4. Mechanism on Successful Cryopreservation of Mouse Blastocysts by One Step Vitrification Procedure(Papers presented at the 38th Annual Meeting)

Abstract

Mouse blastocysts were exposed to a solution containing 20% ethyleneglycol, 20% dimethyl-sulfoxide and 10% 1,3-butanediol (called as VSv) or to a 65% sorbitol solution (called as SS) with or without prior equilibration with solution in half concentration of VSv (called as ESv) for 3 min at room temperature, they were maintained for different time periods to the VSv or SS at room temperature, and they were frozen by immersion into liquid nitrogen. 1) By immersion into LN_2, no ice crystals were produced both inside and outside of mouse blastocysts which were directly (or after equilibration with ESv for 3 min) exposed to VSv or SS for more than 30 sec. 2) The survival assay of embryos after freeze-thawing showed that: a) by direct (or after equilibration with ESv for 3 min) exposure to VSv, high survival (> 90%) was obtained by exposure for 30 sec or 1 min, but survival was gradually reduced by prolonged exposure causing complete injury by exposure for more than 5 min, and: b) by direct (or after equilibra tion with ESv for 3 min) exposure to SS, no survival was provided regardless of the exposure time. 3) The light microscope observation showed that: a) by direct (or after equilibration with ESv for 3 min) exposure to VSv, no distinct shrinkage of embryos was produced, and: b) by direct (or after equilibration with ESv for 3 min) exposure to SS, distinct shrinkage of embryos was produced. 4) The freeze-etching electron microscope observation showed that: a) by direct (or after equilibration with ESv for 3 min) exposure to VSv, only slight clustering of IMPs in the plasma membrance was produced by exposure for 30 sec or 1 min, distinct IMP clustering and vesiculation in the plasma membrane and slight coagulation of cytosol were produced by exposure for 3 min, and complete denaturation of cells was produced by exposure for more than 5 min., and: b) by direct (or after equilibration with ESv for 3 min) exposure to SS, no cellular ultrastructural changes were produced by exposure for at least 10 min. From these results, it was concluded that successful cryopreservation of mouse blastocysts by one step exposure to VSv for a short period resulted from the facts that blastocysts were prevented from severe dehydration due to quick permiation of VSv inside the cells, and in a short exposure time blastocysts were also prevented from chemical toxicity which producedultras tructural changes.