Fig. 3

Association between ovarian stimulation (OS) and DNA methylation of parthenogenetic placenta. A Flow diagram illustrating the parthenogenetic activation of oocytes, their development into blastocysts, and the subsequent transfer to pseudopregnant female mice. B, C The percentage of methylated DNA, hydroxymethylated DNA and the 5-hydroxymethylcytosine (5hmC)/5-methylcytosine (5mC) ratio were measured using an Enzyme-Linked Immunosorbent Assay (ELISA) kit (n = 4). D Cluster heat map of the 1000 most differentially methylated cytosine preceding a guanine base (CpG) sites in placenta tissue DNA samples from 4 mice. Each row represents differentially methylated CpGs (DMCs), and each column represents a sample. E DMCs distribution of OS and Control groups depicted in a volcano plot. Non-significant CpGs are shown in grey color, while hypermethylation and hypomethylation CpGs are shown in red and blue, respectively. F Top 20 KEGG enrichment results of DMCs annotated genes. G The Venn diagram analysis was used to determine the interaction of hypermethylated maternal imprinted genes, placenta-developed genes and transcription factors targeted by estrogen receptor alpha (ERα). H, I Analysis of methylation levels at CpG sites within the differentially methylated region (DMR) in the mesoderm-specific transcript homologue (Mest) promoter, evaluated by bisulfite sequencing PCR (BSP, n = 9). Each row of eight CpG sites within a group represents a single bisulfite-treated clone with methylated CpG (●) or unmethylated CpG (○). J, K Protein expression of ten-eleven translocation 2 (TET2) and MEST was determined using western blot analysis (n = 3). β-Actin was used as an internal control to quantify protein amounts. L Analysis of RNA levels for Tet2 and Mest was performed within each group (n = 4). Data are expressed as the means ± standard deviation (SD), *P < 0.05, **P < 0.01, ***P < 0.001(unpaired two-tailed t test)