Extended Data Fig. 5: Reduced Tet3 expression in mouse oocytes and zygotes, and impaired 5mC oxidation in the zygotic DNA of one-cell embryos from HG females.
From: Maternal inheritance of glucose intolerance via oocyte TET3 insufficiency
a, Principal component analysis (PCA) of the transcriptional profile of MII oocytes identifies a clear separation between control and HG group. b, Scatter plot shows the differentially expressed genes (DEGs) of HG MII oocytes compared to controls. nCount stands for normalized counts (mean of the counts divided by size factors; DESeq2 software package). The dashed lines indicate the 1.5-fold change threshold for identifying DEGs. Red and blue dots denote significantly changed genes (padj < 0.05 and fold change ≥ 1.5) and grey dots denote not significantly changed genes. c, Gene ontology analysis of the downregulated genes in HG MII oocytes from RNA-seq. The red vertical line corresponds to P = 0.05. d, Expression of DNA-modifying enzymes in MII oocytes determined by RNA-seq. Tet3 expression is shown in Fig. 2b. e, Validation of the relative gene expression levels in MII oocytes from HG females by RT-qPCR. Gene expression shown in the left panel is the qPCR result of abundantly expressed genes encoding DNA-modifying enzymes in 10 pooled oocytes using the method of Smart-seq2 pre-amplification. Ctrl and HG: n = 8 and 9 replicates, respectively. The right panel is the confirmation of relative Tet3 expression in 60 pooled oocytes independently isolated using direct RT-qPCR relative to 18S rRNA. f, The HFD-fed and low-dose STZ‑induced type 2 diabetes mellitus (T2DM) mice of 16-week-old showed obesity and increased blood glucose when oocytes were collected. Ctrl, T2DM: n = 14 and 9 mice, respectively. g, Decreased Tet3 mRNA expression in GV oocytes from 16-week-old T2DM mice versus control by RT-qPCR. Ctrl, T2DM: n = 14 and 9 mice (with 5 oocytes pooled per mouse), respectively. h–k, Representative images of anti-TET3 (magenta) and DAPI (blue) staining in mouse zygotes at PN3–4 stage (h) and the quantification of relative fluorescence intensity levels (j). Ctrl, HG: n = 8 and 10, respectively. Representative images of anti-5hmC (red) and anti-5mC (green) staining in mouse zygotes at PN3–4 stage (i) and the quantification of relative fluorescence intensity levels of 5hmC and 5mC (k). Ctrl, HG: n = 15 and 16, respectively. Male and female pronuclei are indicated by their respective sex symbols. PB, polar body. PN, pronucleus. Scale bars, 10 μm. Each data point is based on the level of the TET3 signal relative to the DAPI staining intensity (j) or 5hmC relative to 5mC (k) in the same pronucleus. b–c, P values were calculated by Wald test corrected for multiple testing using the Benjamini and Hochberg method by default (b) and Fisher's Exact test (c). d–g, j–k, Data are presented as the mean ± s.e.m.; two-tailed P values were calculated by the unpaired Student’s t-test. *P < 0.05, **P < 0.01 and ***P < 0.001. Statistical details are in Supplementary Table 5.
