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 cells but that sensitivity is reduced or abolished if vitrified cells are cultured for a short period before subjecting them to hypotonic stress. In the present study, we examined the mechanism by which vitrified two-cell mouse embryos regain their resistance to osmotic swelling by culturing the embryos in the presence of various inhibitors before hypotonic treatment. New synthesis of RNA and proteins during culture was not required for regaining resistance to osmotic swelling because actinomycin D and cycloheximide failed to inhibit restoration. Inhibitors of polymerization of microfilaments and microtubules (cytochalasin B and demecolcine, respectively) also did not affect restoration of resistance to osmotic swelling, suggesting that rearrangement or repolymerization of cytoskeletal components is not involved in this process. On the other hand, brefeldin A and concanamycin A, which inhibit intracellular vesicular transport, strongly suppressed restoration of resistance. These results suggest that the intracellular vesicular transport system plays a crucial role in restoration of resistance of vitrified embryos to osmotic swelling during short-term culture.
Animals Anti-Bacterial Agents