Restoration of resistance to osmotic swelling of vitrified mouse embryos by short-term culture

In cryopreservation of mammalian embryos, embryos can be injured by osmotic swelling during removal of the cryoprotectant after warming. We have shown that vitrified embryos are more sensitive to osmotic swelling than fresh c ells but that sensitivity is reduced or abolished if vitrified cells are cu ltured for a short period before subjecting them to hypotonic stress. In th e present study, we examined the mechanism by which vitrified two-cell mous e embryos regain their resistance to osmotic swelling by culturing the embr yos in the presence of various inhibitors before hypotonic treatment. New s ynthesis of RNA and proteins during culture was not required for regaining resistance to osmotic swelling because actinomycin D and cycloheximide fail ed to inhibit restoration. Inhibitors of polymerization of microfilaments a nd microtubules (cytochalasin B and demecolcine, respectively) also did nor affect restoration of resistance to osmotic swelling, suggesting that rear rangement or repolymerizarion of cytoskeletal components is not involved in this process. On the other hand, brefeldin A and concanamycin A, which inh ibit intracellular vesicular transport, strongly suppressed restoration of resistance. These results suggest that the intracellular vesicular transpor t system plays a crucial role in restoration of resistance of vitrified emb ryos to osmotic swelling during short-term culture. (C) 1999 Academic Press .