Stimulatory effect of Rho-associated coiled-coil kinase (ROCK) inhibitor on revivability of in vitro-produced bovine blastocysts after vitrification.
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.
Hochi
S
S
Graduate School of Science and Technology, Shinshu University, Ueda, Nagano 386-8567, Japan. shochi@shinshu-u.ac.jp
Abdalla
H
H
Hara
H
H
Shimoda
M
M
Morita
H
H
Kuwayama
M
M
Hirabayashi
M
M
D000577:Amides / Q000494:pharmacology*; D000818:Animals; D001755:Blastocyst / Q000187:drug effects / Q000502:physiology; D002417:Cattle; D018929:Cell Culture Techniques; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D046149:Embryo Culture Techniques; D047108:Embryonic Development / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005307:Fertilization in Vitro / Q000662:veterinary; D011725:Pyridines / Q000494:pharmacology*; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors
D000577:Amides; D003470:Culture Media; D004791:Enzyme Inhibitors; D011725:Pyridines; C108830:Y 27632; D054460:rho-Associated Kinases