Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
Department of Microbiology, School of Biology, College of Science, University of Tehran, Tehran, Iran.
Theriogenology. 2022 Apr 15;183:53-60. doi: 10.1016/j.theriogenology.2022.02.013. Epub 2022 Feb 14.
Aflatoxin is considered as one of the most harmful mycotoxins in the world to be found in human food and animal feed. Previous reports have revealed that Aflatoxin B1 (AFB1) may disrupt gamete development through epigenetic modifications as well as promotion of oxidative stress, excessive autophagy, and apoptosis. Therefore, in this study we aimed to address the effects of AFB1 on the meiotic and cytoplasmic maturation, internal reactive oxygen species (ROS) production, mitochondrial membrane potential (ΔΨm), blastocyst formation as well as mRNA alterations for the apoptotic (BAX and Caspase3), anti-apoptotic (BCL2), and DNA methyltransferase (DNMTs) genes in ovine oocytes. To accomplish this, maturation of oocytes was performed in presence of increasing AFB1 concentrations (0, 10, 50, and 100 μM). Meiotic and cytoplasmic maturation, intracellular ROS level, and ΔΨm were evaluated following 24 h of IVM. Embryonic cleavage and blastocyst formation following fertilization were also assessed. We also investigated alterations of BAX, BCL2,Caspase3, DNMT1, DNMT3a, and DMT3b mRNA levels in mature oocytes. In the presence of 50 and 100 μM AFB1, the number of oocytes reaching the metaphase II stage decreased and the oocytes presented with lower intracellular levels of GSH (P < 0.05). Furthermore, intracellular ROS production in matured oocytes reached the highest-level following exposure to 50 and 100 μM of AFB1 (P < 0.05). Reduction of ΔΨm was clearly evident in the AFB1-treated groups (P < 0.05). Rates of cleavage and blastocyst formation decreased in the presence of AFB1 as compared with those recorded in the Control group (P < 0.05). Apoptosis-related gene analysis in AFB1 treated groups (10 and 50 μM) revealed a higher abundance of the BAX and Caspase3 genes, and a lower abundance of the BCL2 gene as compared with the Control group (P < 0.05). Additionally, our data showed that relative abundances of DNMT3b gene decreased in the 10 μM group when compared to the Control group (P < 0.05). We showed that exposure of oocytes to AFB1 leads to a reduced nuclear and cytoplasmic maturation that may ultimately impair the embryonic development in the sheep oocyte. Furthermore, alterations in DNA methylation and initiation of apoptosis through excessive ROS generation could be a prime molecular mechanism responsible for the disruption of oocyte developmental competence in the presence of AFB1 in the ovine model.
黄曲霉毒素被认为是世界上对人类食品和动物饲料危害最大的霉菌毒素之一。先前的报告表明，黄曲霉毒素 B1（AFB1）可能通过表观遗传修饰以及促进氧化应激、过度自噬和细胞凋亡来干扰配子发育。因此，在这项研究中，我们旨在研究 AFB1 对绵羊卵母细胞减数分裂和细胞质成熟、内部活性氧（ROS）产生、线粒体膜电位（ΔΨm）、囊胚形成以及凋亡（BAX 和 Caspase3）、抗凋亡（BCL2）和 DNA 甲基转移酶（DNMTs）基因的 mRNA 改变的影响。为了实现这一目标，在增加的 AFB1 浓度（0、10、50 和 100 μM）存在下进行卵母细胞成熟。在 IVM 24 小时后评估减数分裂和细胞质成熟、细胞内 ROS 水平和 ΔΨm。受精后还评估了胚胎分裂和囊胚形成。我们还研究了成熟卵母细胞中 BAX、BCL2、Caspase3、DNMT1、DNMT3a 和 DMT3b mRNA 水平的改变。在 50 和 100 μM AFB1 的存在下，达到中期 II 阶段的卵母细胞数量减少，并且卵母细胞的 GSH 水平较低（P < 0.05）。此外，成熟卵母细胞中 ROS 的产生在暴露于 50 和 100 μM 的 AFB1 后达到最高水平（P < 0.05）。在 AFB1 处理组中明显可见 ΔΨm 的减少（P < 0.05）。与对照组相比，AFB1 存在时的分裂和囊胚形成率降低（P < 0.05）。在 AFB1 处理组（10 和 50 μM）中，BAX 和 Caspase3 基因的丰度增加，而 BCL2 基因的丰度降低，与对照组相比（P < 0.05）。此外，与对照组相比，我们的数据显示 10 μM 组中 DNMT3b 基因的相对丰度降低（P < 0.05）。我们表明，卵母细胞暴露于 AFB1 会导致核和细胞质成熟减少，这可能最终损害绵羊卵母细胞的胚胎发育。此外，通过过量 ROS 生成引发的 DNA 甲基化和凋亡起始可能是 AFB1 存在时破坏卵母细胞发育能力的主要分子机制。
