Department of Animal Sciences, Chungbuk National University, Chungbuk, Cheongju 361-763, Republic of Korea.
Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Jeonju 55365, Republic of Korea.
Toxicol In Vitro. 2019 Dec;61:104635. doi: 10.1016/j.tiv.2019.104635. Epub 2019 Aug 31.
Thiamethoxam (TMX) is a neonicotinoid insecticide, the residues of which have been detected on various crops. In addition to its specific acetylcholine toxicity to insects, TMX was also found to be toxic to mammals. Moreover, oocytes are vulnerable to reactive oxygen species (ROS). Excessive ROS production can override antioxidant defenses and produce oxidative stress and DNA damage that trigger apoptosis and necrosis in organisms. In this study, we exposed bovine oocytes to TMX during maturation. Microscopic examination showed that 1.6 mM TMX significantly inhibited maturation at the germinal vesicle (GV) and metaphase I (MI) stages. Immunofluorescence staining and enzyme activity analysis revealed that TMX induced a reduction in CDC25 and CDC2 activity. Furthermore, time-lapse tracking and immunofluorescence staining indicated the maintenance of cyclin B in the cytoplasm, persistence of Bub3 at kinetochores, and absence of actin caps after TMX-exposed oocytes reached the MI stage. In addition, metaphase II (MII) oocytes exposed to TMX showed disordered chromosomes and spindles. These oocytes accumulated excess ROS and showed significantly decreased mitochondrial membrane potential and increased apoptotic signals. Parthenogenetic embryos from these oocytes showed decreased percentages of morulae and blastocysts. These results indicate that TMX delays bovine oocyte progression to MI stage, blocks them at the MI stage, triggers disordered chromosomes and spindles at MII stage, and ultimately results in MII oocytes with poor cleavage ability and inhibited development to morulae and blastocysts.
噻虫嗪（TMX）是一种新烟碱类杀虫剂，已在各种作物上检测到其残留。除了对昆虫具有特定的乙酰胆碱毒性外，TMX 还被发现对哺乳动物有毒。此外，卵母细胞易受活性氧（ROS）的影响。过量的 ROS 产生会破坏抗氧化防御系统，导致氧化应激和 DNA 损伤，从而引发细胞凋亡和坏死。在本研究中，我们在牛卵母细胞成熟过程中暴露于 TMX。显微镜检查显示，1.6 mM 的 TMX 显著抑制了卵母细胞在生发泡（GV）和第一次减数分裂中期（MI）阶段的成熟。免疫荧光染色和酶活性分析显示，TMX 诱导 CDC25 和 CDC2 活性降低。此外，延时追踪和免疫荧光染色表明，TMX 暴露的卵母细胞到达 MI 阶段后，cyclin B 仍保留在细胞质中，Bub3 仍保留在动粒上，没有形成肌动蛋白帽。此外，暴露于 TMX 的第二次减数分裂中期（MII）卵母细胞表现出染色体和纺锤体排列紊乱。这些卵母细胞积累了过量的 ROS，线粒体膜电位显著降低，凋亡信号增加。来自这些卵母细胞的孤雌胚胎的桑葚胚和囊胚比例降低。这些结果表明，TMX 延迟了牛卵母细胞向 MI 阶段的进展，使其在 MI 阶段停滞，在 MII 阶段引发染色体和纺锤体紊乱，最终导致 MII 卵母细胞的分裂能力差，并抑制其向桑葚胚和囊胚的发育。
