Abstract

Maternal obesity can impair embryo development and offspring health, yet the mechanisms responsible remain poorly understood. In a high-fat diet (HFD)-based female mouse model of obesity, we identified a marked reduction of Stella (also known as DPPA3 or PGC7) protein in oocytes. Starting with this clue, we found that the establishment of pronuclear epigenetic asymmetry in zygotes from obese mice was severely disrupted, inducing the accumulation of maternal 5-hydroxymethylcytosine modifications and DNA lesions. Furthermore, methylome-wide sequencing analysis detected global hypomethylation across the zygote genome in HFD-fed mice, with a specific enrichment in transposon elements and unique regions. Notably, overexpression of Stella in the oocytes of HFD-fed mice not only restored the epigenetic remodeling in zygotes but also partly ameliorated the maternal-obesity-associated developmental defects in early embryos and fetal growth. Thus, Stella insufficiency in oocytes may represent a critical mechanism that mediates the phenotypic effects of maternal obesity in embryos and offspring.

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Change history

  • Correction 27 February 2018

    In the version of this article originally published, the positions of Wenjie Shu and Qiang Wang in the author list were reversed and incorrect images were displayed in the HTML for Supplementary Figs. 1–12. The errors have been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We are grateful to T. Schedl (Washington University in St. Louis School of Medicine) for a valuable discussion that led to the experiment design reported in this paper and his critical reading of the manuscript. We also thank Q.-Y. Sun and Z.-B. Wang (Chinese Academy of Sciences) for providing plasmids and Y.-S. Guo (Nanjing Medical University) for help with the proteomic assay. This work was supported by National Key Scientific Research Projects of China (2014CB943200 to Q.W.), the National Key Research and Development Program of China (2017YFC1001500 to Q.W.), the National Natural Science Foundation of China (no. 31571543 to Q.W.), a Major Research Plan of the National Natural Science Foundation of China (no. U1435222 to W.S.), a Major Research Plan of the National Key R&D Program of China (no. 2016YFC0901600 to W.S.), and the National High Technology Research and Development Program of China (no. 2015AA020108 to W.S.).

Author information

Author notes

  1. These authors contributed equally: Longsen Han, Chao Ren and Ling Li.

  2. These authors jointly directed this work: Qiang Wang and Wenjie Shu.

Affiliations

  1. State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, China

    • Longsen Han
    • , Ling Li
    • , Juan Ge
    • , Haichao Wang
    • , Xuejiang Guo
    •  & Qiang Wang
  2. Department of Biotechnology, Beijing Institute of Radiation Medicine, Beijing, China

    • Chao Ren
    •  & Wenjie Shu
  3. College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, China

    • Xiaoyan Li
  4. Institute of Stem Cell and Regenerative Biology, College of Animal Science and Technology & College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China

    • Yi-Liang Miao
  5. Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, MO, USA

    • Kelle H. Moley

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Contributions

Q.W., W.S. and K.H.M. conceived the project; X.G. contributed to the proteomic experiment; L.H., L.L., X.L. and H.W. performed the experiments on embryo staining and methylation validation; C.R. and W.S. contributed to the sequencing and bioinformatics analysis; L.H., L.L., J.G. and Y.M. performed IVF, microinjection and embryo transfer experiments; and Q.W. and W.S. wrote and K.H.M. revised the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Wenjie Shu or Qiang Wang.

Integrated supplementary information

  1. Supplementary Figure 1 High-fat diet leads to obesity, glucose intolerance and insulin resistance in female mice.

    a, Female mice receiving a normal diet (ND) or high-fat diet (HFD) for 16 weeks. bf, Mice were assessed for body fat (b), body weight (c), blood glucose (d) and serum insulin (e) during GTT, and blood glucose during ITT (f) (n = 10 for ND mice; n = 12 for HFD mice). Data are expressed as mean ± s.d. Student’s t test (two-tailed) was used for statistical analysis.

  2. Supplementary Figure 2 Effects of maternal HFD on ovulation and litter size in mice.

    a, Maternal HFD did not significantly affect the average number of ovulated eggs in mice, although there was a slight reduction (n = 8 for ND mice; n = 8 for HFD mice). b, Average litter size was significantly reduced in HFD mice compared to ND mice when these mice were mated with normal males (n = 8 for ND mice; n = 8 for HFD mice). Student’s t test (two-tailed) was used for statistical analysis. Error bars, s.d.; center values, mean; n.s., not significant.

  3. Supplementary Figure 3 Female ob/ob mice exhibit obesity, glucose intolerance and insulin resistance.

    a, Eight-week-old female wild-type (WT) and ob/ob mice (n = 8 for WT mice; n = 10 for ob/ob mice). be, Mice were assessed for body fat (b), body weight (c), and blood glucose (d) and serum insulin (e) during GTT. Student’s t test (two-tailed) was used for statistical analysis. Error bars, s.d.; center values, mean; n.s., not significant.

  4. Supplementary Figure 4 Maternal obesity does not significantly affect Stella mRNA abundance in oocytes.

    Relative quantification of Stella mRNA levels in MII oocytes from ND and HFD mice or WT and ob/ob mice was conducted by real-time RT–PCR. Data are expressed as means ± s.d. from three independent experiments. Student’s t test (two-tailed) was used for statistical analysis. Center values, mean; n.s., not significant.

  5. Supplementary Figure 5 Rapid loss of 5mC in the maternal genome of zygotes from ob/ob mice.

    a, Representative images of zygotes from wild-type (WT) and ob/ob mice stained with anti-5mC (red) and anti-5hmC (green) antibodies. ♂ and ♀ indicate the paternal pronucleus and maternal pronucleus, respectively. Arrowheads indicate 5mC loss and 5hmC accumulation in the maternal pronuclei of ob/ob zygotes. PB, polar body. Scale bars, 25 μm. b,c, Quantification of the relative levels of 5mC and 5hmC in both pronuclei of zygotes. Results are presented as signal intensity in paternal and maternal pronuclei (left axis) or a ratio of paternal over maternal signal (right axis). Each data point represents a zygote (n = 12 biologically independent zygotes for each group). Student’s t test (two-tailed) was used for statistical analysis. Error bars, s.d.; center values, mean; n.s., not significant.

  6. Supplementary Figure 6 Maternal HFD has no effects on the intensity of 5mC staining in mouse oocytes.

    a, Representative images of MII oocytes from ND and HFD mice stained with anti-5mC antibody (5mC, green). DNA was counterstained with propidium iodide (red). PB, polar body. Scale bars, 25 μm. b, Quantification of the relative levels of 5mC in oocytes. Each data point represents an oocyte (n = 14 biologically independent oocytes for each group). Student’s t test (two-tailed) was used for statistical analysis. Error bars, s.d.; center values, mean; n.s., not significant.

  7. Supplementary Figure 7 Methylation status of histone H3K9me2 in oocytes and zygotes from ND and HFD mice.

    a, Representative images of MII oocytes from ND and HFD mice stained with H3K9me2 antibody (H3K9me2; green). DNA was counterstained with propidium iodide (red). b, Representative images of zygotes from ND and HFD mice stained with H3K9me2 antibody (H3K9me2, green; DNA, red). ♂ and ♀ indicate the paternal pronucleus and maternal pronucleus, respectively. PB, polar body. Scale bars, 25 μm. c,d, Quantification of the H3K9me2 fluorescence in MII oocytes (n = 12 biologically independent oocytes for each group) and the maternal pronuclei of zygotes (n = 12 biologically independent zygotes for each group), with the ND values set to one. Student’s t test (two-tailed) was used for statistical analysis. Error bars, s.d.; center values, mean; n.s., not significant.

  8. Supplementary Figure 8 TET3 expression in oocytes from ND and HFD mice.

    a, The relative levels of Tet3 mRNA in MII oocytes obtained from ND and HFD mice were determined by real-time RT–PCR, with Gapdh as an internal control. Data are expressed as the means ± s.d. from three independent experiments. Student’s t test (two-tailed) was used for statistical analysis. Center values, mean; n.s., not significant. b, TET3 protein levels in MII oocytes from ND and HFD mice were evaluated by western blot, with actin as a loading control. The experiments were repeated three times independently with similar results. Band intensity was calculated using ImageJ software, and the ratio of TET3/actin expression was normalized and values are indicated.

  9. Supplementary Figure 9 Abnormal γ-H2AX accumulation in the maternal pronuclei of zygotes from HFD mice.

    a, Representative images of γ-H2AX (green) and nuclear (red) staining on ND zygotes, HFD zygotes, HFD zygotes injected with PBS or Stella mRNA. ♂ and ♀ indicate the paternal pronucleus and maternal pronucleus, respectively. Arrows indicate the apparent γ-H2AX accumulation in the maternal pronuclei of HFD zygotes. PB, polar body. Scale bars, 25 μm. b, Quantification of the numbers of γ-H2AX foci in maternal and paternal pronuclei. Each data point represents a zygote (n = 11 for ND, n = 10 for HFD, n = 11 for HFD + PBS, and n = 12 for HFD + Stella, biologically independent zygotes). Statistical analyses were performed with one-way ANOVA with Tukey’s post hoc test. Adjustments were made for multiple comparisons. Error bars, s.d.; center values, mean; n.s., not significant.

  10. Supplementary Figure 10 Knockdown of endogenous TET3 protein in MII oocytes.

    Specifically designed siRNAs for TET3 were microinjected into MII oocytes. Samples (100 oocytes per lane) were processed for western blot analysis; actin was served as a loading control. The experiments were repeated three times independently with similar results. Band intensity was calculated using ImageJ software, and the ratio of TET3/actin expression was normalized and values are indicated. Images have been cropped; please compare to Supplementary Data.

  11. Supplementary Figure 11 TET3-dependent 5hmc and γ-H2AX accumulation in HFD zygotes contributes to early embryonic developmental defects.

    a, Representative images of 5mC (red) and 5hmC (green) staining on ND zygotes, ND + DMOG zygotes, HFD zygotes and HFD + DMOG zygotes. b,c, Quantification of the levels of 5hmC and 5mC signal in the paternal and maternal pronuclei of zygote (n = 10 biologically independent zygotes for each group). d, Representative images of γ-H2AX (green) and nuclear (red) staining on ND zygotes, ND + DMOG zygotes, HFD zygotes and HFD + DMOG zygotes. e, Quantification of the numbers of γ-H2AX foci in maternal and paternal pronuclei. ♂ and ♀ indicate the paternal and maternal pronucleus, respectively. PB, polar body. Scale bars, 25 μm. Each data point represents a zygote (n = 12 biologically independent zygotes for each group). f, The percentage of zygotes that successfully progressed to the blastocyst stage during in vitro culture. Data are presented as the means ± s.d. from three independent experiments (total number of embryos analyzed: n = 90 for ND, n = 112 for ND + DMOG, n = 95 for HFD and n = 92 for HFD + DMOG). In e and f, statistical analyses were performed with one-way ANOVA with Tukey’s post hoc test. Adjustments were made for multiple comparisons. In all other panels, Student’s t test (two-tailed) was used for statistical analysis. Error bars, s.d.; center values, mean; n.s., not significant.

  12. Supplementary Figure 12 Global DNA hypomethylation across different genomic features in zygotes from HFD mice.

    Violin plots showing the methylation levels of the major repetitive elements in zygotes from ND and HFD mice. Mean methylation levels are indicated by the numerical value and green cross. Statistical analyses were performed by bootstrap test.

  13. Supplementary Figure 13 Heat map analysis of gene promoters differentially methylated between ND and HFD zygotes.

    DNA methylation level is colored from yellow to red to indicate low to high. The right panels show the enriched GO terms of biological processes.

  14. Supplementary Figure 14 Overexpression of exogenous Stella protein in MII oocytes.

    PBS (control group) or exogenous Myc-Stella mRNA (overexpression group) was microinjected into MII oocytes. Oocyte samples were processed for western blot analysis (100 oocytes per lane), probing with anti-Myc and anti-Stella antibody, respectively. Tubulin served as a loading control. Images have been cropped; please compare to Supplementary Data. The experiments were repeated three times independently with similar results.

  15. Supplementary Figure 15 Effects of Stella overexpression on the methylation status of zygotes in HFD mice.

    a,c,e,g, Graphical representation of the methylation pattern at the example loci of RLTR10-int, Gab1, Gstp1 and Park2 in ND (top, blue) and HFD (bottom, red) zygotes. The regions highlighted by green boxes were chosen for further validation. b,d,f,h, Bisulfite sequencing for verifying the methylation status of RLTR10-int, Gab1, Gstp1 and Park2 in ND zygotes, HFD zygotes, and HFD zygotes injected with PBS or Stella mRNA. Open and filled circles represent unmethylated and methylated CpGs, respectively. The percentages of methylated CpGs are shown below each panel. The experiments were repeated three times independently with similar results.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–15 and Supplementary Tables 1–6

  2. Life Sciences Reporting Summary

  3. Supplementary Data

    Uncropped western blot images

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https://doi.org/10.1038/s41588-018-0055-6