抗冷冻蛋白Ⅲ对玻璃化冷冻小鼠卵母细胞线粒体的影响

doi:10.16431/j.cnki.1671-7236.2023.08.015

摘要/Abstract

摘要： 【目的】探索抗冷冻蛋白 Ⅲ(antifreeze protein Ⅲ,AFP Ⅲ)对玻璃化冷冻小鼠卵母细胞线粒体的保护作用。【方法】以5~8周龄昆明雌性小鼠为试验材料,将收集的小鼠MⅡ期卵母细胞随机分为空白对照组、冷冻对照组(经过玻璃化冷冻-解冻过程)和AFP Ⅲ添加组(在冷冻平衡液及冷冻液中均添加 500 ng/mL AFP Ⅲ),利用线粒体绿色荧光探针(Mito-Tracker green,MTG)检测线粒体分布及含量,JC-1荧光染色检测线粒体膜电位,溶酶体红色荧光探针(Lyso-Tracker red,LTR)检测线粒体与溶酶体共定位,二氯荧光素二乙酸(DCFH-DA)检测活性氧(ROS)水平,ATP Assay Kit 检测线粒体ATP含量。【结果】与空白对照组相比,玻璃化冷冻后小鼠卵母细胞的线粒体含量显著降低,部分线粒体呈现异常的散点状分布,且线粒体-溶酶体共定位情况明显增多,线粒体膜电位显著降低(P＜0.05);同冷冻对照组相比,AFP Ⅲ添加组的小鼠卵母细胞线粒体含量显著升高,异常分布的线粒体和线粒体-溶酶体共定位情况减少,线粒体膜电位显著升高(P＜0.05)。与空白对照相比,玻璃化冷冻后小鼠卵母细胞的ROS含量显著升高而ATP含量显著降低(P＜0.05);与冷冻对照组相比,AFP Ⅲ添加组ROS含量显著下降而ATP含量显著升高(P＜0.05)。【结论】AFP Ⅲ 有助于改善玻璃化冷冻小鼠卵母细胞的氧化应激反应,对冻融卵母细胞线粒体具有保护作用。

关键词: 抗冷冻蛋白 Ⅲ(AFP Ⅲ); 线粒体; 卵母细胞; 玻璃化冷冻

Abstract: 【Objective】 In order to explore the protective effect of antifreeze protein Ⅲ (AFP Ⅲ) on mitochondria of vitrified mouse oocytes. 【Method】 Kunming female mice aged 5-8 weeks were used as test materials,the collected mouse oocytes at MⅡ stage were randomly divided into blank control group,frozen control group and AFP Ⅲ adding group (500 ng/mL AFP Ⅲ was added to frozen balance solution and frozen solution),Mito-Tracker green (MTG) was used to detect the distribution and content of mitochondria,JC-1 fluorescence staining was used to detect mitochondrial membrane potential,Lyso-Tracker red (LTR) was used to detect the co-localization of mitochondria and lysosomes,dichlorofluorescein diacetic acid (DCFH-DA) was used to detect the level of ROS,and ATP Assay Kit was used to detect the content of mitochondrial ATP.【Result】 Compared with the blank control group,the content of mitochondria in mouse oocytes were decreased significantly after vitrification,some mitochondria showed abnormal scattered distribution,the co-localization of mitochondria and lysosomes were increased,and the mitochondrial membrane potential was decreased significantly (P＜0.05).Compared with the frozen control group,the mitochondrial content of mouse oocytes in AFP Ⅲ group was significantly increased,the abnormal distribution of mitochondria and the co-localization of mitochondria lysosomes were reduced,and the mitochondrial membrane potential was significantly increased (P＜0.05).Compared with the blank control group,the ROS content of mouse oocytes was significantly increased and ATP content was significantly decreased after vitrification (P＜0.05). Compared with the frozen control group,the ROS content of AFP Ⅲ group was significantly decreased and ATP content was significantly increased (P＜0.05).【Conclusion】 AFP Ⅲ could improve the oxidative stress response of vitrified mouse oocytes and protect mitochondria of frozen and thawed oocytes.

Key words: antifreeze protein Ⅲ (AFP Ⅲ); mitochondrion; oocyte; vitrification

中图分类号:

S852.16

刘可可, 薛梦琦, 唐非台, 杜倩笙, 王欣雨, 董胤余, 王晓丽. 抗冷冻蛋白Ⅲ对玻璃化冷冻小鼠卵母细胞线粒体的影响[J]. 中国畜牧兽医, 2023, 50(8): 3180-3188.

LIU Keke, XUE Mengqi, TANG Feitai, DU Qiansheng, WANG Xinyu, DONG Yinyu, WANG Xiaoli. Effect of AFP Ⅲ on Mitochondria of Vitrified Mouse Oocytes[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(8): 3180-3188.

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