Fuku E J, Liu J, Downey B R
Department of Animal Science, McGill University, Québec, Canada.
Mol Reprod Dev. 1995 Feb;40(2):177-85. doi: 10.1002/mrd.1080400206.
Abattoir-derived oocytes were exposed to a concentrated cryoprotectant solution (DAP213: 2 M DMSO, 1 M acetamide, 3 M propanediol, and 10% FCS in TCM199) for 1.5 or 5 min at the germinal vesicle (GV) stage or after maturation in vitro (IVM). Their viability was assessed by in vitro fertilization (IVF) and culture (IVC) to blastocysts. To investigate the effect of DAP213 on the ultrastructure, GV and IVM oocytes were processed for transmission electron microscopy (TEM) before (control) or after exposure to the cryoprotectant. DAP213 induced profound ultrastructural modifications to the microvilli and mitochondria, resulted in large vesicle formation, and, most significantly, caused the premature release of the cortical granules (CG). In IVM oocytes exposed to the cryoprotectant for 5 min, exocytosis of CG into the perivitelline space was common and the IVF rate was reduced (P < .05). After exposure for 5 min, GV oocytes displayed clusters of CG comparable to controls, but after IVM-IVF, polyspermy rate was increased (P < .05). Furthermore, treated GV oocytes showed a reduced rate of cleavage and blastocyst formation and an increased percentage of oocytes exhibiting alterations in organelles, whereas the viability and ultrastructure of IVM oocytes treated for 1.5 min was not different from controls. These observations demonstrate that 1) cortical granule kinetics is one of the key elements controlling fertilizability of bovine oocytes treated with cryoprotectant, and 2) GV oocytes are more sensitive to the cryoprotectant than those that have already been matured in vitro.
将屠宰场来源的卵母细胞在生发泡（GV）期或体外成熟（IVM）后，于浓缩冷冻保护剂溶液（DAP213：2 M二甲基亚砜、1 M乙酰胺、3 M丙二醇和10%胎牛血清于TCM199中）中暴露1.5或5分钟。通过体外受精（IVF）和培养（IVC）至囊胚来评估其活力。为研究DAP213对超微结构的影响，在暴露于冷冻保护剂之前（对照）或之后，对GV和IVM卵母细胞进行透射电子显微镜（TEM）处理。DAP213引起微绒毛和线粒体的超微结构发生深刻改变，导致形成大囊泡，最显著的是导致皮质颗粒（CG）过早释放。在暴露于冷冻保护剂5分钟的IVM卵母细胞中，CG向卵周隙的胞吐作用很常见，且IVF率降低（P < 0.05）。暴露5分钟后，GV卵母细胞显示出与对照相当的CG簇，但经过IVM-IVF后，多精受精率增加（P < 0.05）。此外，处理过的GV卵母细胞显示出较低的卵裂率和囊胚形成率，以及细胞器出现改变的卵母细胞百分比增加，而处理1.5分钟的IVM卵母细胞的活力和超微结构与对照无差异。这些观察结果表明：1）皮质颗粒动力学是控制经冷冻保护剂处理的牛卵母细胞受精能力的关键因素之一；2）GV卵母细胞比已经在体外成熟的卵母细胞对冷冻保护剂更敏感。
