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

Abstract

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 mM 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.

ROCK activity regulates functional tight junction assembly during blastocyst formation in porcine parthenogenetic embryos

“…Based on our findings and previous reports regarding the role of ROCK in blastocyst formation ( Choi et al, 2012 ; Kawagishi et al, 2004 ; Zhang et al, 2014 ), we speculated that inhibition of the cavitation onset in Y-27632-treated embryos is caused by mislocalization or AJ and TJ proteins and impaired TJ assembly. Thus, we examined localization and expression levels of CXADR, OCLN, TJP1, and CDH1 mRNA and protein molecules, which are essential for the integrity of the AJ-TJ complex, by using ICC and qRT-PCR at the blastocyst stage.…”

“…4A ). A possible explanation of defective compaction and impaired blastocyst formation is that the two ROCK isoforms, ROCK1 and ROCK2, have different stage-specific expression patterns and subcellular localizations during preimplantation development and, therefore, can have different biological activities ( Zhang et al, 2014 ). ROCK1 is predominantly detected in the cytoplasm and localized mainly at the cell–cell boundaries at the four-cell to morula stages.…”

“…They regulate cell migration, intercellular adhesion, cell polarity cytokinesis, and cell proliferation by affecting actin cytoskeleton reorganization and microtubule dynamic ( Etienne-Manneville & Hall, 2002 ; Olson & Sahai, 2008 ; Ridley, 1999 ). The biological roles and expression patterns of ROCK in mammalian embryo development have been intensively studied by pharmacological disruption of ROCK activity by the specific ROCK inhibitor Y-27632 and by using Real-Time qRT-PCR (Quantitative Reverse Transcription Polymerase Chain Reaction) and immunocytochemistry (ICC) approaches to visualize expression of ROCK genes and proteins ( Duan et al, 2014 ; Kawagishi et al, 2004 ; Zhang et al, 2014 ). These studies demonstrated that early embryo developmental competence was affected in a dose/stage-dependent manner when mouse and porcine embryos were exposed to Y-27632 (100 µM in mouse, 5, 10, and 15 µM in porcine).…”

Rho Kinase as a Target for Cerebral Vascular Disorders

“…Since the Rho signaling equilibrium in the central nervous system is both delicate and dynamic, and the independent functional roles of ROCK1 versus ROCK2 are increasingly evidenced (e.g., [168–171]), it would be reasonable to begin a therapeutic evaluation with preclinical testing of the safety/efficacy of Rho kinase inhibitors with varying potencies and varying ROCK1 versus ROCK2 selectivities. The patented Rho kinase inhibitor BA-1049 [172] is one inhibitor that shows potential for testing in these indications, due to its increased selectivity for Rock2 (the isoform that is more highly expressed in the central nervous system [20] and has been suggested to be dominant in endothelial cells [21,22]).…”

Transcription Factor Sp1 Promotes the Expression of Porcine ROCK1 Gene

“…As a downstream effector of the small GTP-binding protein Rho, rho-associated, coiled-coil containing protein kinase ( ROCK ) acts as a molecular switch controlling a variety of cellar functions, such as the regulation of stress fiber formation, actin polymerization and so on [ 1 , 2 ]. ROCK1 and ROCK2 , two isoforms of ROCK , have distinct roles and cannot be replaced by each other [ 3 ].…”