Department of Stem Cell and Regenerative Biotechnology, Humanized Pig Research Center (SRC), Konkuk University, Seoul, Korea.
Department of Cell Biology, College of Medicine, Konyang University, Daejeon, Korea.
Theranostics. 2017 Oct 17;7(19):4735-4752. doi: 10.7150/thno.21662. eCollection 2017.
: Dimethyl sulfoxide (DMSO) is commonly used as a solvent for water-insoluble substances, a vehicle for drug therapy, and a cryoprotectant for cultured cells. DMSO induced embryonic defects and its mechanism of action remains unclear. The rationale is based on the assumption that DMSO supplementation should induce long-term negative effects on both pre- and post-implantation embryo development. DMSO induced oxidative stress, ER stress, autophagy, mitophagy, signaling responsible genes and proteins were determined by RT-qPCR, Western blotting, immunofluorescence, and confocal microscopy. DMSO induced mitochondrial dysfunction was measured by transmission electron microcopy and JC-1 assay. Apoptosis was estimated using TUNEL and comet assay. Post-implantation embryo developmental capability was estimated by implantation site and fetus numbers. Exposure to DMSO induced an early oxidative stress response within 0.5 to 2 h in 1-cell zygotes by disrupting the balance of pro- and anti-oxidants. Notably, DMSO-treated 2-cell embryos showed increased expression of unfolded protein response genes such as and . As a result, the development of many embryos is arrested at the 2-cell, 4-cell, or morula stages in a dose-dependent manner. Further, DMSO-induced endoplasmic reticulum stress increased mitochondrial Ca levels, induced mitochondrial depolarization/dysfunction, and induced apoptotic cell death via the JNK/ATF2-dependent pathway. Consequently, treatment with DMSO increased the expression of autophagy initiation-, phagophore elongation-, and autophagosome formation-related genes, as well as localization of PINK1/Parkin, which are the main mediators of mitophagy, in mitochondria. Interestingly, DMSO causes cytotoxic effects in preimplantation embryos by inducing extensive mitophagy and autophagy. Especially, DMSO treatment decreased the inner cell mass and trophectoderm cell numbers as well as mRNA expression of and in developed blastocysts, which decreased the implantation and developmental rates of full-term offspring after being transferred into pseudopregnant mice. These results provide a significant contribution to finding effective protective agents to combat DMSO mediated reproductive toxicity for application in human embryos in the near future.
二甲基亚砜（DMSO）通常用作水不溶性物质的溶剂、药物治疗的载体和培养细胞的冷冻保护剂。DMSO 诱导胚胎缺陷，其作用机制尚不清楚。其基本原理是假设 DMSO 的补充应该对植入前和植入后胚胎发育产生长期的负面影响。DMSO 诱导氧化应激、内质网应激、自噬、线粒体自噬、负责信号的基因和蛋白质通过 RT-qPCR、Western blot、免疫荧光和共聚焦显微镜确定。通过透射电子显微镜和 JC-1 测定来测量 DMSO 诱导的线粒体功能障碍。通过 TUNEL 和彗星试验估计细胞凋亡。通过植入部位和胎儿数量估计植入后胚胎的发育能力。暴露于 DMSO 在 1 细胞合子中在 0.5 到 2 小时内引起早期氧化应激反应，破坏了氧化剂和抗氧化剂的平衡。值得注意的是，DMSO 处理的 2 细胞胚胎显示出未折叠蛋白反应基因如 和 的表达增加。结果，许多胚胎以剂量依赖的方式在 2 细胞、4 细胞或桑椹胚阶段发育停滞。此外，DMSO 诱导的内质网应激增加线粒体 Ca 水平，导致线粒体去极化/功能障碍，并通过 JNK/ATF2 依赖性途径诱导细胞凋亡死亡。因此，DMSO 处理增加了自噬起始、吞噬体伸长和自噬体形成相关基因的表达，以及 PINK1/Parkin 的定位，PINK1/Parkin 是线粒体自噬的主要介导物。有趣的是，DMSO 通过诱导广泛的线粒体自噬和自噬对植入前胚胎产生细胞毒性作用。特别是，DMSO 处理降低了内细胞团和滋养外胚层细胞数量以及发育中的囊胚中 和 的 mRNA 表达，这降低了转入假孕小鼠后的全期后代的植入和发育率。这些结果为寻找有效的保护剂来对抗 DMSO 介导的生殖毒性，以便在不久的将来应用于人类胚胎提供了重要贡献。
