Differential methylation of pluripotency gene promoters in in vitro matured and vitrified, in vivo-matured mouse oocytes.

ABSTRACT

OBJECTIVE: To assess the methylation patterns of four pluripotency gene promoters in mouse oocytes after in vivo maturation, in vitro maturation (IVM), and vitrification followed by IVM. DESIGN: Experimental study. SETTING: Research laboratory. ANIMAL(S) Three populations of metaphase II mouse oocytes were analyzed after in vivo maturation, IVM, and vitrification followed by IVM (V-IVM). Cumulus cells and blastocyst embryos were controls. INTERVENTION(S) The CpG methylation patterns (overall and CpG specific) in the promoters of four pluripotency genes (Oct4, Nanog, Foxd3, and Sox2) were analyzed for each cell type by traditional DNA bisulfite sequencing. MAIN OUTCOME MEASURE(S) Differences for overall methylation were evaluated using the Student's t-test and for individual CpG sites by χ2 analysis. RESULT(S) Significantly lower levels of overall methylation in promoters of Oct4 (25%) and Sox2 (4.5%) were noted in V-IVM oocytes compared with in vivo-matured oocytes (62.5% and 8.5%, respectively). Cumulus cell promoters were generally hypomethylated at Nanog, Foxd3. and Sox2, but hypermethylated at Oct4. CONCLUSION(S) The methylation status of Oct4 and Sox2 promoters of V-IVM mouse oocytes are altered when compared with in vivo-matured oocytes. The biological risk and significance of these changes are unknown and this study indicates caution and that further analyses are warranted.