邻苯二甲酸酯对小鼠植入前胚胎体外发育的影响及作用机制的研究
发布时间：2019-03-29 07:52
【摘要】：邻苯二甲酸酯(phthalate esters, PEs),是一类普遍使用的化学工业品,主要用作增塑剂,其中邻苯二甲酸二乙基己基酯(Di-2-ethylhexyl phthalate, DEHP)和邻苯二甲酸二丁酯(Dibutyl phthalate, DBP)是使用较为广泛的增塑剂。邻苯二甲酸-单-乙基己基酯(Mono-2-ethylhexyl phthalate, MEHP)和邻苯二甲酸单丁酯(Mono-n-butyl phthalate, MBP)分别是DEHP和DBP的主要代谢物。使用过程中,邻苯二甲酸酯会通过各种途径进入环境中,人类可经由食品、水和土壤接触邻苯二甲酸酯。现已发现邻苯二甲酸酯具有很强的生殖和发育毒性,能损害生物的生育力。然而,目前我们对于MEHP和MBP对植入前胚胎发育的影响仍知之甚少。 为了研究MEHP对植入前胚胎发育的影响,我们用0,10-5,10-4,10-3M MEHP处理受精卵,发现10-3M MEHP会引起小鼠胚胎发生2-细胞阻滞。10-3M MEHP暴露24h后,小鼠胚胎细胞内活性氧水平显著升高。然而,抗氧化物(CAT和SOD)可以降低胚胎细胞中活性氧的水平,提高胚胎的存活率,但是没有恢复发生2-细胞阻滞胚胎的发育。进一步的实验结果表明,MEHP暴露后,只有死亡胚胎的凋亡水平升高了,但活的2-细胞阻滞的胚胎凋亡水平没有明显变化。这些结果提示ROS水平的升高和胚胎细胞凋亡并不是发生2-细胞发育胎阻滞的主要原因。通过分析胚胎基因组激活(embryonic genome activation, EGA)的关键基因(Hsc70, MuERV-L, Hsp70.1, eIF-1A和Zscan4)以及母源效应基因(OCT4和SOX2),我们发现胚胎2-细胞到4-细胞阶段,MEHP暴露显著降低了基因Hsc70、MuERV-L和SOX2的表达水平,提高了Hsp70.1、eIF-1A、Zscan4和OCT4的表达水平。添加抗氧化物(CAT和SOD)没能改变这些基因的表达趋势。由此可见,MEHP-诱导的胚胎发育阻滞是由母体向胚胎转换异常介导的,而不是氧化应激和细胞凋亡。 为了研究MBP对植入前胚胎发育的影响,我们用0,10-5,10-4,10-3M MBP处理受精卵,发现10-3和10-4M MBP暴露96h,囊胚的数量均显著降低,当延迟培养24h时,即120h,10-3M MBP暴露组桑椹胚继续发育到囊胚的胚胎数量显著高于对照组,但96h和120h囊胚的总数仍显著低于对照组,并且10-3M MBP暴露显著降低了孵化囊胚的数量,这些结果显示了10-3M MBP会损害小鼠植入前胚胎的发育潜能,10-4M MBP暴露只是延缓了胚胎的发育进程。10-3M MBP暴露48h后,胚胎细胞内ROS水平显著升高,并通过促进线粒体释放细胞色素c提高了凋亡水平。免疫荧光染色分析显示MBP处理以剂量依赖(10-5,10-4,10-3M)的方式降低了DNA甲基化水平。这些结果显示MBP可能通过诱导氧化应激、细胞凋亡或抑制DNA甲基化降低胚胎的发育潜能。 总之,本研究首次揭示了PEs暴露能对植入前胚胎发育造成损害。MEHP能够引起胚胎2-细胞发育阻滞,其主要是由于EGA启动失败和母源效应基因表达异常引起,与ROS和凋亡水平无显著相关性。MBP能够降低植入前胚胎的发育潜能,其主要与ROS和细胞凋亡相关,还可能与DNA甲基化水平的降低有关。
[Abstract]:Phthalate (phthalate esters, PEs), is a widely used chemical industry, mainly used as plasticizer, in which diethylhexyl phthalate (Di-2-ethylhexyl phthalate, DEHP) and dibutyl phthalate (Dibutyl phthalate,) DBP) is a widely used plasticizer. Monoethyl hexyl phthalate (Mono-2-ethylhexyl phthalate, MEHP) and monobutyl phthalate (Mono-n-butyl phthalate, MBP) are the main metabolites of DEHP and DBP, respectively. During use, phthalates enter the environment through various channels, and humans can access phthalates through food, water and soil. It has been found that phthalates have strong reproductive and developmental toxicity, which can damage the fertility of organisms. However, little is known about the effects of MEHP and MBP on the development of preimplantation embryos. In order to study the effect of MEHP on preimplantation embryo development, we treated fertilized eggs with 0, 10 ~ 5, 10 ~ 4, 10 ~ 3 M MEHP, and found that 10 ~ (3) M MEHP induced 2-cell arrest in mouse embryos. 24 hours after exposure to 10-3 M MEHP, we found that 10-3 M MEHP induced 2-cell block in mouse embryos. The level of reactive oxygen species (Ros) in mouse embryo cells increased significantly. However, antioxidants (CAT and SOD) decreased the level of reactive oxygen species (Ros) in embryonic cells and increased the survival rate of embryos, but did not resume 2-cell block of embryo development. The further results showed that only the apoptotic level of dead embryos increased after MEHP exposure, but there was no significant change in the apoptotic level of live 2-cell arrest embryos. These results suggest that the increase of ROS level and embryonic cell apoptosis are not the main causes of 2-cell fetal block. By analyzing the key genes that activate (embryonic genome activation, EGA) in the embryonic genome (Hsc70, MuERV-L, Hsp70.1, eIF-1A and Zscan4) and maternal effectors (OCT4 and SOX2), we found that embryonic 2-cell to 4-cell stage. MEHP exposure significantly decreased the expression level of gene Hsc70,MuERV-L and SOX2, and increased the expression level of Hsp70.1,eIF-1A,Zscan4 and OCT4. The addition of antioxidants (CAT and SOD) did not change the expression trend of these genes. It can be seen that MEHP- induced embryonic development block is mediated by abnormal transformation from mother to embryo, not oxidative stress and apoptosis. In order to study the effect of MBP on the development of preimplantation embryos, we treated the fertilized eggs with 0, 10, 5, 10, 3 M MBP. It was found that the number of blastocysts decreased significantly after exposure to 10, 10 and 4 M MBP for 96 hours, and the number of blastocysts decreased significantly at 24 hours of delayed culture, that is, 120 hours. The number of blastocysts continued to develop into blastocysts in the group exposed to 10 m MBP was significantly higher than that in the control group, but the total number of blastocysts at 96 h and 120 h was still significantly lower than that in the control group, and the number of hatched blastocysts was significantly decreased after exposure to 10 m MBP. These results showed that 10 m MBP could damage the developmental potential of mouse preimplantation embryos, and 10 m MBP exposure only delayed the development of embryos. 48 h after exposure to 10-3 M MBP, the level of ROS in embryonic cells increased significantly. The level of apoptosis was increased by promoting the release of cytochrome c from mitochondria. Immunofluorescence staining showed that MBP treatment reduced the level of DNA methylation in a dose-dependent manner (10 ~ 5, 10 ~ 4, 10 ~ 3 M). These results suggest that MBP may reduce the developmental potential of embryos by inducing oxidative stress, apoptosis or inhibition of DNA methylation. In conclusion, this study revealed for the first time that PEs exposure can cause damage to preimplantation embryo development. MEHP can cause embryonic 2-cell development block, mainly due to the failure of EGA initiation and abnormal expression of maternal effector genes. MBP can decrease the developmental potential of preimplantation embryos, which is mainly related to ROS and apoptosis, and may also be related to the decrease of DNA methylation level.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R114
本文编号：2449316
[Abstract]:Phthalate (phthalate esters, PEs), is a widely used chemical industry, mainly used as plasticizer, in which diethylhexyl phthalate (Di-2-ethylhexyl phthalate, DEHP) and dibutyl phthalate (Dibutyl phthalate,) DBP) is a widely used plasticizer. Monoethyl hexyl phthalate (Mono-2-ethylhexyl phthalate, MEHP) and monobutyl phthalate (Mono-n-butyl phthalate, MBP) are the main metabolites of DEHP and DBP, respectively. During use, phthalates enter the environment through various channels, and humans can access phthalates through food, water and soil. It has been found that phthalates have strong reproductive and developmental toxicity, which can damage the fertility of organisms. However, little is known about the effects of MEHP and MBP on the development of preimplantation embryos. In order to study the effect of MEHP on preimplantation embryo development, we treated fertilized eggs with 0, 10 ~ 5, 10 ~ 4, 10 ~ 3 M MEHP, and found that 10 ~ (3) M MEHP induced 2-cell arrest in mouse embryos. 24 hours after exposure to 10-3 M MEHP, we found that 10-3 M MEHP induced 2-cell block in mouse embryos. The level of reactive oxygen species (Ros) in mouse embryo cells increased significantly. However, antioxidants (CAT and SOD) decreased the level of reactive oxygen species (Ros) in embryonic cells and increased the survival rate of embryos, but did not resume 2-cell block of embryo development. The further results showed that only the apoptotic level of dead embryos increased after MEHP exposure, but there was no significant change in the apoptotic level of live 2-cell arrest embryos. These results suggest that the increase of ROS level and embryonic cell apoptosis are not the main causes of 2-cell fetal block. By analyzing the key genes that activate (embryonic genome activation, EGA) in the embryonic genome (Hsc70, MuERV-L, Hsp70.1, eIF-1A and Zscan4) and maternal effectors (OCT4 and SOX2), we found that embryonic 2-cell to 4-cell stage. MEHP exposure significantly decreased the expression level of gene Hsc70,MuERV-L and SOX2, and increased the expression level of Hsp70.1,eIF-1A,Zscan4 and OCT4. The addition of antioxidants (CAT and SOD) did not change the expression trend of these genes. It can be seen that MEHP- induced embryonic development block is mediated by abnormal transformation from mother to embryo, not oxidative stress and apoptosis. In order to study the effect of MBP on the development of preimplantation embryos, we treated the fertilized eggs with 0, 10, 5, 10, 3 M MBP. It was found that the number of blastocysts decreased significantly after exposure to 10, 10 and 4 M MBP for 96 hours, and the number of blastocysts decreased significantly at 24 hours of delayed culture, that is, 120 hours. The number of blastocysts continued to develop into blastocysts in the group exposed to 10 m MBP was significantly higher than that in the control group, but the total number of blastocysts at 96 h and 120 h was still significantly lower than that in the control group, and the number of hatched blastocysts was significantly decreased after exposure to 10 m MBP. These results showed that 10 m MBP could damage the developmental potential of mouse preimplantation embryos, and 10 m MBP exposure only delayed the development of embryos. 48 h after exposure to 10-3 M MBP, the level of ROS in embryonic cells increased significantly. The level of apoptosis was increased by promoting the release of cytochrome c from mitochondria. Immunofluorescence staining showed that MBP treatment reduced the level of DNA methylation in a dose-dependent manner (10 ~ 5, 10 ~ 4, 10 ~ 3 M). These results suggest that MBP may reduce the developmental potential of embryos by inducing oxidative stress, apoptosis or inhibition of DNA methylation. In conclusion, this study revealed for the first time that PEs exposure can cause damage to preimplantation embryo development. MEHP can cause embryonic 2-cell development block, mainly due to the failure of EGA initiation and abnormal expression of maternal effector genes. MBP can decrease the developmental potential of preimplantation embryos, which is mainly related to ROS and apoptosis, and may also be related to the decrease of DNA methylation level.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R114
【参考文献】
相关期刊论文 前1条
1 王立鑫;杨旭;;邻苯二甲酸酯毒性及健康效应研究进展[J];环境与健康杂志;2010年03期
，本文编号：2449316
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