玻璃化冷冻对猪MⅡ期卵母细胞及其DNA的影响

doi:10.16431/j.cnki.1671-7236.2021.07.023

摘要/Abstract

摘要： 研究旨在筛选最适合猪MⅡ期卵母细胞玻璃化冷冻的冷冻液，并探究玻璃化冷冻对猪MⅡ期卵母细胞DNA的影响。选取目前应用最多的7种冷冻液（分别为1、2、3、4、5、6、7组），将MⅡ期卵母细胞随机分为8组，其中对照组直接进行孤雌激活，其余7组分别进行7种冷冻液处理后不经液氮冷冻直接于解冻液中解冻，解冻后进行孤雌激活，通过卵裂率、囊胚率和囊胚细胞数的统计结果筛选出最适冷冻液；应用筛选的3种冷冻液，进行猪MⅡ期卵母细胞玻璃化冷冻，解冻后恢复2 h，统计卵母细胞形态正常率，孤雌激活44~48 h统计卵裂率；应用透射电子显微镜观察正常MⅡ期卵母细胞与玻璃化冷冻-复苏后的MⅡ期卵母细胞超微结构的变化；将猪MⅡ期卵母细胞随机分成对照组、冷冻液处理组和冷冻组，应用彗星电泳技术检测玻璃化冷冻对卵母细胞DNA的损伤。结果发现，与对照组相比，除5组卵裂率、1组囊胚率显著降低（P<0.05）外，其余各组卵裂率、囊胚率均差异不显著（P>0.05）；各组间囊胚细胞数均低于对照组，但差异均不显著（P>0.05），3、6、7组卵裂率和囊胚率较高；玻璃化冷冻-解冻后，7组卵母细胞的形态正常率、卵裂率均显著低于3、6组（P<0.05），6组卵裂率高于3组；MⅡ期卵母细胞移入预处理液中后可见明显的皱缩，移入冷冻液中迅速脱水，解冻后可见卵母细胞透明带断裂，胞质皱缩、分布不均；透射电子显微镜下，冷冻后猪MⅡ期卵母细胞透明带及细胞膜损伤，微绒毛严重损伤甚至消失，皮质颗粒排列在质膜下且数量减少，脂滴形态破坏、形成空泡，内质网与脂滴的联系损坏，线粒体肿胀、嵴不明显；彗星电泳发现，与对照组相比，冷冻液处理组头部DNA、尾部DNA和Olive尾矩值均差异不显著（P>0.05），有彗星拖尾现象；冷冻组头部DNA损伤、尾部DNA损伤与Olive尾矩值均显著高于冷冻液处理组和对照组（P<0.05），有明显彗星拖尾现象。结果表明，以二甲基亚砜（DMSO）和乙二醇（EG）为主要成分的冷冻液适于猪MⅡ期卵母细胞玻璃化冷冻；玻璃化冷冻对猪MⅡ期卵母细胞超微结构及其DNA存在一定损伤作用，其损伤机制有待进一步研究。

关键词: DNA损伤; 冷冻液; 玻璃化冷冻; 卵母细胞

Abstract: The aim of this study was to screen the most suitable cryoprotectant for porcine MⅡ oocytes and explore the effect of vitrification on DNA of porcine MⅡ stage oocytes. The oocytes of MⅡ stage were randomly divided into 8 groups. The oocytes of the control group were parthenogenetically activated directly, while the oocytes of the other 7 groups were treated with the most used 7 kinds of cryoprotectant, respectively, and then thawed in the cryoprotectant directly without liquid nitrogen freezing, and parthenogenetically activated after thawing. The suitable cryoprotectant was screened through the statistical results of cleavage rate, blastocyst rate and blastocyst cell number, and porcine MⅡ oocytes were vitrificated using the three selected cryoprotectants. After thawing, porcine MⅡ oocytes were recovered for 2 h, and the normal rate of oocytes morphology and the cleavage rate were acalculated. The ultrastructural changes of porcine MⅡ oocytes after vitrification were observed by transmission electron microscopy. Porcine MⅡ oocytes were randomly divided into control group, cryoprotectant treat group and vitrification group, and the DNA damage of oocytes by vitrification was detected by comet assay. The results showed that compared with control group, the cleavage rate of group 5 and blastocyst rate of group 1 were significantly decreased (P<0.05), but there were no significant differences in the cleavage rate and blastocyst rate of the other groups (P>0.05). The number of blastocyst cells in control group was higher than that of the other groups, but the differences were not significant (P>0.05). The cleavage rate and blastocyst rate in groups 3, 6 and 7 were higher. After vitrification and thawing, the morphological normal rate and cleavage rate of oocytes in group 7 were significantly lower than those in groups 3 and 6 (P<0.05), and the cleavage rate in group 6 was higher than that in group 3. The MⅡ oocytes were obviously shrinkage after being transferred into the pretreatment solution, and quickly dehydrated after being transferred into the freezing solution. After thawing, the zona pellucida of oocytes was broken, and the cytoplasm was shrunk and unevenly distributed. Under transmission electron microscope, the zona pellucida and cell membrane of porcine MⅡ oocytes were damaged after vitrification, microvilli were seriously damaged or even disappeared, cortical granules were arranged under the plasma membrane and decreased in number, morphology of lipid droplets were destroyed and vacuoles were formed, the connection between endoplasmic reticulum and lipid droplets were damaged, mitochondria swelled and cristae were not obvious. Comet assay showed that there were no significant differences in head DNA, tail DNA and Olive tail moment between control group and cryoprotectant-treated group (P>0.05),and there was comet tailing. The head DNA damage, tail DNA damage and Olive tail moment values of vitrification group were significantly higher than those of the cryoprotectant treated group and control group (P<0.05), and there was obvious comet tailing. The results showed that the cryoprotectant with DMSO and EG as main components were suitable for vitrification of porcine MⅡ oocytes, vitrification could damage the ultrastructure and DNA of porcine MⅡ oocytes to some extent, and the damage mechanism needed further study.

Key words: DNA damage; cryoprotectants; vitrification; oocyte

中图分类号:

S852.16

薛梦琦, 周悦, 刘可可, 王欣雨, 董胤余, 唐小川, 王晓丽. 玻璃化冷冻对猪MⅡ期卵母细胞及其DNA的影响[J]. 中国畜牧兽医, 2021, 48(7): 2475-2483.

XUE Mengqi, ZHOU Yue, LIU Keke, WANG Xinyu, DONG Yinyu, TANG Xiaochuan, WANG Xiaoli. Effect of Vitrification on MⅡ Oocytes and Their DNA of Porcine[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(7): 2475-2483.

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