Kwon Jeongwoo, Kim Nam-Hyung, Choi Inchul
Department of Animal Science, Chungbuk National University , Cheongju Chungbuk , South Korea.
Department of Animal and Dairy Sciences, Chungnam National University , Daejeon Chungnam , South Korea.
PeerJ. 2016 Apr 11;4:e1914. doi: 10.7717/peerj.1914. eCollection 2016.
The Rho-associated coiled-coil-containing protein serine/threonine kinases 1 and 2 (ROCK1 and ROCK2) are Rho subfamily GTPase downstream effectors that regulate cell migration, intercellular adhesion, cell polarity, and cell proliferation by stimulating actin cytoskeleton reorganization. Inhibition of ROCK proteins affects specification of the trophectoderm (TE) and inner cell mass (ICM) lineages, compaction, and blastocyst cavitation. However, the molecules involved in blastocyst formation are not known. Here, we examined developmental competence and levels of adherens/tight junction (AJ/TJ) constituent proteins, such as CXADR, OCLN, TJP1, and CDH1, as well as expression of their respective mRNAs, after treating porcine parthenogenetic four-cell embryos with Y-27632, a specific inhibitor of ROCK, at concentrations of 0, 10, 20, 100 µM for 24 h. Following this treatment, the blastocyst development rates were 39.1, 20.7, 10.0, and 0% respectively. In embryos treated with 20 µM treatment, expression levels of CXADR, OCLN, TJP1, and CDH1 mRNA and protein molecules were significantly reduced (P < 0.05). FITC-dextran uptake assay revealed that the treatment caused an increase in TE TJ permeability. Interestingly, the majority of the four-cell and morula embryos treated with 20 µM Y-27643 for 24 h showed defective compaction and cavitation. Taken together, our results indicate that ROCK activity may differentially affect assembly of AJ/TJs as well as regulate expression of genes encoding junctional proteins.
含Rho相关卷曲螺旋的蛋白丝氨酸/苏氨酸激酶1和2（ROCK1和ROCK2）是Rho亚家族GTP酶的下游效应器，通过刺激肌动蛋白细胞骨架重组来调节细胞迁移、细胞间粘附、细胞极性和细胞增殖。抑制ROCK蛋白会影响滋养外胚层（TE）和内细胞团（ICM）谱系的分化、致密化和囊胚空化。然而，参与囊胚形成的分子尚不清楚。在此，我们用浓度为0、10、20、100μM的ROCK特异性抑制剂Y-27632处理猪孤雌生殖四细胞胚胎24小时后，检测了其发育能力以及粘附/紧密连接（AJ/TJ）组成蛋白（如CXADR、OCLN、TJP1和CDH1）的水平，以及它们各自mRNA的表达。经过这种处理后，囊胚发育率分别为39.1%、20.7%、10.0%和0%。在用20μM处理的胚胎中，CXADR、OCLN、TJP1和CDH1 mRNA及蛋白分子的表达水平显著降低（P<0.05）。FITC-葡聚糖摄取试验表明，该处理导致TE TJ通透性增加。有趣的是，用20μM Y-27643处理24小时的大多数四细胞和桑葚胚胚胎显示出致密化和空化缺陷。综上所述，我们的结果表明，ROCK活性可能对AJ/TJs的组装有不同影响，并调节编码连接蛋白的基因表达。
