Inhibition of Rho-associated coiled coil-containing kinase (ROCK) has been reported to promote differentiation of neuronal cells. Here, we examined the effect of Y-27632, a ROCK inhibitor, on the outgrowth of neurites in PC12 cells. Y-27632 caused a rapid induction of neurite outgrowth in PC12 cells in a time-dependent manner. The neurite outgrowth, triggered by Y-27632, was accompanied by Rac1 activation, and was attenuated by Rac1 inhibitor NSC23766, in a concentration-dependent manner. Y-27632 also induced an increase in the production of reactive oxygen species (ROS). Pretreatment with N-acetylcysteine, an ROS scavenger, inhibited the ROS generation and neurite outgrowth in response to Y-27632. These results indicate that the activation of Rac1 and the generation of ROS contribute to the neurite outgrowth triggered by Y-27632 in PC12 cells.

The blastocyst should initiate the dynamic changes in morphology and gene expression during hatching and implantation. Blastocyst morphogenesis includes two major events as the formation of blastocoel cavity for lineage differentiation into trophectoderm and inner cell mass, and the blastocyst hatching for implantation. However, there is little known about the relation of dynamic morphogenesis in blastocyst with hatching and implantation potential. In this study, we investigated effects of the dynamic morphogenesis in blastocyst on hatching and implantation potential by outgrowth assay. The cumulative time between each stages was calculated and analyzed. The feature of contraction was evaluated as follows: the number of contractions and the period of circumference was measured. The percentage of reduction during contraction was classified as weak when it was less than 20% and as strong when 20% or more. Compared to embryos of hatching group, embryos of non-hatching group were significantly delayed time at the compacted morula stage by 375.3 min (p<0.05) and at the early blastocyst stage by 650.1 min (p<0.01), respectively. Compared to blastocysts of outgrowth group, blastocysts of non-outgrowth group were significantly delayed at the compacted morula by 404.0 min (p<0.01) and at the early blastocyst stage by 535.4 min (p<0.01), respectively. There is no significant difference in the feature of contraction between hatching and non-hatching groups. However, blastocyst of outgrowth group showed more number of weak contraction and less number of strong contractions, compared with blastocysts of non-outgrowth group (p<0.01). Period of circumference was not significantly different in hatching and outgrowth process. These results suggested that time of blastocoel formation and number of weak contraction in blastocysts were closely related to hatching and outgrowth potential. Dynamic changes of blastocyst formation and contraction could be useful markers to select embryos for predicting the success implantation and pregnancy in human ART program.