Insulin-like peptide 3 facilitates sheep oocytes in vitro maturation and subsequent early embryo development.

Oocyte in vitro maturation (IVM) is a critical step for in vitro embryo production. Compared with the in vivo system, conventional IVM lacks paracrine support, resulting in reduced oocyte quality. Insulin-like peptide 3 (INSL3), an ovarian paracrine factor, has been shown to be involved in follicular development and oocyte maturation. However, whether INSL3 plays a role in sheep oocyte IVM remains unclear. This study investigated the effects of INSL3 on IVM of sheep oocytes and subsequent embryonic development. Our results showed that supplementation with 10 ng/mL INSL3 significantly increased the polar body extrusion (PBE) rate compared with the control group (77.71% vs. 52.74%, P < 0.05), reaching levels comparable to the EGF group. INSL3 also enhanced cumulus expansion and upregulated expansion-related genes (PTX3, HAS2, PTGS2, TNFαIP6). In addition, INSL3 reduced intracellular reactive oxygen species (ROS), improved mitochondrial function, and decreased early apoptosis, as indicated by a lower BAX/BCL-2 ratio. Following in vitro fertilization, oocytes matured with 10 ng/mL INSL3 exhibited higher cleavage (78.62% vs. 67.68%), morula (52.64% vs. 36.64%), and blastocyst rates (35.07% vs. 25.28%) than the control group (P < 0.05), with results comparable to EGF. In conclusion, INSL3 supplementation during IVM improves oocyte quality and developmental competence in sheep, potentially through regulation of cumulus cell function, oxidative status, and mitochondrial activity, and may serve as a promising factor in sheep embryo production technologies.