Distinct roles of ROCK1 and ROCK2 during development of porcine preimplantation embryos.

Cell-to-cell contact mediated by cell adhesion is fundamental to the compaction process that ensures blastocyst quality during embryonic development. In this study, we first showed that Rho-associated coiled-coil protein kinases (ROCK1 and ROCK2) were expressed both in porcine oocytes and IVF preimplantation embryos, playing different roles in oocytes maturation and embryo development. The amount of mRNA encoding ROCK1 and the protein concentration clearly increased between the eight-cell and morula stages, but decreased significantly when blastocysts were formed. Conversely, ROCK2 was more abundant in the blastocyst compared with other embryonic stages. Moreover, immunostaining showed that ROCK1 protein distribution changed as the embryo progressed through cleavage and compaction to the morula stage. Initially, the protein was predominantly associated with the plasma membrane but later became cytoplasmic. By contrast, ROCK2 protein was localized in both the cytoplasm and the spindle rotation region during oocyte meiosis, but in the cytoplasm and nucleus as the embryo developed. In addition, ROCK2 was present in the trophectoderm cells of the blastocyst. Treatment with 15 μM Y27632, a specific inhibitor of ROCKs, completely blocked further development of early four-cell stage embryos. Moreover, we did not detect the expression of ROCK1 but did detect ROCK2 expression in blastocysts. Moreover, lysophosphatidic acid an activator of ROCKs significantly improved the rates of blastocyst formation. These data demonstrate that ROCKs are required for embryo development to the blastocyst stage. Together, our results indicate that ROCK1 and ROCK2 may exert different biological functions during the regulation of compaction and in ensuring development of porcine preimplantation embryos to the blastocyst stage.