Investigation of the effect of dna methylation on pluripotency in the inner cell mass of mice embryos

Objective: Stem cells are the cells with the highest potential to differentiate and develop into other cells. The most important sources of embryonic stem cells are pre-implantation blastocyst inner cell masses. These cells synthesize some transcription factors unique to them and not found in other cells. These factors are the main controllers of the cell's ability to differentiate into other cells or to renew themselves. In our study, we aimed to see the effect of DNA methylation, one of the epigenetic cell regulators, on the expression of Nanog, which is one of the embryonic stem cell markers, and thus its direct effects on the pluripotency of cells. Method: In our study, embryos were obtained from Balb/c female mice by uterine dissection and horn washing at embryonic 3.5 days and treated in 50 microliter droplets. Methylation and pluripotency levels determined respectively with 5-Methylcytosine and Nanog immunofluorescence by applying methylation inhibitor to total blastocyst groups and inner cell masses obtained by immunosurgery. Results were analyzed by ImageJ. Results: Immunofluorescence images were evaluated and significant increases were observed in both 5-Methylcytosine and Nanog expressions in the 0,1 µM inhibitor treated groups in the total blastocyst groups. In the group treated with 1 µM inhibitor, the decrease in 5-Methylcytosine expression was not found to be significant compared to the control groups, while there was a significant decrease in Nanog expression. In the immunosurgery groups, both 5-Methylcytosine and Nanog expressions were decreased in the 0,1 µM inhibitor group compared to the control groups. Conclusion: In this study, the effect of methylation on pluripotency was investigated in total blastocyst and inner cell masses obtained by immunosurgery. Changes in 5-methylcytosine and Nanog expression levels show that these cell groups have different responses to the inhibitor. It has been observed that there are changes in the intracellular localization of nanog expression. Our study may contribute to studies on pluripotent stem cell cultures.