Boonkusol Duangjai, Faisaikarm Tassanee, Dinnyes Andras, Kitiyanant Yindee
Department of Anatomy, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
Reprod Fertil Dev. 2007;19(2):383-91. doi: 10.1071/rd06097.
The purpose of the present study was to investigate the effects of two vitrification procedures on developmental capacity and ultrastructural changes of matured swamp buffalo oocytes. In vitro-matured oocytes were vitrified by using 35 and 40% ethylene glycol as vitrification solution for solid surface vitrification (SSV) and in-straw vitrification (ISV), respectively. Survival rate of vitrified-warmed oocytes, evaluated on the basis of ooplasm homogeneity, oolemma integrity and zona pellucida intactness, as well as parthenogenetic blastocyst rates of vitrified-warmed oocytes were significantly higher with SSV (89.3 and 13.6%, respectively) than ISV (81.8 and 5.5%, respectively). However, they were still significantly lower than that of control oocytes (100 and 34.2%, respectively). For examining the ultrastructural changes, fresh, VS-exposed (ISV and SSV), and vitrified-warmed (ISV and SSV) oocytes were processed for transmission electron microscopy. In VS-exposed oocytes, reduction of microvilli abundance and damage of mitochondrial membrane were found only in the ISV group. In vitrified-warmed oocytes, however, it was clear that both methods of vitrification induced profound ultrastructural modifications to microvilli, mitochondria, oolemma and cortical granules as well as to the size and position of vesicles. Damaged mitochondria were, however, more abundant in ISV vitrified oocytes than in SSV vitrified oocytes, which correlated with the developmental data, showing the superiority of the SSV method. The present study demonstrated the feasibility of vitrification of in vitro-matured swamp buffalo oocytes.
本研究的目的是探讨两种玻璃化冷冻方法对成熟沼泽水牛卵母细胞发育能力和超微结构变化的影响。体外成熟的卵母细胞分别使用35%和40%的乙二醇作为玻璃化溶液，进行固体表面玻璃化冷冻（SSV）和细管玻璃化冷冻（ISV）。根据卵质均匀性、卵膜完整性和透明带完整性评估，玻璃化冷冻-解冻后卵母细胞的存活率以及玻璃化冷冻-解冻后卵母细胞的孤雌胚泡率，SSV法（分别为89.3%和13.6%）显著高于ISV法（分别为81.8%和5.5%）。然而，它们仍显著低于对照卵母细胞（分别为100%和34.2%）。为了检查超微结构变化，对新鲜的、暴露于玻璃化溶液（ISV和SSV）的以及玻璃化冷冻-解冻（ISV和SSV）的卵母细胞进行透射电子显微镜检查。在暴露于玻璃化溶液的卵母细胞中，仅在ISV组发现微绒毛数量减少和线粒体膜损伤。然而，在玻璃化冷冻-解冻的卵母细胞中，很明显两种玻璃化冷冻方法均对微绒毛、线粒体、卵膜和皮质颗粒以及囊泡的大小和位置诱导了深刻的超微结构改变。然而，ISV玻璃化冷冻的卵母细胞中受损线粒体比SSV玻璃化冷冻的卵母细胞中更丰富，这与发育数据相关，表明SSV方法具有优越性。本研究证明了体外成熟沼泽水牛卵母细胞玻璃化冷冻的可行性。
