Fuku E, Xia L, Downey B R
Department of Animal Science, McGill University, Quebec, Canada.
Cryobiology. 1995 Apr;32(2):139-56. doi: 10.1006/cryo.1995.1013.
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.
从屠宰场获取的卵巢中回收的卵母细胞，在冷冻保护剂溶液（DAP213：含2 M二甲基亚砜、1 M乙酰胺、3 M丙二醇和10%胎牛血清的199组织培养基）中暴露少于20秒，然后在生发泡（GV）期或体外成熟（IVM）后进行玻璃化冷冻。通过受精和体外培养至囊胚期来评估存活率。为了确定超微结构变化，一些解冻后形态正常的玻璃化冷冻卵母细胞在去除DAP213后立即进行透射电子显微镜处理。使用一步和三步冷冻保护剂稀释程序时，玻璃化冷冻的IVM卵母细胞的分裂率分别为4.5%和6.7%。采用三步程序时，有4个（3%）卵母细胞发育到八细胞期，但无一形成囊胚。GV卵母细胞均未分裂，而对照组中有66.7%（78/117）发育到二细胞期，其中19.2%（15/78）形成囊胚。玻璃化冷冻诱导了GV卵母细胞微绒毛、线粒体、囊泡形成和卵质中的超微结构发生深刻改变，而这些结构在IVM卵母细胞中通常保存得更好。在GV和IVM卵母细胞中，三步程序后细胞器的完整性相对比一步程序后维持得更好。IVM卵母细胞因玻璃化冷冻导致透明带（ZP）的变化与卵质中皮质颗粒减少有关。由于先前的研究表明，短期暴露于DAP213不会导致IVM卵母细胞的ZP改变，这些发现表明低温导致的ZP损伤可能是由于皮质颗粒过早释放所致。
