| Phthalates (PEs) are a family of industrial chemicals that have been used as plasticizers. Among phthalates, the di-(2-ethylhexyl) phthalate (DEHP) and dibutyl phthalate (DBP) are most widely used PEs. During use, they leach, migrate, or evaporate into environment over time and humans are potentially exposed to PEs through food, water, and soil. They are thought as an environmental contaminant, which is known to cause many serious diseases, especially in reproductive system. Mono-2-ethylhexyl phthalate (MEHP) and Mono-n-butyl phthalate (MBP) are metabolite of DEHP and DBP respectively. However, little is known about the effect of MEHP and MBP on preimplantation embryo development.In order to study the effect of MEHP on preimplantation embryos of mouse, embryos were treated by0,10-5,10-4,10-3M MEHP. We found that the development of mouse2-cell embryo was blocked by10-3M MEHP. A significant increase in the level of reactive oxygen species (ROS) was observed in arrested2-cell embryo following10-3M MEHP treatment for24h. However, antioxidants, catalase (CAT), and superoxide dismutase (SOD), reduced intracellular ROS and protected MEHP-exposed embryos from death but failed to return the arrested embryos. Further experiments demonstrated that the level of apoptosis was not altered in live arrested2-cell embryo and increased in dead arrested2-cell embryo after MEHP treatment, which implied that ROS and apoptosis were not related with2-cell block.During analysis of the indicators of embryonic genome activation (EGA) initiation (Hsc70, MuERV-L, Hsp70.1, eIF-1A, and Zscan4) and maternal-effect genes (OCT4and SOX2), we found that MEHP treatment could significantly decline Hsc70, MuERV-L mRNA level and SOX2protein level, and markedly enhance Hsp70.1, eIF-1A, Zscan4mRNA level, and OCT4protein level at2-cell to4-cell stage. Supplementation of CAT and SOD did not reverse the expression tendency of EGA related genes.In order to study the effect of MBP on preimplantation embryos of mouse, embryos were treated by0,10-5,10-4,10-3M MBP. We found the rate of blastocyst formation was significantly reduced following96h exposure to10-4and10-3M MBP compared with the control group. After96h culture, the blastocysts were removed and the remaining embryos were cultured for a further24h. After120h in culture, the rate of blastocyst formation was obviously increased in the10-3M MBP-treated group. However, exposure to10-3M MBP significantly decreased the total blastocyst rate (i.e. the sum of blastocysts at96and120h) and the formation of hatching/hatched embryos compared with that in the control group. These findings suggested that treatment of embryos with10"3M MBP impaired developmental competency, whereas exposure to10-4M MBP delayed the progression of preimplantation embryos to the blastocyst stage. Furthermore, reactive oxygen species (ROS) levels in embryos were significantly increased following treatment with10"3M MBP. In addition,10-3M MBP increased apoptosis via the release of cytochrome c, whereas immunofluorescent analysis revealed that exposure of preimplantation embryos to MBP concentration-dependently (10-5,10-4and10-3M) decreased DNA methylation.Collectively, this study demonstrates for the first time that there is a possible relationship between PEs exposure and developmental failure in preimplantation embryos. An intracellular redox imbalance and apoptosis are involved in this process. But, MEHP-induced2-cell block is mediated by the failure of EGA onset and maternal-effect genes, not oxidative stress and apoptosis. Redox imbalance and apoptosis are related with MBP-induced reductions in the developmental competency of preimplantation embryos. In addition, reductions in DNA methylation may be another mechanism. |
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