| In the study of mammalian embryo development in space, due to the limited aircraft carrying and the particularity of space environment, we must apply the sealed-culture system(including liquid-tight and gas-tight) to support embryo development in space. Sealed-culture means culture medium is aerated with reference gas of definite gas ratios in advance, so the standard gas is dissolved into the cultue medium onwards. It would provide the requisite gas for embryo development. Load the aerated medium and early embryo quickly into the sealed-culture container and seal the container immediately. Our research group had preliminarily identified the basis culture medium, culture density, reference gas ratio and aeration time for the culture medium. In addition, we also identified the appropriate growth factor supplement protocol for embryo development in sealed-culture system, based on the expression of early embryo development related growth factors and their receptors. In this study, we further optimized the sealed-culture system which could support early embryo development in vitro by screening the optimal culture temperature range and feral bovine serum(FBS) concentration. We analyzed the relative expression levels of primary transcription factors of embryos in optimized sealed-culture system by real-time fluorescence quantification PCR as well, to explore whether the optimized sealed-culture system could support normal early embryo development.Following results have been obtained:1. We aerated culture medium with reference gas containing 5% O2, 5% CO2, 90% N2 for 130 min before embryo culture. After that we added 0.1 ng/mL EGF and 1 ng/mL IGF-1 to the aerated culture medium and then cultured mouse 2-cell embryos in sealed-culture system under different temperature(36, 36.5, 37, 37.5, 38℃) immediately. Analyze the results of embryo development rates, total blastomere number cultured for 72 h and 96 h, and blastomere apoptosis detection results cultured for 96 h to screen the optimal temperature range for mouse early embryo development in sealed-culture system. The results indicated that embryo development rate and total blastomere number both reached higher level when culture temperature was 36.5 ℃ or 37℃, and blastomere apoptosis rate showed significantly lower level, comparing to the other groups(P<0.05). When culture temperature was 36℃, embryo development rate decreased and development block tended to be obvious. When culture temperature were 37.5 ℃and 38 ℃, embryo blastomere fragmented and shrinked, and blastomere apoptosis rates were significantly higher than the other groups(P<0.05). Thus, culture temperature ranged between 36.5~37 ℃could better support mouse 2-cell embryos developing to blastocyst and hatched blastocyst stage in sealed-culture system. According to these results, we choose temperature control system of 36.8±0.2℃ when designing hardware for the research satellite.2. We aerated different culture medium(added 2.5%, 5%, 7.5%, 10%, 15%, 20% FBS, respectively) with reference gas containing 5% O2, 5% CO2, 90% N2 for 130 min before embryo culture. After that we add 0.1 ng/mL EGF and 1 ng/mL IGF-1 to the aerated culture medium and then cultured mouse 2-cell embryos in sealed-culture system under 37℃ immediately. Analyze the results of embryo development rates, total blastomere number cultured for 72 h and 96 h, and blastomere apoptosis detection results cultured for 96 h to screen the optimal serum concentration for mouse early embryo development in sealed-culture system. The results indicated that blastocyst formation rate cultured for 72 h, hatched blastocyst rate cultured for 96 h and total blastomere number all reached higher level when serum concentration was 7.5% or 10%, and blastomere apoptosis rate showed significantly lower level, comparing to the other groups(P<0.05); but there was no significant difference between the two groups(P>0.05). When serum concentration was 5% or 15%, blastocyst formation rate and total blastomere number were significantly lower than those of 7.5% serum concentration group, and blastomere apoptosis rate were significantly higher than 7.5% serum concentration group(P<0.05). When serum concentration was 2.5%, embryos showed degradation partially and blastomere apoptosis rate cultured for 96 h was significantly higher than other groups(P<0.05). When serum concentration was 20%, blastocyst formation rate cultured for 72 h was significantly lower than other groups(P<0.05), and embryos showed obvious slow development issue. According to the above results, adding 7.5%~10% fetal bovine serum to embryo culture medium could better support mouse 2-cell embryos developing to blastocyst and hatched blastocyst stage in sealed-culture system. In follow-up experiments, we choose sealed-culture system of culture medium adding 7.5% fetal bovine serum for mouse 2 cell embryo.3. According to the results above, we aerated culture medium(added 7.5% FBS) with reference gas containing 5% O2, 5% CO2, 90% N2 for 130 min before embryo culture. After that we add 0.1 ng/mL EGF and 1 ng/mL IGF-1 to the aerated culture medium and then cultured mouse 2-cell embryos in sealed-culture system under 37℃ immediately. We employed real-time fluorescence quantification PCR to compare the relative expression level of development related transcription factors Oct4, Nanog, Sox2 and Cdx2 among embryos cultured in sealed-culture for 72 h, embryos cultured in micro-drop for 72 h and embryos produced in vivo. Results showed that the relative expression level of Oct4, Nanog, Sox2 and Cdx2 showed no significant difference between embryos cultured in sealed-culture and produced in vivo(P>0.05). The relative expression level of Oct4 and Sox2 was slightly lower compared to the in vivo group, but the difference was not significant(P>0.05); the relative expression level of Oct4 and Sox2 was significantly lower than the in vivo group(P<0.05). According to the above results, mouse embryos cultured in optimized sealed-culture system have better developmental potential, and this system could support normal early embryo development under sealed-culture condition. |
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