细胞松弛素B改善冷冻引起的猪卵母细胞皮质颗粒迁移障碍

doi:10.11843/j.issn.0366-6964.2024.05.018

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (5): 1999-2010.doi: 10.11843/j.issn.0366-6964.2024.05.018

细胞松弛素B改善冷冻引起的猪卵母细胞皮质颗粒迁移障碍

李婉君^1,2, 徐皆欢^2,3,4, 何孟纤², 孔钰婷^1,2, 张德福^2,3,4, 戴建军^2,3,4*

1. 上海海洋大学水产与生命学院, 上海 201306;
2. 上海市农业科学院畜牧兽医研究所上海市农业遗传育种重点实验室, 上海 201106;
3. 农业农村部畜禽资源(猪)评价利用重点实验室, 上海 201106;
4. 上海种猪工程技术研究中心, 上海 201302

收稿日期:2023-12-11 出版日期:2024-05-23 发布日期:2024-05-27
通讯作者: 戴建军，主要从事动物遗传育种研究，E-mail：blackman0520@126.com
作者简介:李婉君(1999-)，女，河南商丘人，硕士生，主要从事动物胚胎工程研究，E-mail：qiujun0516@sina.com
基金资助:
国家重点研发计划(2021YFD1200301)；国家自然基金青年基金(32202640)；上海市“科技创新行动计划”启明星培育(扬帆专项)(23YF1439000)

Cytochalasin B Alleviates the Migration Disorder of Cortical Particle Caused by Vitrification in Porcine Oocytes

LI Wanjun^1,2, XU Jiehuan^2,3,4, HE Mengxian², KONG Yuting^1,2, ZHANG Defu^2,3,4, DAI Jianjun^2,3,4*

1. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China;
2. Shanghai Key Laboratory of Agricultural Genetics and Breeding, Institute of Animal Husbandry and Veterinary Science, Shanghai Academy of Agricultural Sciences, Shanghai 201106, China;
3. Key Laboratory of Livestock and Poultry Resources(Pig) Evaluation and Utilization, Ministry of Agriculture and Rural Affairs, Shanghai 201106, China;
4. Shanghai Engineering Research Center of Breeding Pig, Shanghai 201302, China

Received:2023-12-11 Online:2024-05-23 Published:2024-05-27

摘要/Abstract

摘要： 旨在揭示冷冻后的猪GV期卵母细胞体外成熟过程中细胞骨架与皮质颗粒迁移之间的相互关系和影响。所采集猪卵巢样品来自上海市嘉定区五丰上食屠宰场的健康经产母猪，每次采样选用体重70~90 kg、生产性能良好的浦东白母猪300头左右采取卵巢样品，采集卵巢数量120~150枚·次^-1，挑选胞质分布均匀且紧密的GV期卵母细胞按试验要求随机分为3组，每组设3个重复，每组至少30枚细胞，利用细胞骨架稳定剂细胞松弛素B (cytochalasin B，CB)冻前处理猪GV期卵母细胞，冷冻解冻后经体外成熟培养，检测卵母细胞存活率、各时间点微丝与皮质颗粒荧光共定位情况、卵丘扩散程度、第一极体排出率、谷胱甘肽(glutathione，GSH)水平和发育能力等指标。结果显示，冷冻前用CB处理可有效提高GV期卵母细胞解冻后存活率(44.11% vs. 27.91%, P＜0.01)。在新鲜卵母细胞成熟过程中，可观察到皮质颗粒与微丝存在共迁移。冷冻会引起皮质颗粒-微丝迁移异常，而冷冻前CB的添加会缓解冷冻引起的迁移障碍，表现为CB处理组的微丝与皮质颗粒均成功迁移至质膜的比例更高，迁移均匀分布于质膜效果更好。冷冻卵母细胞体外成熟后，CB预处理的第一极体排出率((26.79±2.37)% vs. (8.13±0.30)%，P＜0.01)、GSH水平(23.12±2.65 vs. 7.27±0.79, P＜0.01)和孤雌激活后的卵裂率((20.91±2.84)% vs. (5.64±0.37)%, P＜0.01)与囊胚率((5.00±0.03)% vs. (0.41±0.01)%, P＜0.05)均有显著提高。本研究表明，微丝细胞骨架对卵母细胞体外成熟过程中胞内皮质颗粒由内部向质膜迁移有一定的调节作用，且两者存在一定共定位关系，通过冻前添加CB孵育可有效影响细胞微丝骨架与皮质颗粒的迁移程度，减轻了冷冻引起的细胞骨架异常分布，缓解了由冷冻引起的皮质颗粒-微丝迁移障碍，进而提升细胞抗冻能力，表现为促进胞质成熟的同时，提高了冷冻卵母细胞存活率、核成熟率与胚胎体外发育潜能。

关键词: 皮质颗粒, 微丝, 玻璃化冷冻, 猪卵母细胞

Abstract: The purpose of this study was to reveal the interrelationships and effects of cytoskeleton and cortical granule migration during in vitro maturation of frozen porcine GV-stage oocytes. Pig ovary samples were collected from healthy multiparous sows.For each sampling, about 300 Pudong White sows weighing 70-90 kg with good production performance were used to take ovarian samples, the number of ovaries collected was 120-150 per time, and the GV stage oocytes with homogeneous and compact cytoplasmic distribution were selected and randomly divided into 3 groups according to the experimental requirements, and 3 replicates were set up in each group, with at least 30 cells in each group, and cytoskeleton stabilizers, cytochalasin B (CB) were used to treat the frozen porcine oocytes before freezing. Porcine GV oocytes were treated with cytoskeleton stabilizer CB before freezing, and then matured in vitro after freezing and thawing. Oocyte survival, fluorescence co-localization of microfilaments and cortical granules at each time point, degree of spreading of the oval mound, rate of discharge of the first polar body, level of glutathione (GSH), and developmental ability were examined. The result showed that treatment with CB before freezing effectively increased the survival rate of GV stage oocytes after thawing (44.11% vs. 27.91%, P<0.01). During the maturation of fresh oocytes, co-migration of cortical granules with microfilaments was observed. Freezing caused abnormal cortical granule-microfilament migration, and the addition of CB prior to freezing alleviated the freezing-induced migration obstacles, as evidenced by a higher proportion of both microfilaments and cortical granules successfully migrating to the plasma membrane in the CB-treated group, and a better homogeneous distribution of migration to the plasma membrane. After in vitro maturation of frozen oocytes, CB pretreatment was associated with a higher rate of discharge of first polar body ((26.79±2.37)% vs. (8.13±0.30)%, P<0.01), GSH levels (23.12±2.65 vs. 7.27±0.79, P<0.01), cleavage rate after parthenogenetic activation ((20.91±2.84)% vs. (5.64±0.37)%, P<0.05) and blastocyst rate ((5.00±0.03)% vs. (0.41±0.01)%, P<0.05) were significantly increased. The present study showed that the microfilament cytoskeleton has a certain regulatory effect on the migration of intracellular cortical particles from the interior to the plasma membrane during oocyte maturation in vitro, and there exists a certain co-localization relationship between the two, and that the addition of CB incubation before freezing can effectively affect the degree of migration of the cellular microfilament cytoskeleton and cortical particles, mitigate the abnormal distribution of the cytoskeleton induced by freezing, and ease the migration obstruction of cortical particles and microfilaments induced by freezing, thereby enhancing cellular resistance, which is manifested as promoting cytoplasmic maturation, improving the survival rate of frozen oocytes, nuclear maturation rate and embryo development potential in vitro.

Key words: cortical granules, microfilament, vitrification, pig oocyte

中图分类号:

S828.3

李婉君, 徐皆欢, 何孟纤, 孔钰婷, 张德福, 戴建军. 细胞松弛素B改善冷冻引起的猪卵母细胞皮质颗粒迁移障碍[J]. 畜牧兽医学报, 2024, 55(5): 1999-2010.

LI Wanjun, XU Jiehuan, HE Mengxian, KONG Yuting, ZHANG Defu, DAI Jianjun. Cytochalasin B Alleviates the Migration Disorder of Cortical Particle Caused by Vitrification in Porcine Oocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 1999-2010.

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细胞松弛素B改善冷冻引起的猪卵母细胞皮质颗粒迁移障碍

Cytochalasin B Alleviates the Migration Disorder of Cortical Particle Caused by Vitrification in Porcine Oocytes

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