Ultrastructural Changes in Bovine Oocytes Cryopreserved by Vitrification

Abstract

Oocytes recovered from abattoir-derived ovaries were exposed to a cryoprotectant 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 normal after thawing were immediately processed for transmission electron microscopy after DAP213 removal. Cleavage rates for vitrified IVM oocytes were 4.5 and 6.7% using one-step and three-step cryoprotectant dilution procedures, respectively. Four (3%) oocytes developed to the eight-cell stage with the three-step procedure, but none formed blastocysts. None of the GV oocytes cleaved, while 66.7% (78/117) of controls developed to the two-cell stage and 19.2% (15/78) of those became blastocysts. Vitrification induced profound ultrastructural modifications in microvilli, mitochondria, vesicle formation, and the ooplasm of GV oocytes, whereas these structures were generally better preserved in IVM oocytes. The integrity of cell organelles was relatively better maintained following the three-step than after the one-step procedure in both GV and IVM oocytes. Changes in the zona pellucida (ZP) of IVM oocytes due to vitrification were associated with fewer cortical granules in the ooplasm. Since previous work showed that short-term exposure to DAP213 did not cause ZP alterations in IVM oocytes, these findings suggest that ZP damage due to low temperatures may result from the premature release of cortical granules.