| Mammalian early embryonic development and differentiation is a complex process, the regulation mechanism is one of the difficult and hot research issues in recent years.Mammalian preimplantation embryo development in vitro often appear'development block phenomenon', occurring during vary periods. In vitro, mouse embryo development is blocked at 2-cell stage, which means that in vitro culture conditions, cleavage of mouse eggs are paused at 2-cell stage after the first division, and can not continue to divide. Despite many ways to do research to find the reasons of causing 2-cell block, the molecular mechanism of the phenomenon of blocking remains poorly understood.MicroRNAs (miRNAs) are newly found 19-25nt single stranded noncoding RNAs, and were distributed widely in Eukaryotes from C.elegans to human beings. miRNAs have been shown to control temporal gene expression by downregulating mRNAs transcribed during previous developmental stages.In recent years, a large number of studies have shown that, miRNAs express in mouse preimplantation embryos, and the expression was decreased significantly in 2-cell stage, suggesting that the miRNAs play an important role in the process of embryonic genome activation. However, by the four-cell stage, miRNA abundance increases again relative to two-cell embryos, but the consequences of this change is not clear, and the impact on embryonic development is also not clear. And, the mouse embryo development is blocked at 2-cell stage, so we hypothesized that miRNA expression changes during this period may be closely related to embryonic development block.Therefore, the research to be carried out is the expression of miRNAs in the 2-cell and 4-cell stage mouse embryos, in order to find the basis of which miRNAs may play a role in preimplantation embryo development. Objective: To establish the system of mice superovulation, mating, and embryo collection, to investigate the expression of miRNAs in mouse 2-cell and 4-cell stage embryos, and to explore the role and significance of miRNAs in the preimplantation embryo development.Methods and Results:Part1: The technologies of miRNA amplifying and miRNA microarray were applied to screen the miRNAs that expressed differentially in the mouse embryos between 1.5 days (2-cell) after fertilization and 2.5 days (4-cell) after fertilization. By microRNA microarray data analysis, we found a total of 192 (> 2-fold) miRNAs with significantly different expression. Of these, 122 miRNAs expressing at 4-cell embryos were significantly increased compared with 2-cell embryos, and 70 miRNAs expressing in 4-cell embryos were significantly lower than 2-cell embryos.Part2: Fluorescent quantitative RT-PCR method was used to validate the expression levels of mmu-miR-467h,mmu-miR-466d-3p,mmu-miR-292-5p, mmu- miR-154, mmu-miR-2145 and mmu-miR-706 in mice 2-cell and 4-cell embryos, and we found that the six miRNAs'expression levels were consistent with the chip, which may demonstrate the relationship of miRNAs with the growth and development of early mouse embryos.Conclusion: By successfully establishing the system of superovulation, mating, and embryo collection in mice and the application of miRNA microarray technology, we screened to find differentially expressed microRNAs in the mouse preimplantation 2-cell and 4-cell embryos. MiRNA expression changes occurred again after embryonic genome activation. Through the analysis of the levels and functions of these differentially expressed miRNAs, it is suggesting that miRNAs may play an important role in embryonic development and differentiation of embryonic cells by regulating some of the important and development-related gene expression in the embryonic development.And, it is important for further understanding the molecular mechanisms of embryonic development block. |
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