甘氨酸提高水貂卵母细胞玻璃化冷冻保存效率的关键机制

doi:10.11843/j.issn.0366-6964.2025.07.020

摘要/Abstract

摘要：

旨在利用小分子渗透剂甘氨酸具有优化细胞体积调控的作用机理，探究水貂GV期卵母细胞玻璃化冷冻/解冻以及体外成熟(in vitro maturation, IVM)过程中添加甘氨酸对提高卵母细胞冷冻保存效率的影响。本试验在水貂繁殖季节(2~3月份)取1~3岁性成熟雌性水貂的卵巢，收集的卵母细胞随机分成3组，分别为对照组、冷冻组以及甘氨酸组，每组至少20枚卵母细胞，每次试验至少重复3次。在玻璃化冷冻、解冻以及随后的体外成熟过程中添加5 mmol·L^-1甘氨酸，检测卵母细胞的存活率，体外成熟培养后细胞的核成熟率、线粒体和皮质颗粒分布情况、卵母细胞的活性氧(ROS)水平、细胞凋亡以及组蛋白修饰。利用qRT-PCR和免疫荧光分析凋亡相关基因和DNMT1表达。研究表明，经甘氨酸处理后的玻璃化冷冻方法显著提高了水貂卵母细胞冷冻后的生物学功能。主要表现为玻璃化以及IVM过程中添加甘氨酸组与玻璃化未添加组的卵母细胞相比，存活率和核成熟率显著提高(P < 0.05)，成熟卵母细胞的线粒体损伤，皮质颗粒易位，ROS水平发生减少(P < 0.05)；细胞凋亡率降低，促细胞凋亡相关基因CASP3和BAX的mRNA相对表达水平降低(P < 0.05)，BCL-2/BAX比值升高(P < 0.05)；此外，玻璃化冷冻后水貂MII期卵母细胞的DNMT1转录水平(P < 0.05)、组蛋白H3K9甲基化表达水平得到明显改善(P < 0.05)。综上所述，在玻璃化冷冻过程中添加或去除冷冻保护剂所造成的卵母细胞渗透损伤可通过添加甘氨酸调控卵母细胞体积稳态，提高水貂卵母细胞玻璃化冷冻/解冻后的发育能力。

关键词: 玻璃化冷冻, 水貂卵母细胞, 甘氨酸, 体积调控

Abstract:

This study aimed to investigate the effects of glycine, a small-molecule permeant that optimizes cellular volume regulation, on improving the cryopreservation efficiency of mink germinal vesicle (GV) stage oocytes during vitrification, thawing, and subsequent in vitro maturation (IVM). Ovaries were collected from sexually mature female minks (1-3 years old) during the breeding season (February-March).The collected oocytes were randomly divided into 3 groups: control (fresh oocytes), vitrified without glycine (vitrified group), and vitrified with 5 mmol·L^-1 glycine (vitrified+Gly group), each group contained at least 20 oocytes, each experiment was repeated at least 3 times. Glycine of 5 mmol·L^-1 was supplemented during vitrification, thawing, and IVM. Post-thaw assessments included survival rate, nuclear maturation rate, mitochondrial integrity, cortical granule distribution, reactive oxygen species (ROS) levels, apoptosis, and histone modifications (H3K9 methylation). Quantitative PCR and immunofluorescence were employed to analyze apoptosis-related genes (CASP3, BAX, BCL-2) and DNA methyltransferase 1 (DNMT1) expression. Glycine supplementation during vitrification and IVM significantly enhanced the biological function of cryopreserved oocytes. Compared to the non-glycine vitrified group, glycine-treated oocytes exhibited higher survival rate (P < 0.05) and nuclear maturation rates (P < 0.05). Mitochondrial damage and cortical granule mislocalization were reduced (P < 0.05), while ROS levels decreased (P < 0.05). Apoptosis rates declined with downregulated CASP3 and BAX expression (P < 0.05) and an elevated BCL-2/BAX ratio (P < 0.05). Additionally, glycine improved DNMT1 transcription (P < 0.05) and H3K9 methylation levels (P < 0.05) in MII oocytes post-vitrification. In summary, oocytes osmosis damage caused by addition or removal of cryoprotectants during vitrification can regulate oocyte volume homeostasis by addition of glycine, thereby improving the developmental ability of vitrification mink oocytes.

Key words: vitrification, mink oocytes, glycine, volume regulation

中图分类号:

S814

唐毓, 张颖, 杨镒峰, 薛海龙, 刘理想, 许保增. 甘氨酸提高水貂卵母细胞玻璃化冷冻保存效率的关键机制[J]. 畜牧兽医学报, 2025, 56(7): 3265-3277.

TANG Yu, ZHANG Ying, YANG Yifeng, XUE Hailong, LIU Lixiang, XU Baozeng. Mechanisms of Glycine Improving Vitrification Cryopreservation Efficiency of Mink Oocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(7): 3265-3277.

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基因 Gene	参考序列 Reference sequence	引物序列(5′→3′) Primers sequence	产物大小/bp Amplicon size
GAPDH	XM_044227464	GGTTGTCTCCTGCGACTTCA; ATTCCTTGGAGGCCATGTGG	168
BAX	XM_059151377	CCGAGCTGATCAGGACCATC; ATAGTAGGAGAGGAGGCCGT	106
CASP3	XM_044225635	ATTATTCAGGCCTGCCGAGG; GGCATACAGGAAGTCCGCTT	114
BCL-2	XM_044242855	TTGAGTTCGGTGGGGTCATG; CTGGATCCAGGTGTGCAAGT	118
DNMT1	XM_044254246	CAGAGTGGGAATGGCAGAG; GTCTGCCTGGTAGTTTGCCT	138

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