial membrane potential, uniform cortical granule distribution, and increased secretion of CNP and estradiol (E2) (P < 0.05). Integrated multi-omics and experimental assays revealed that UBE2T upregulated mTOR protein expression, thereby increasing CNP level to maintain meiotic arrest. Furthermore, an optimized two-step IVM protocol featuring a 4h pre-maturation co-culture with MGCs significantly improved oocyte quality, evidenced by increased mitochondrial DNA copy number compared with conventional IVM (P < 0.05). Oocytes matured under this optimized protocol exhibited enhanced subsequent embryonic developmental competence, yielding blastocysts with significantly higher total cell numbers and upregulated expression of key pluripotency and maternal effect genes (P < 0.05). This study identifies the MGC-specific UBE2T-mTOR-CNP signaling axis as a critical driver of meiotic arrest, offering an evidence-based strategy to improve ovine oocyte IVM efficiency and embryonic development.