ULTRASTRUCTURAL-CHANGES IN BOVINE OOCYTES CRYOPRESERVED BY VITRIFICATION

Oocytes recovered from abattoir-derived ovaries were exposed to a cryo protectant solution (DAP213: 2 M DMSO, 1 M acetamide, 3 M propanediol, and 10% fetal calf serum in tissue culture medium 199) for less than 20 s and vitrified either at the germinal vesicle (GV) stage or after maturation in vitro (IVM). Survival was assessed by fertilization and culture in vitro to the blastocyst stage. To identify ultrastructural changes, some of the vitrified oocytes that were morphologically norma l after thawing were immediately processed for transmission electron m icroscopy after DAP213 removal. Cleavage rates for vitrified IVM oocyt es were 4.5 and 6.7% using one-step and three-step cryoprotectant dilu tion procedures, respectively. Four (3%) oocytes developed to the eigh t-cell stage with the three-step procedure, but none formed blastocyst s. None of the GV oocytes cleaved, while 66.7% (78/117) of controls de veloped to the two-cell stage and 19.2% (15/78) of those became blasto cysts. Vitrification induced profound ultrastructural modifications in microvilli, mitochondria, vesicle formation, and the ooplasm of GV oo cytes, whereas these structures were generally better preserved in IVM oocytes. The integrity of cell organelles was relatively better maint ained following the three-step than after the one-step procedure in bo th GV and IVM oocytes. Changes in the zona pellucida (ZP) of IVM oocyt es due to vitrification were associated with fewer cortical granules i n the ooplasm. Since previous work showed that short-term exposure to DAP213 did not cause ZP alterations in IVM oocytes, these findings sug gest that ZP damage due to low temperatures may result from the premat ure release of cortical granules. (C) 1995 Academic Press, Inc.