The Study Of The Impaction Of Sealed Culture Condition For The Development Of Early Mouse Embryos And The Methylation Status Of Igf2/H19 Imprinting Control Region

The development study of embryos in space has become an important part in the research of life in space. Embryo cultured in sealed is the basic condition for the embryo development studies in space. As the gas-tight and liquid-tight restrictions, the sealed-culture should choose the most suitable density conditions, gas phase, medium composition et al, which could maintain long-term cultured for mammalian early embryos. Therefore the appropriate density of embryos cultured (embryo number/ culture medium volume), which will meet the balance between the nutritional and metabolic waste in the development of embryos, is the primary research topic. Second, because the sensitivity of mouse early embryos to the environment and the epigenetics model of genes related to development are easily changed with the development environment, especially the methylation imprinting of Igf2/H19 imprinting control region are easy to change for the impaction of manipulation in vitro and the culture condition in vitro. So this main study focus to approach initially the influence of the development of 2-cell mouse embryos and the methylation levels of Igf2/H19 imprinting control region under sealed culture condition, and to study and explorate the optimal condition of sealed culture system for further in-depth, so as to supply the new research tools and the mew data on earth for the space biology study of mammalian.1. On the condition of sealed culture set in the study which is composed by 200μL CZB culture medium filled the high pure reference gas of 5%O2, 5%CO2, 90%N2, analysis the development rates of 2-cell mouse embryos respectively cultured for the four time point (24 h, 48 h, 72 h, 96 h), under the 3 different densities that embryo numbers : culture medium volumes is respective 1:4, 1:2, 1:1, and using the development rates of the corresponding stages embryos by general culture in vitro as a standard. The study confirm that the culture destiny of 1:2 is much fitter for 2-cell mouse embryos, comparing 1:1 and 1:4; meanwhile, the development rates and speeds of embryos in sealed culture are lower than the results of embryos in general culture, but the development rates of hatching embryos are almost same(75.1%,78.1%), so this sealed culture condition could be used to culture 2-cell mouse embryos in vitro.2. Using the bisulfite sequencing PCR (BSP) to analysis the methylation levels of Igf2/H19 ICR in 8-cell embryos, morula and blastocyst, these embryos are respectively come from 2-cell mouse embryos after cultured 24 h, 48 h and 72 h in sealed culture, using the methylation levels of the corresponding stages embryos after general culture in vitro as control and the methylation levels of the corresponding stages embryos in vivo as a standard. The study confirmed that the methylation levels of Igf2/H19 ICR of all stages embeyos in sealed culture condition all were lower than the results of general cultured in vitro, and significantly lower than the results of development in vivo, so the sealed culture could make the methylation levels of Igf2/H19 imprinting control region of mouse preimplantation embryos decreased. And the study confirmed that the methylation level of Igf2/H19 imprinting control region could be as a molecular target to detect the development status of the mammal early embryos.