IWR-1 targets β-catenin degradation to regulate blastocyst formation and TE development in ovine IVF and SCNT embryos via the RhoA/ROCK-YAP1 signaling axis.

Low blastocyst formation rates and defective trophectoderm (TE) development in ovine somatic cell nuclear transfer (SCNT) embryos represent a critical bottleneck limiting the practical application of cloning technology. The Wnt/β-catenin signaling pathway exerts central functions in lineage segregation of mammalian early embryos; however, its dynamic expression pattern and the outcomes of targeted intervention have not been fully elucidated in ovine embryos. This study aimed to explore the regulatory effects of the Wnt pathway inhibitor IWR-1 on blastocyst formation and TE development in ovine in vitro fertilization (IVF) and SCNT embryos, and further dissect its downstream molecular mechanism. Immunofluorescence (IF) results revealed that β-catenin displayed specific high expression at the morula stage of ovine preimplantation embryos. Treatment of Day 4 morulae with 5 μM IWR-1 highly significantly increased blastocyst developmental rates in ovine IVF and SCNT embryos. IWR-1 dually inhibited β-catenin expression at both transcriptional and protein levels; it exerted no significant effect on the inner cell mass marker SOX2, but specifically upregulated the TE marker CDX2. Proteomic screening and subsequent validation confirmed that IWR-1 markedly activated the expression of RhoA and its downstream ROCK, promoted YAP1 protein accumulation, and simultaneously elevated the transcript abundances of Hippo pathway downstream target genes CCN1 and CCN2. In conclusion, our findings suggest that IWR-1 specifically promotes TE differentiation in ovine IVF and SCNT blastocysts, possibly through targeted degradation of β-catenin and activation of the RhoA/ROCK-YAP1 signaling axis, consequently increasing blastocyst developmental rates. This study provides evidence for a novel cross-regulatory mechanism between the Wnt pathway and RhoA/ROCK-YAP1 pathway in ovine early embryos, and provides an experimental basis for optimizing the in vitro culture (IVC) system of ovine embryos and improving the developmental capacity of SCNT embryos.