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

【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的表达均显著上调(P<0.05),抑凋亡蛋白 BCL2 的表达显著下调(P<0.05),提示早期凋亡和自噬的发生。【结论】FB_(1)对猪卵母细胞体外成熟及其激活后胚胎发育具有明显的毒性损伤作用,致使减数分裂周期阻滞,纺锤体结构紊乱、微丝分布减少和线粒体损伤,其毒性作用机制与诱导卵母细胞凋亡和自噬有关。

【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 spindle abnormal rate(P<0.01) and a decrease in actin distribution at the plasma membrane (P<0.05). Further studies showed that, compared with control group, the mitochondrial membrane potential was significantly decreased (P<0.05), impairing mitochondrial function. At the same time, the rate of early apoptosis was obviously increased (P<0.01), and the level of autophagy was also significantly increased (P<0.01) in FB_(1)-treated oocytes. Western blotting analysis showed that the expressions of pro-apoptotic protein BAX and autophagy protein LC3A/B II in FB_(1)-treated oocytes were significantly up-regulated (P<0.05), and the expression of anti-apoptotic protein BCL2 was significantly down-regulated (P<0.05), indicating the occurrence of early apoptosis and autophagy.【Conclusion】FB_(1)had obvious toxic effects on porcine oocyte maturation in vitro and embryo development after activation, resulting in meiotic cycle arrest,spindle structure disorder, actin distribution reduction and mitochondrial injury, and its toxic mechanism might be related to the induction of apoptosis and autophagy in oocytes.