ULTRASTRUCTURE OF IVM-IVF BOVINE BLASTOCYSTS VITRIFIED AFTER EQUILIBRATION IN GLYCEROL 1,2-PROPANEDIOL USING 2-STEP AND 16-STEP PROCEDURES
ULTRASTRUCTURE OF IVM-IVF BOVINE BLASTOCYSTS VITRIFIED AFTER EQUILIBRATION IN GLYCEROL 1,2-PROPANEDIOL USING 2-STEP AND 16-STEP PROCEDURES
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DOI：
10.1006/cryo.1994.1051
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发表时间：
1994-10-01
期刊：
影响因子：
2.7
通讯作者：
NAGAI, T
中科院分区：
文献类型：
--
作者：
KUWAYAMA, M;FUJIKAWA, S;NAGAI, T
使用冷冻复制技术，进行了两项实验来研究玻璃化冷冻后牛囊胚的存活率和超微结构外观之间的关系。在实验1中，从体外成熟和体外受精（IVM-IVF）牛卵母细胞获得的囊胚在玻璃化溶液（VS；22.5%甘油+22.5%1,2-丙二醇）中分步平衡，共16步共18分钟（16步法）或用10%甘油+20%平衡1,2-丙二醇 10 分钟，然后暴露于 VS（两步法）。然后将胚泡放入液氮中进行玻璃化。随后将所有样品在 37°C 水浴中解冻，并与单层卵丘细胞一起体外培养。通过16步法平衡的囊胚存活率为83.3%（25/30）。相比之下，两步法没有囊胚存活 (0/30)。在实验2中，对通过16步法和2步法玻璃化的囊胚进行冷冻复制观察。所有囊胚的细胞质、囊腔或细胞外区域均未观察到冰晶，证实发生了完全玻璃化。通过 16 步法平衡的囊胚质膜几乎没有观察到超微结构变化。相反，在通过两步法平衡的囊胚的质膜中经常观察到小囊泡和明显的膜内颗粒（IMP）聚集。这些结果表明，经过 16 步平衡后成功冷冻保存囊胚不仅需要完全玻璃化，还需要将对质膜的超微结构损伤降至最低。 (C) 1994 年学术出版社
Two experiments were conducted to investigate the relationship between survival rates and ultrastructural appearance, using the freeze-replica technique, of bovine blastocysts after vitrification. In experiment 1, blastocysts obtained from in vitro-matured and in vitro-fertilized (IVM-IVF) bovine oocytes were either equilibrated in a stepwise manner in a vitrification solution (VS; 22.5% glycerol + 22.5% 1,2-propanediol) using 16 steps for 18 min in total (the 16-step method) or equilibrated with 10% glycerol + 20% 1,2-propanediol for 10 min and then exposed to VS (the 2-step method). The blastocysts were then vitrified by plunging them into liquid nitrogen. All samples were subsequently thawed in a water bath at 37 degrees C and cultured in vitro with a monolayer of cumulus cells. The survival rate obtained for blastocysts equilibrated by the 16-step method was 83.3% (25/30). In contrast, no blastocysts survived by the 2-step method (0/30). In experiment 2, freeze-replica observations were carried out on blastocysts vitrified by the 16-step method and the 2-step method. In all the blastocysts, no ice crystals were observed in the cytoplasm, blastocoelic cavity, or extracellular areas, which confirmed the occurrence of complete vitrification. Little ultrastructural change was observed in the plasma membrane of the blastocysts equilibrated by the 16-step method. In contrast, small vesicles and distinct intramembrane particle (IMP) aggregation were frequently observed in the plasma membranes of blastocysts equilibrated by the 2-step method. These results indicate that the successful cryopreservation of blastocysts following the 16-step equilibration requires not only complete vitrification, but also minimization of ultrastructural damage to the plasma membrane. (C) 1994 Academic Press, Inc.