Graduate School of Science and Technology, Shinshu University, Ueda, Nagano 386-8567, Japan.
Theriogenology. 2010 May;73(8):1139-45. doi: 10.1016/j.theriogenology.2010.01.015. Epub 2010 Feb 19.
Inhibition of Rho-associated coiled-coil kinase (ROCK) activity promoted recovery and growth of frozen-thawed human embryonic stem cells. The primary objective was to determine if a ROCK inhibitor (Y-27632) in post-thaw culture medium improved revivability of vitrified IVP bovine blastocysts. Expanding or expanded blastocysts (7 d after IVF) were vitrified (minimum volume cooling procedure, using a Cryotop) in 15% ethylene glycol, 15% DMSO and 0.5M sucrose. When post-warm blastocysts were cultured in mSOF medium, survival rate (re-expansion of blastocoel at 24h of culture) was improved (P<0.05) by the addition of 10 microM Y-27632 (94.9+/-2.4%, mean+/-SEM) compared to a control (78.0+/-6.0%). Conversely, after 48 h of culture, there were no significant differences in hatching rate (62.8+/-11.1 vs. 59.6+/-9.4%) and mean total cell number (135.2+/-13.1 vs. 146.7+/-13.3). In non-vitrified IVP bovine blastocysts, the hatching rate on Day 9 was improved by Y-27632 (91.7+/-3.8 vs. 54.7+/-8.9%, P<0.05), with no difference in mean total cell number of blastocysts (230.0+/-23.0 vs. 191.2+/-22.2, P=0.23). In an additional experiment, Y-27632 was added to culture medium on either Day 0, Day 2, or Day 4 (and remained present until Day 8), resulting in no improvement in blastocyst yield compared to a control group (7.5+/-2.1, 31.4+/-2.3, 36.2+/-3.2, and 28.6+/-6.9%, respectively). In conclusion, adding a ROCK inhibitor to post-thaw culture medium improved revivability of IVP bovine blastocysts after vitrification and warming.
抑制 Rho 相关卷曲螺旋激酶（ROCK）活性促进了冷冻解冻的人类胚胎干细胞的恢复和生长。主要目的是确定在解冻后培养基中添加 ROCK 抑制剂（Y-27632）是否能提高玻璃化 IVP 牛囊胚的复活率。将扩展或扩展的囊胚（受精后 7 天）在 15%乙二醇、15%DMSO 和 0.5M 蔗糖中进行玻璃化（使用 Cryotop 的最小体积冷却程序）。当解冻后的囊胚在 mSOF 培养基中培养时，与对照组（78.0+/-6.0%）相比，添加 10μM Y-27632（94.9+/-2.4%，均值+/-SEM）可提高囊胚的存活率（培养 24 小时时囊胚腔的重新扩张）（P<0.05）。相反，培养 48 小时后，孵化率（62.8+/-11.1%比 59.6+/-9.4%）和平均总细胞数（135.2+/-13.1 比 146.7+/-13.3）无显著差异。在未玻璃化的 IVP 牛囊胚中，Y-27632 可提高囊胚在第 9 天的孵化率（91.7+/-3.8%比 54.7+/-8.9%，P<0.05），但囊胚的总细胞数无差异（230.0+/-23.0 比 191.2+/-22.2，P=0.23）。在另一个实验中，在第 0 天、第 2 天或第 4 天（直到第 8 天）向培养基中添加 Y-27632，但与对照组相比，囊胚产量没有提高（分别为 7.5+/-2.1、31.4+/-2.3、36.2+/-3.2 和 28.6+/-6.9%）。总之，在玻璃化和解冻后培养基中添加 ROCK 抑制剂可提高 IVP 牛囊胚的复活率。
