Extended Data Fig. 11: Exogeneous TET3 expression in HG MII oocytes suppresses the maternal effect on DNA methylation and offspring susceptibility to metabolic impairments.
From: Maternal inheritance of glucose intolerance via oocyte TET3 insufficiency
a, Schematic diagram of TET3 rescue experiment relying on mRNA injection into HG MII oocytes. b, Representative images of 5mC (green) and 5hmC (red) staining in Mat-WT, Mat-KO and HG zygotes at PN5 stage which were derived from oocytes that were injected with H2O, wildtype Tet3 or Tet3-mut mRNA at MII stage. Male and female pronuclei are indicated by their respective sex symbols. Scale bar, 10 μm. c, BS analysis of the H19 imprinted control region in the male and female pronuclei isolated from aphidicolin-treated zygotes of PN3–4 stage. d, BS analysis of the Gck promoter region in the female pronuclei isolated from aphidicolin-treated zygotes of PN3–4 stage. c–d, Open and filled circles represent unmethylated and methylated CpG sites, respectively. e–f, Pyrosequencing analysis of the Gck promoter methylation in mouse blastocysts (e) and average methylation levels in e (f). Mat-KO + Tet3-mut, Mat-KO + Tet3: n = 6 and 7 replicates (with 5 blastocysts pooled for each replicate). g, Average methylation levels in Figure 4g. Ctrl, HG + Tet3, HG + Tet3-mut: 4, 5 and 4 replicates (with 5 blastocysts pooled for each replicate), respectively. h, Gck mRNA expression in pancreatic islets of male offspring at 1 year of age. Ctrl, HG + Tet3, HG + Tet3-mut: n = 8, 8 and 10 mice, respectively. i, Western blot analysis of GCK in pancreatic islets of offspring males at 1 year of age. Each sample was pooled islets from 2–3 mice. Gel source data are presented in Supplementary Figure 1. j–k, GTT of male offspring at 24 weeks of age (j) and the corresponding AUCs (k). Mat-KO + Tet3-mut, Mat-KO + Tet3: n = 12 and 14 mice. l, GTT of male offspring at 24 weeks of age after HFD feeding. Ctrl, HG + Tet3, HG + Tet3-mut: n = 10, 12 and 11 mice, respectively. P values for comparisons of HG + Tet3-mut and Ctrl are presented using *, HG + Tet3 and HG + Tet3-mut using †. m, The corresponding AUCs of the blood glucose level in Figure 4h and Extended Data Fig. 11l. e–m, Data are presented as the mean ± s.e.m.; two-tailed P values were calculated by the unpaired Student’s t-test (e–f, k) or one-way repeated-measurements ANOVA followed by the post hoc unpaired t-test (k), one-way repeated-measurements ANOVA followed by the post hoc unpaired t-test with HG + Tet3-mut (l) and one-way ANOVA followed by the unpaired Student’s t-test with HG + Tet3-mut (g, h, m). *P < 0.05, **P < 0.01 and ***P < 0.001; †P < 0.05. Statistical details are in Supplementary Table 5.
