3种冷冻方法对猪MⅡ期卵母细胞线粒体分布和损伤的影响

摘要

The purpose of this study was to detect the mitochondria distribution and ultrastructure changes that resulted from vitrified porcine Mil-stage oocytes with three vitrification techniques. Through transmission electron microscopy CTEM) , R-123 (Rhodamine-123) staining and in vitro development, mitochondria distribution and ultrastructure were observed. The result showed that; (1) Given the survival rate of FDA (fluorescein diacetate)-DAPI (4' ,6'-diamidino-2-pheny-lindole dihydrochloride) staining and the cleavage rate after parthenogenetic activation, CLV (Cryoloop vitrification) method (72. 00%, 7. 22%) got the best results. OPS (Open Pulled Straw,60. 00%, 4.85%) and straw (42.22%, 0%) methods were followed by; (2) The rate of normal mitochondria distribution in CLV group (52. 24%) was higher than that in the two others groups (OPS, 48. 65%; straw, 37. 68%) , but there was no significant differences among them (P>0. 05) ; (3) Vague mitochondria in thawed oocytes were observed in the ultrastructure of vitrified oocytes from TEM, and some mitochondrial ridges were reduced or even disappeared. The result indicated that mitochondria was damaged greatly in the freezing process and CLA method could improve the freezing speed and minimize such damages.%本研究旨在探讨3种冷冻方法对猪MⅡ期卵母细胞玻璃化冷冻后线粒体的分布和超微结构变化的影响.通过透射电镜、Rhodamine-123 (R-123)荧光染色和体外发育观察,结果显示,(1)无论是FDA-DAPI复染存活率,还是孤雌激活卵裂率,CLV (Cryoloop vitrification)法(72.00%,7.22％)效果最好,OPS(Open Pulled Straw)法(60.00％,4.85％)次之,straw法(42.22％,0％)最低；(2)CLV法经R-123染色后,卵母细胞线粒体的正常分布率(52.24％)要比其它2组要高(OPS,48.65％；straw,37.68%),但三者之间没有显著差异(P＞0.05)；(3)透射电镜超微结构表明,冻后卵母细胞线粒体变得粗糙和模糊,有的线粒体嵴减少甚至消失.结果表明,冷冻过程中卵母细胞线粒体的分布和形态损伤严重,CLV法可提高冷冻速率,增强冻后卵母细胞的发育能力,降低冷冻造成的卵母细胞线粒体异常分布比例.