Effect of cytochalasin B and demecolcine on freeze-thaw survival of murine embryos in vitro

Abstract

The effects of cytochalasin B (CB), a microfilament inhibitor, and demecolcine (DC), a microtubule inhibitor, on freeze-thaw survival and culture survival of early cleavage stage mouse embryos, was evaluated. In the first experiment, eight-cell mouse embryos were frozen in dimethylsulfoxide (DMSO) or DMSO + 0.1 microgram/ml DC + 7.5 micrograms/ml CB. In the second experiment, eight-cell embryos were dehydrated and cultured in the presence of either DMSO, DMSO + DC, DMSO + CB, or DMSO + DC + CB for 45 min prior to rehydration and culture to stimulate the osmotic and chemical changes encountered during the dehydration and rehydration procedures, but without the consequences of freezing and thawing. In the third experiment, additional eight-cell embryos were frozen in either DMSO, DMSO + DC, DMSO + CB, or DMSO + DC + CB. The survival of embryos frozen in DMSO (75%) was significantly higher (P less than 0.01) than that of embryos frozen in DMSO + DC + CB (55%). No differences (P = 0.55) were observed after a 48-hr culture period in the development of embryos dehydrated, cultured, and rehydrated but not frozen. Embryos frozen in the presence of both DC and CB had a lower (P = 0.06) survival rate (55%) than that of embryos frozen in the presence of DMSO, DC, or CB (approximately 70%). These results suggest that both microfilaments and microtubules have a role in maintaining the structural integrity of the plasma membrane during the freeze-thaw process and that the loss of either loss does not seem to be detrimental to survival, but the loss of both results in lower survival.