The Establish Of The Sealed Culture System For 4 Cell Mouse Embryos And The Screening For Suitable Culture Condition

As one of the most important parts of the research for life in space, the study of embryo development in space has received particular attention. But the general culture system couldn't support the embryo development in a vacuum and weightless situation. So we must choose a sealed-culture method (include gas-tight and liquid-tight) to support embryos'development in space. Sealed-culture means culture medium was treated by definite proportion reference gas with O₂, CO₂ and N₂ before embryo culture. One of the most important factors in sealed-culture system is the density of embryo in the medium because we can't refresh gas and nutrition during the culture. This study mainly concern on the screening of reference gas, culture medium and embryo density to build a system that can support embryo development in vitro and for the research of the influence on embryo develop in space.1. On the condition of sealed-culture set in the study, two kinds of medium (mDPBS and CZB) were chose to be culture medium and two kinds of reference gas composed of 5% O₂, 5% CO₂, 90% N₂ or 10% O₂, 5% CO₂, 85% N₂ to culture mouse 4 cell stage embryos under the liquid volume of 200μL. Through the analysis of development rates of the embryos under 3 different densities that embryo numbers : culture medium volumes were 1:1, 1:2 and 1:4 to judge the culture effect in different sealed-culture group.When culture medium treated by the reference gas with 5% O₂ proportion, the result indicates that embryo development in mDPBS and CZB were similar. The blastosphere growth rate in 1:1 culture group has no significant difference from the control group (P>0.05). But the hatching rate has significant lower than the control group (P<0.05). The blastosphere growth rate and the hatching rate in 1:2 culture group have no significant difference from the control group (P>0.05). The blastosphere growth rate and the hatching rate in 1:4 culture group are significant lower than the control group (P<0.05).When culture medium treated by the reference gas with 10% O₂ proportion, the result indicates that embryo development in mDPBS and CZB were similar. The blastosphere growth rate and the hatching rate in 1:1 culture group have no significant difference from the control group (P>0.05). The blastosphere growth rate and the hatching rate in 1:2 and 1:4 culture groups are significant lower than the control groups (P<0.05).When culture medium not treated by the reference gas, the blastosphere growth rate and the hatching rate in 1:1, 1:2 and 1:4 culture groups are significant lower than the control group (P<0.05).This result proves that embryo couldn't develop well in sealed-culture system with low density, and embryo couldn't develop well in the medium which not treated by the reference gas.2. According to the result above, culture mouse 4 cell stage embryo by mDPBS and CZB when the culture medium treated by the reference gas with 5% O₂ proportion to screening suitable culture medium for sealed-culture. The result indicates that the total blastomere numbers of embryo cultured in mDPBS was significant lower than embryo cultured in CZB. Embryos The morphology of embryos cultured in mDPBS had worse than in CZB. It proves that embryos could develop better in CZB than in mDPBS.3. According to the result above, use CZB as the culture medium, which treated by the reference gas of 5% O₂ proportion or 10% O₂ proportion, to culture the 4 cell stage embryo in vitro. Comparing the total blastomere numbers and the apoptosis rate of the embryos, then detecting the peroxide production and the express of hypoxia-inducible factor 1 alpha (HIF-1α) protein in embryos cultured with1:1 and 1:2 density to screening suitable embryo density for sealed-culture.When culture medium treated by the reference gas with 5% O₂ proportion, the result indicates that the embryo has significant lower total blastomere numbers of 1:1 culture group than that in control group with same density (P<0.05), and apoptosis rate is significant higher than control group (P<0.05). The embryo cultured in 1:2 group have no significant difference of total blastomere numbers and apoptosis rate from control group with same density (P>0.05). When culture medium treated by the reference gas with 10% O₂ proportion, the embryo cultured in 1:1 group have no significant difference of total blastomere numbers and apoptosis rate from control group with same density (P>0.05). The embryo cultured in 1:2 group have significant difference of total blastomere numbers and apoptosis rate from control group with same density (P<0.05).When detecting the peroxide production in embryos during the early period of sealed-culture., we found out that 1:1 and 1:2 culture groups have a higher production of peroxide than control group when cultured in medium treated by the reference gas with 10% O₂ proportion in 12 h. But this situation has not been found in groups cultured in medium treated by the reference gas with 5% O₂ proportion. We can infer that when culture medium treated by the reference gas with 10% O₂ proportion, embryos developed in sealed-culture system may show peroxy-injury in the early culture period.When culture medium treated by the reference gas with 5% O₂ proportion, HIF-1αprotein express in embryos was detected in 60 h in 1:1 culture, and detected out in 84 h in1:2 culture group. When culture medium treated by the reference gas with 10% O₂ proportion, HIF-1αprotein express in embryo was detected in 60 h in 1:1 culture group, and HIF-1αprotein express was not detected in 1:2 culture group.In conclusion, in the study of the sealed culture of mouse 4 cell stage embryo, we have determined CZB as optimal culture medium, and 1:2 density of embryos cultured in 5% O₂ reference gas treated medium as the suitable culture condition.