钌红对玻璃化冷冻牛卵母细胞线粒体Ca2+的调控研究

doi:10.16431/j.cnki.1671-7236.2017.05.025

摘要/Abstract

摘要：

玻璃化冷冻会严重损伤哺乳动物卵母细胞的线粒体功能，进而极大地限制了其解冻后的发育能力。为此，本试验设置3个钌红（RR）处理组，即牛卵母细胞用含0.5、1、2 μmol／L RR的玻璃化冷冻液进行冷冻，解冻后放入含0.5、1、2 μmol／L RR的体外成熟液中继续培养0.5 h，同时，新鲜卵母细胞一部分不进行冷冻，一部分用不含RR的冷冻液进行玻璃化冷冻，分别作为新鲜对照组和玻璃化冷冻对照组，然后共检测5组牛卵母细胞线粒体Ca²⁺水平、ATP含量及孤雌激活后胚胎的发育能力，进而研究RR对玻璃化冷冻牛卵母细胞线粒体Ca²⁺水平的调控作用。结果显示：①玻璃化冷冻显著提高了牛卵母细胞中线粒体Ca²⁺水平（P＜0.05），而2 μmol／L RR处理组线粒体Ca²⁺水平显著低于冷冻对照组（P＜0.05），但与新鲜组相比无显著差异（P＞0.05）；②玻璃化冷冻显著降低了牛卵母细胞中ATP含量（P＜0.05），2 μmol／L RR处理组卵母细胞中ATP含量显著高于冷冻对照组及0.5、1 μmol/L RR处理组（P＜0.05）；③玻璃化冷冻对照组卵裂率、囊胚率显著低于新鲜对照组（P＜0.05），1 μmol／L处理组卵裂率、囊胚率与新鲜对照组相比无显著差异（P＞0.05）。综上所述，RR处理能显著抑制解冻后牛卵母细胞线粒体Ca²⁺流入，保护线粒体功能，提高其发育能力。本试验结果为正向调控玻璃化冷冻卵母细胞线粒体Ca²⁺水平，进而提高其发育能力，促进玻璃化冷冻卵母细胞的广泛应用提供了参考依据。

关键词: 牛; 卵母细胞; 玻璃化冷冻; 钌红; 线粒体Ca²⁺

Abstract:

Studies have shown that the developmental ability of vitrified mammalian oocytes is greatly impacted due to the serious damage of mitochondrial function induced by vitrification process. To solve this problem,in vitro matured bovine oocytes were divided into five groups,three of which were first vitrified using vitrification solution containing 0.5,1, 2 μmol/L ruthenium red (RR), then survived oocytes after thawing were cultivated in vitro maturation medium with the same concentration of RR as above for 0.5 h,meanwhile,the remained two groups of oocytes were as control, and one group was vitrified using vitrification solution without RR (vitrified control group) and the other group had no any treatment (fresh control group).Then the mitochondrial Ca²⁺ (mCa²⁺) level,ATP content and the developmental capacity of parthenogenetic embryos of oocytes in the five groups were detected to explore the effect of RR on mCa²⁺ level of vitrified bovine oocytes. The results showed that:①Vitrification significantly increased mCa²⁺ concentration in bovine oocytes (P <0.05), while RR significantly decreased mCa²⁺ concentration in vitrified bovine oocytes and there was no significant difference between fresh control group and 2 μmol/L RR group (P >0.05);②Vitrification significantly decreased ATP content in bovine oocytes (P <0.05),and the ATP content of 2 μmol/L RR group was significantly higher than that of vitrified control group and 0.5,1 μmol/L RR groups (P <0.05);③The cleavage rate and blastocyst rate of fresh control group were significantly higher than those of vitrified control group (P <0.05),while were no significant difference with 1 μmol/L RR group (P >0.05). In conclusion,the results showed that RR treatment could maintain the normal concentration of mCa²⁺ in vitrified bovine oocytes,protect the function of mitochondria and improve developmental competence of oocytes, which would provid theoretical basis to positively regulate the concentration of mCa²⁺,eventually improve the developmental competence and promote the extensive application of vitrified oocytes.

Key words: bovine; vitrification; oocytes; ruthenium red; mCa²⁺

中图分类号:

S823

王娜, 李崇阳, 朱化彬, 郝海生, 王皓宇, 闫长亮, 杜卫华, 王栋, 刘岩, 庞云渭, 赵学明. 钌红对玻璃化冷冻牛卵母细胞线粒体Ca²⁺的调控研究[J]. 《中国畜牧兽医》, 2017, 44(5): 1431-1437.

WANG Na, LI Chong-yang, ZHU Hua-bin, HAO Hai-sheng, WANG Hao-yu, YAN Chang-liang, DU Wei-hua, WANG Dong, LIU Yan, PANG Yun-wei, ZHAO Xue-ming. Regulation of Mitochondrial Ca²⁺ in Vitrified Bovine Oocytes by Ruthenium Red[J]. , 2017, 44(5): 1431-1437.

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钌红对玻璃化冷冻牛卵母细胞线粒体Ca²⁺的调控研究

Regulation of Mitochondrial Ca²⁺ in Vitrified Bovine Oocytes by Ruthenium Red

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