微囊藻毒素-LR对猪体外成熟卵母细胞氧化损伤与凋亡的影响

doi:10.11843/j.issn.0366-6964.2021.010

摘要/Abstract

摘要： 旨在探讨微囊藻毒素-LR（microcystin-LR，MC-LR）对体外成熟猪卵母细胞的毒性损伤及其潜在损伤机制。本研究从屠宰场所获得的猪卵巢中收集GV期卵母细胞，将其随机分为4组，在卵母细胞体外成熟培养液中分别添加0、20、40和60 μg·mL^-1 MC-LR，研究MC-LR对卵母细胞第一极体（PbI）排出、纺锤体结构以及细胞内活性氧（ROS）、早期凋亡蛋白（Annexin V）及谷胱甘肽过氧化物酶（GSH-Px）活力的影响，并采用RT-qPCR分析氧化应激和凋亡相关基因SOD1、SOD2、CAT、GSH-Px、Bax和Bcl2 mRNA的表达变化情况，试验重复3次。结果发现，MC-LR处理导致猪卵母细胞PbI排出率呈浓度依赖性下降，当MC-LR添加浓度达40 μg·mL^-1以上时，卵母细胞成熟率极显著下降（P<0.01），且纺锤体结构异常率极显著升高（P<0.001）；进一步研究发现，MC-LR处理后卵母细胞ROS水平极显著升高（P<0.01），而GSH-Px活性极显著降低（P<0.01），并伴随着抗氧化相关基因SOD1、CAT及GSH-Px mRNA表达极显著下调（P<0.01）；细胞凋亡检测表明，MC-LR处理可导致卵母细胞早期凋亡率极显著增加（P<0.01），凋亡相关基因Bax/Bcl2比值极显著升高（P<0.001）。研究结果表明，MC-LR可诱导猪卵母细胞纺锤体结构异常，并引发氧化损伤与细胞凋亡，最终导致卵母细胞成熟能力下降。

关键词: 微囊藻毒素-LR, 猪, 卵母细胞, 纺锤体, 氧化应激, 细胞凋亡

Abstract: The aim of this study was to investigate the toxic damage effect of microcystin-LR (MC-LR) on porcine oocytes in vitro and its potential mechanism. The GV oocytes were collected from porcine ovaries obtained from a local slaughterhouse and randomly assigned into 4 groups, 0, 20, 40 and 60 μg·mL^-1 MC-LR were added into maturation medium in vitro, respectively. Then, the effects of MC-LR on the first polar body (PbI) extrusion, spindle structure, reactive oxygen species (ROS), membrane mucin V(Annexin V) and glutathione peroxidase (GSH-Px) activity were examined, respectively. The mRNA expression of oxidative stress and apoptosis related genes (SOD1, SOD2, CAT, GSH-Px, Bax and Bcl2) were also analyzed by RT-qPCR, and three replicates were performed for each experiment. The result showed that the PbI extrusion rate of porcine oocytes decreased in a concentration-dependent manner after MC-LR treatment. When the MC-LR concentration reached more than 40 μg·mL^-1, the oocyte maturation rate extremely significantly decreased(P<0.01), and the spindle structure abnormality rate also extremely significantly increased(P<0.001). Further studies showed that, after MC-LR treatment, ROS levels in oocytes were extremely significantly increased (P<0.01), while GSH-Px activity was extremely significantly decreased (P<0.01), accompanied by extremely significant down-regulation of antioxidant related genes SOD1, CAT and GSH-Px mRNA expressions(P<0.01). Apoptosis detection showed that MC-LR treatment could extremely significantly increase the early apoptosis rate of oocytes (P<0.01), and the ratio of Bax/Bcl2 was extremely significantly increased (P<0.001). The results indicate that MC-LR can induce abnormal spindle structure of porcine oocytes, lead to oxidative stress and apoptosis, and eventually result in a decline of porcine oocyte maturation ability.

Key words: microcystin-LR, porcine, oocyte, spindle, oxidative stress, apoptosis

中图分类号:

S828.3

石凤垚, 李文慧, 赵红宇, 王阳, 庄瑞雪, 芮荣, 剧世强. 微囊藻毒素-LR对猪体外成熟卵母细胞氧化损伤与凋亡的影响[J]. 畜牧兽医学报, 2021, 52(1): 98-106.

SHI Fengyao, LI Wenhui, ZHAO Hongyu, WANG Yang, ZHUANG Ruixue, RUI Rong, JU Shiqiang. Effects of MC-LR on Oxidative Stress and Apoptosis of Porcine Oocytes in Vitro[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(1): 98-106.

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