| Arsenic exposure on the female reproductive system has significant toxicity, especially the human reproductive cells, are very sensitive to chemical substances. The preimplantation embryo stage is the earliest stage of human life. During this time, the embryo passes through distinct metabolic phases, undergoing changes in the rate of protein synthesis, energyre quirements, and amino acid uptake. It is also the the most sensitive period to internal and external environmental changes. Preimplantation embryos may be more susceptible to arsenic, and its fate is closely to women's fertility, pregnancy outcome and offspring fitness.In fact, many young women live in arsenic exposed environment for a long time before marriage. However, the researches of preimplantation embryo from pregestational arsenic exposed immature females are not fully studied. In this study,3-5 weeks old sex immature female mice were injected intraperitoneally with 8 mg/kg sodium arsenite (NaAsO2) once every other day for 8 times. And then the PMSG-hCG superovulation, in vitro fertilization and in vitro culture of embryo were used to evaluate the effect of pregestational arsenic exposure on mouse fertility. Our data show that pregestational arsenic exposure results in decreased ovulability and embryonic retardation at 1-2 cell stage. Compared with the control group embryos, these blocked embryos increased reactive oxygen species (ROS) about twice, marked the release of cytochrome c, nuclear apoptosis rate increased 36%. All these results indicate that pregestational arsenic exposure affects the development of preimplantation embryo, which also predicts that pregestational arsenic exposure maybe results in pregnancy problems of women of childbearing age.Many possible modes of arsenic action have been proposed, including chromosomal abnormalities, altered DNA repair and methylation patterns, altered cell proliferation, abnormal gene amplification, and the inhibition of p53 and telomerase. Much more researches indicate that arsenic exerts its toxicity by generating ROS. However, how the redox imbalance generation link to arsenic exposure is still not well understood. The results of pregestational arsenic exposure have shown that arsenic exerts its toxicity through generating reactive oxygen species (ROS). To understand the detailed mechanism of arsenic intervention, sodium arsenite was directly exposed to the 2-cell embryos cultured in vitro, which is easy to study the detailed molecullar mechanism of arsenic intervention.According to the literature, p66Shc is a kind generating enzyme of ROS, it is translocated into the mitochondrial intermembrane space, where it interacts with reduced cytochrome c to produce H2O2 and opern the permeability transition pores that allow the release of ROS into the cytosol. We find that high dose arsenic exposure induces the intracellular level of ROS for three times and the expression level of p66Shc protein increased one time. Moreover, the ROS scavager, N-acetyl-L-cysteine (NAC), effectively decreases p66Shc levels in embryos challenged with 1uM arsenic, suggesting that the p66Shc protein is an ROS-response protein. In one word, p66Shc may respond to arsenic tress by inducing excessive ROS generation in a positive feedback signaling pathway. Glutathione (GSH) is an important antioxitant for protectin cells against oxidative injuries. Unlike somatic cells, preimplantation embryos cannot synthesize GSH de novo. Therefore, we also find that the embryonic GSH level is sharply decreased due to arsenic exposure. Here we propose that high-level arsenite induces severe redox imbalance by decreasing the levels of glutathione and increasing the levels of ROS through the oxidative stress adaptor p66Shc; as a results, it is the cytochrome c-caspase apoptotic pathway but not apoptosis - inducing factor (AIF) which is actvited and embryo viability decreases.Detailed biochemical analysis has shown that the luminal fluids of both the oviduct and uterus contain significant levels of free amino acids, which may have a physiological role in preimplantation development. Therefore, when embryos are stressed, metabolism is immediately perturbed. It has been previously shown that amino acid depletion/appearance (turnover) during cleavage predicts the ability of spare human embryos to develop into the blastocyst stage in vitro. We analsize the changes of amino acid level in embryonic culture through reversed-phase chromatography (RP-HPLC), suggesting the changes of intracellular aimno acid metabolism indirectly. We demonstrated that the extracellular amino acid metabolism, especially the cytotoxic and antioxidative amino acids, is more perturbed upon exposure to low-level arsenite than high-level arsenite, which may be the reason for the embryonic delay induced by low arsenic exposure.Furthermore, An antioxidant, N-acetyl-L-cysteine, improves the development of arsenite-exposed embryos by reducing intracellular ROS and adjusting amino acid metabolism, suggesting that increasing the intracellular antioxidant level may have preventive or therapeutic effects on arsenic-induced embryonic toxicity.The thesis of this study is the effect and mechanism of arsenic treatment in the early pregnancy stage. The results suggest that the development of preimplantation embryo is delayed or blocked due to the low level arsenic exposure. The p66Shc-linked redox imbalance and abnormal extracellular amino acid metabolism were thinked to be the detailed reasons for preimplantation embryo developmental block or delay. Thus, the arsenic exposure of pre-pregnancy and early pregnancy should be given more attention for the women's reproductive health and the well-being of future generations.
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