伏马毒素B1对猪体外成熟卵母细胞凋亡与自噬的影响  

Effects of FB_(1) on Apoptosis and Autophagy of Porcine Oocytes in vitro Maturation

作　　者：李文慧 贺依静 姜瑶 赵红宇 彭磊 李佳 芮荣[1] 剧世强[1] LI WenHui;HE YiJing;JIANG Yao;ZHAO HongYu;PENG Lei;LI Jia;RUI Rong;JU ShiQiang(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095)

机构地区：[1]南京农业大学动物医学院,南京210095

出　　处：《中国农业科学》2022年第6期1241-1252,共12页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31972759,31572589);南京农业大学2019年大学生创新创业训练专项计划(S20190021);南京农业大学“课程思政”示范项目(KCSZ2020044)。

摘　　要：【目的】探究伏马毒素 B_(1)(fumonisin B_(1),FB_(1))对猪卵母细胞体外成熟的影响及其潜在的作用机制, 为临床有效防治FB_(1)所致的生殖毒性损伤提供理论参考。【方法】采集猪卵丘-卵母细胞复合体(cumulus oocyte complexes, COCs)进行随机分组,在体外成熟培养过程中分别用不同浓度 FB_(1)(0、10、20 和 30 μg·mL^(-1))处理 44 h 后,统计卵母细胞第一极体(first polar body, PB1)排出和激活后胚胎发育情况;通过免疫荧光染色结合共聚焦显微镜技术进一步检测 FB_(1)对卵母细胞减数分裂进程和细胞骨架结构的影响;为进一步探究 FB_(1)对猪卵母细胞毒性损伤的作用机制,分别采用 JC-1、Annexin V-FITC和 LC3A/B 荧光染色检测各组卵母细胞内线粒体功能、早期凋亡和自噬水平,并在此基础上,进一步通过 Western blotting分析了凋亡/自噬相关蛋白的表达情况。【结果】FB_(1)处理对卵母细胞成熟具有明显的抑制作用,PB1 排出率呈浓度依赖性下降,当 FB_(1)浓度达到 20 μg·mL^(-1)以上时,PB1 排出率显著降低(P<0.01),并使卵母细胞孤雌激活后胚胎的卵裂率及囊胚率均显著降低(P<0.01),对卵母细胞的发育潜力有一定的损伤作用。细胞周期分析结果表明,FB_(1)处理还会导致减数分裂周期进程紊乱,使阻滞在生发泡破裂期(germinal vesicle breakdown,GVBD)的卵母细胞比例显著升高(P<0.01)成功发育至第二次减数分裂中期(metaphase II,MII)细胞比例明显下降(P<0.01),同时,卵母细胞中纺锤体异常的比例显著升高(P<0.01)、胞膜上的微丝分布显著减少(P<0.05)。进一步的研究结果表明,与对照组相比,FB_(1)处理组卵母细胞线粒体膜电位明显降低(P<0.05),线粒体功能受损,同时,FB_(1)处理组卵母细胞早期凋亡率显著增加(P<0.01),细胞自噬水平也显著升高(P<0.01)。Western blotting 分析结果显示,FB_(1)处理组卵母细胞中促凋亡蛋白 BAX 和自噬蛋白 LC3A/B II的�【Objective】The purpose of this study was to investigate the effects of fumonisin B_(1)(FB_(1)) on porcine oocyte in vitro maturation and its potential mechanism, so as to provide the theoretical reference for effective prevention and treatment of reproductive toxicity injury caused by FB_(1)in the clinic.【Method】Porcine cumulus oocyte complexes (COCs) were collected and randomly divided into groups, and treated with different concentrations of FB_(1)(0, 10, 20 and 30 μg·mL^(-1)) for 44 h during in vitro maturation, respectively. Then, the first polar body (PB1) extrusion and embryo development after activation of oocytes were analyzed, and the effects of FB_(1)on meiotic progression and cytoskeleton structure of oocytes were further detected by immunofluorescence staining and combined with confocal microscopy. In order to further explore the mechanism of FB_(1)on toxic injury of porcine oocytes, the JC-1, Annexin V-FITC and LC3A/B fluorescence staining were used to detect mitochondrial function,early apoptosis and autophagy levels in oocytes, respectively. On this basis, the expression of apoptosis/autophagy related to proteins were further analyzed by Western blotting.【Result】FB_(1)treatment had significant inhibitory effects on oocyte maturation, while PB1extrusion rate decreased in a concentration-dependent manner. When the concentration of FB_(1)reached more than 20 μg·mL^(-1), the PB1 extrusion was significantly decreased (P<0.01), and the cleavage rate and blastocyst rate of the embryos after oocytes parthenogenetic activation were significantly decreased (P<0.01), damaging the development potential of oocytes to a certain extent.And the cell cycle analysis showed that FB_(1)treatment also disordered the meiotic cycle progression, resulting in a significant increase in the proportion of oocytes that arrested at germinal vesicle breakdown (GVBD) (P<0.01), and a significant decrease in the proportion of oocytes that successfully developed to the Metaphase II (MII) (P<0.01), with an increase in

关 键 词：猪卵母细胞 伏马毒素B_(1)细胞骨架 凋亡 自噬 

分 类 号：S859.8[农业科学—临床兽医学]