Differential roles of Stella in the modulation of DNA methylation during oocyte and zygotic development

a Diagram illustrating the BS-seq procedure for genome-wide methylation analysis. Individual parental pronuclei and ovulated oocytes were collected, and DNA was bisulfite converted, followed by library preparation and high-throughput sequencing. b Distribution of the average methylation level across 20-kb windows in oocyte, female pronucleus (♀PN), male pronucleus(♂PN) from StellaΔ and WT mice. Boxplot illustrates the median (red bar), mean (green cross), 25/75 percentage range (box), maximum and minimum (whiskers), and extreme values (red dots outside box). c Density plot of the average methylation level across 20-kb windows in oocyte, ♀PN, ♂PN from StellaΔ and WT mice. d–g Violin plots show the methylation levels for four genomic features in oocytes from StellaΔ and WT mice. The green cross indicates the mean methylation levels. Bootstrap test was used to for statistical analysis. h Total number of DMRs identified between oocyte from StellaΔ and WT mice. The proportions of hyper- and hypo-DMRs are presented in circular plot. i PN4 zygotes from WT and StellaΔ mice were stained with anti-5mC (red) and anti-5hmC (green) antibodies. Arrows indicate the gain of 5hmC in the maternal PN of StellaΔ zygotes. PB, polar body. Scale bar, 20？μm. j Quantification of 5hmC fluorescence intensity in female pronuclei of zygotes. Each data point represents one maternal PN in zygotes (n？=？10 for each group). A Student’s t-test was used for statistical analysis. k Scatter plot illustrates the difference in DNA methylation level across 20-kb windows in the groups as indicated. X-axis represents the difference in DNA methylation level between oocyte and ♀PN from StellaΔ mice, and Y-axis represents the difference in DNA methylation level between oocyte and ♀PN from WT mice. The red dots denote the 20-kb windows with large difference (<5%) in DNA methylation level in StellaΔ group compared with WT group. Green line X-Y=26.31 indicates the threshold corresponding to 5%. l Density plot of the difference in average DNA methylation level across 20-kb windows between oocyte and ♀PN from StellaΔ and WT mice. m Screenshot of Csmd1 gene, as an example of a gene whose methylation is protected by Stella following fertilizatio