Effect of single step culturesystem on Insulin/IGF system expression in mice embryo
Background: The single step culture system forces embryos to adapt in culture media without considering the differences in embryo stages needs and requirements which could cause alteration in gene expression or alteration in DNA methylation pattern in genes involved in embryo development such as Insulin/IGF system. Moreover, the single step culture system increases the potential for ammonium accumulation in culture media which negatively affects the Insulin/IGF system gene expression and DNA methylation level. Hypothesis: Single step culture media causes detrimental alteration in gene expression and DNA methylation of the Insulin/IGF system in blastocyst embryo more than sequential culture media. Methods: 220 fertilized oocytes will be retrieved from mice oviduct. The zygotes will be evenly divided into three groups. The embryos in the first group will culture in a sequential culture system (G1&G2) media for five days. In the second group, embryos will culture in single step culture system (Advanced KSOM) for five days. The embryos in the third group will culture in single step culture system (Advanced KSOM) for five days, but media will renew on day 2. On day 5, embryos will lyse to extract mRNA and DNA and will use RNA-seq to analyses gene expression of Insulin/IGF system, and whole-genome bisulfite sequencing to analyses the DNA methylation level. moreover, the ammonium level of culture media will be measured using dry chemistry technology. Expected results: There is significant change in Insulin/IGF system gene expression in embryos cultured in a single step culture media compared to embryos cultured in a sequential culture media. There is alteration in DNA methylation pattern in embryo culture in single step culture media compared to embryos cultured in a sequential culture system. additionally, the ammonium levels significantly increase in group two, where embryos culture in single step media for five days without renewing the media.