ROCK signalling pathway is required to maintain blastomere number and support blastocyst formation in ovine embryos

In mammalian embryos, the first cell lineage segregation, occurring at the morula stage, gives rise to the inner cell mass (ICM) and trophectoderm (TE). Although the underlying mechanisms, including blastomere polarization and Rho-associated kinase (ROCK) signaling, have been extensively characterized in mouse embryos, their conservation across species remains unclear. This study aimed to characterize the timing of blastomere polarization in ovine embryos and to assess the role of ROCK signaling in polarization, lineage specification, and blastocyst formation. We show that blastomere polarization in sheep begins as early as day (D) 2.5, at the 5-8-cell stage, and progresses until completion in embryos with more than 16 cells. Based on this timing, embryos were treated from D2 with increasing concentrations (5, 10, 20, 40, and 100 μM) of the ROCK inhibitor Y-27632. ROCK inhibition impaired blastocyst formation in a dose-dependent manner. Low concentrations (5-20 μM) delayed development, whereas higher concentrations (40-100 μM) significantly reduced or completely abolished blastocyst formation. These effects were associated with reduced cell number, particularly within the trophectoderm (TE) lineage in D8 blastocysts. Early TE specification was not affected, as nuclear localization of YAP1 remained unchanged in D5 morulae. In contrast, blastomere polarization was disrupted only at high Y-27632 concentrations. Together, these findings indicate that ROCK signaling is dispensable for lineage specification in sheep embryos but is required to maintain blastomere number and support development to the blastocyst stage. These results challenge previous findings in mouse embryos linking ROCK-dependent polarization to lineage segregation and highlight species-specific differences in early embryonic development.