Epi-mutations for spermatogenic defects by maternal exposure to di(2-ethylhexyl) phthalate
- Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University, Japan
- Graduate School of Life Sciences, Tohoku University, Japan
- Department of Bioscience, Faculty of Life Sciences, Tokyo University of Agriculture, Japan
- Department of Informative Genetics, Environment and Genome Research Center, Graduate School of Medicine, Tohoku University, Japan
- Graduate School of Medicine, Tohoku University, Japan
Figures
Figure 1 with 1 supplement
Histological analysis of testicular tubules of C57BL/6 offspring after prenatal di(2-ethylhexyl) phthalate (DEHP) exposure.
(A, B) Representative images of testicular tubules in F1 (A) and F2 (B). Abnormal testicular tubules were categorized as ‘Type A’ (presence of various size of vacuoles; indicated by arrows), ‘Type …
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Figure 1—source data 1
Numerical source data of abnormal testicular tubule rates (Figure 1C) and ratios of abnormal tubule types (Figure 1D), and data for Kolmogorov–Smirnov test for Figure 1C.
- https://cdn.elifesciences.org/articles/70322/elife-70322-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Histological analysis of the testicular tubules of prenatally di(2-ethylhexyl) phthalate (DEHP)-exposed offspring from C57BL/6 females mated with Oct4-deltaPE-GFP transgenic males.
(A, B) Representative images of testicular tubules in F1 (A) and F2 (B). Abnormal testicular tubules were categorized as ‘Type A’ (presence of various size of vacuoles; indicated by arrows) and …
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Figure 1—figure supplement 1—source data 1
Numerical source data of abnormal testicular tubule rates (Figure 1—figure supplement 1C), ratios of abnormal tubule types (Figure 1—figure supplement 1D), and multinucleated cells (Figure 1—figure supplement 1F), and data for Kolmogorov–Smirnov test for Figure 1—figure supplement 1C, F.
- https://cdn.elifesciences.org/articles/70322/elife-70322-fig1-figsupp1-data1-v1.xlsx
Figure 2
Purification of germ cells from E19.5 and adult testis.
(A) The bleeding schema of the experiments. (B) A representative FACS histogram of E19.5 testis. GFP-positive germ cells in the R3 gate (green color), and GFP-negative testicular somatic cells in …
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Figure 2—source data 1
Numerical source data for relative gene expression of Gfra1, Scp1, and Acrv1, and data for Kolmogorov–Smirnov test for Figure 2D.
SPG: spermatogonia; SPC: spermatocytes; RS: round spermatids.
- https://cdn.elifesciences.org/articles/70322/elife-70322-fig2-data1-v1.xlsx
Figure 3 with 1 supplement
Methylome of testicular cells of F1 after maternal exposure to di(2-ethylhexyl) phthalate (DEHP).
(A) Heatmap of promoter DNA methylation levels in the testicular cell populations prenatally exposed to oil as vehicle control (O) or DEHP (D) (clustering distance: Euclidean; clustering method: …
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Figure 3—source data 1
Analytical codes of reduced representation bisulfite sequencing for Figure 3.
- https://cdn.elifesciences.org/articles/70322/elife-70322-fig3-data1-v1.docx
Figure 3—figure supplement 1
Chromosomal positions of differentially promoter-methylated genes in E19.5 germ and testicular somatic cells.
Red arrowhead: more than 5% increase in methylation in the di(2-ethylhexyl) phthalate (DEHP) group compared to the oil group; blue arrowhead: more than 5% decrease in methylation in the DEHP group …
Figure 4 with 2 supplements
Gene expression and promoter methylation of spermatogenesis-related genes hypermethylated in both E19.5 germ cells and F1 spermatogonia following prenatal di(2-ethylhexyl) phthalate (DEHP) exposure.
(A) Relative mRNA levels in prenatally oil- or DEHP-treated adult F1 spermatogonia, as determined by RT-qPCR. Values of oil group are defined as 1.0. Values are plotted as mean ± SEM for samples …
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Figure 4—source data 1
Numerical source data for relative gene expression (Figure 4A) and DNA methylation status (Figure 4D), and data for Kolmogorov–Smirnov test for Figure 4A.
- https://cdn.elifesciences.org/articles/70322/elife-70322-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Transcriptome of testicular germ cells after prenatal di(2-ethylhexyl) phthalate (DEHP) exposure.
(A) A heatmap of TMM-normalized RNA-seq read count values in E19.5 and adult germ cell populations of F1 prenatally exposed to oil (O) or DEHP (D) (clustering distance: Euclidean; clustering method: …
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Figure 4—figure supplement 1—source data 1
Analytical codes of RNA-seq for Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/70322/elife-70322-fig4-figsupp1-data1-v1.docx
Figure 4—figure supplement 2
Gene expression and promoter methylation of Hist1h2ba, Sycp1, and Taf7l in F2 spermatogonia.
(A) Relative mRNA levels determined by RT-qPCR in prenatal oil- or di(2-ethylhexyl) phthalate (DEHP)-treated adult F2 spermatogonia. Values are plotted as mean ± SEM of spermatogonia samples …
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Figure 4—figure supplement 2—source data 1
Numerical source data for relative gene expression Figure 4—figure supplement 2A, D and DNA methylation status (Figure 4—figure supplement 2C), and data for Kolmogorov–Smirnov test for Figure 4—figure supplement 2A, D. SPG: spermatogonia.
- https://cdn.elifesciences.org/articles/70322/elife-70322-fig4-figsupp2-data1-v1.xlsx
Figure 5
Changes in DNA methylation of the candidate epi-mutated genes in di(2-ethylhexyl) phthalate (DEHP) (red) and oil (blue)-treated E19.5 and adult F1 germ cell populations.
(A–C) Values of promoter methylation of each replicate (reduced representation bisulfite sequencing [RRBS], n = 2) of Hist1h2ba (A), Sycp1 (B), and Taf7l (C) are plotted by open red or blue circles, …
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Figure 5—source data 1
Numerical source data of DNA methylation level for Figure 5A–C.
SPG: spermatogonia; SPC: spermatocytes; RS: round spermatids.
- https://cdn.elifesciences.org/articles/70322/elife-70322-fig5-data1-v1.xlsx
Figure 6
The effects on gene expression of promoter methylation in the candidate epi-mutated loci.
(A) Gene structures of Hist1h2ba, Sycp1, and Taf7l. Exons are indicated by blue bars, and the promoter regions (defined as ± 500 bp from transcription start site) used for firefly luciferase (Luc) …
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Figure 6—source data 1
Numerical source data for relative luciferase activity and data for Kolmogorov–Smirnov test for Figure 6B–D.
- https://cdn.elifesciences.org/articles/70322/elife-70322-fig6-data1-v1.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Mus musculus, males) | Oct4-delta PE-GFP | PMID:10646797 | C57BL/6J background | |
| Cell line (Human) | HEK293T | ATCC RRID:CVCL_0063 | CRL-3216 | |
| Antibody | Anti-Cleaved Caspase-3 (rabbit polyclonal) | CST RRID:AB_2341188 | Cat# 9661 | (1:400) |
| Recombinant DNA reagent | pCpGL (plasmid) | PMID:17965610 | http://www.ag-rehli.de/materials.html | |
| Commercial assay or kit | EZ DNA Methylation-Gold Kit | Zymo Research | Cat# D5005 | |
| Commercial assay or kit | NEBNext Ultra II RNA Library Prep Kit for Illumina | BioLabs | Cat#E7770S | |
| Commercial assay or kit | Dual-Luciferase Reporter Assay System | Promega | Cat#E1910 | |
| Chemical compound, drug | DEHP | Sigma | Cat#80030 | |
| Chemical compound, drug | Corn oil | Sigma | Cat#C8267 | |
| Software, algorithm | Bismark | PMID:21493656 | v0.10.1 | https://www.bioinformatics.babraham.ac.uk/projects/bismark/ |
| Software, algorithm | TopHat2 | PMID:23618408 | V2.1.0 | https://ccb.jhu.edu/software/tophat/index.shtml |
| Software, algorithm | Bowtie2 | PMID:22388286 | V2.2.6.0 | http://bowtie-bio.sourceforge.net/index.shtml |
| Software, algorithm | featureCounts | PMID:24227677 | Packaged in Subread v1.5.0-p2 | http://subread.sourceforge.net/ |
| Software, algorithm | edgeR | PMID:19910308 | V3.13 | https://bioconductor.org/packages/release/bioc/html/edgeR.html |
Additional files
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Supplementary file 1
Methylation levels (%) of various genomic regions.
- https://cdn.elifesciences.org/articles/70322/elife-70322-supp1-v1.xlsx
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Supplementary file 2
List of hyper- (DEHP-oil [%] > 5) and hypo- (DEHP-oil [%] < −5) methylated genes in E19.5 testicular germ cells and somatic cells for Figure 3.
- https://cdn.elifesciences.org/articles/70322/elife-70322-supp2-v1.xlsx
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Supplementary file 3
List of hyper- (DEHP-oil [%] > 5) and hypo- (DEHP-oil [%] < −5) methylated genes in spermatogonia, spermatocyte, and round spermatid for Figure 3.
- https://cdn.elifesciences.org/articles/70322/elife-70322-supp3-v1.xlsx
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Supplementary file 4
List of down- and upregulated genes in E19.5 testicular germ cells, and spermatogonia, spermatocyte, and round spermatid in adult testis (log2-fold-change > 1 or < −1, FDR < 0.05) for Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/70322/elife-70322-supp4-v1.xlsx
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Supplementary file 5
Primers used in this study (for Figure 2, Figure 4, Figure 6, and Figure 4—figure supplement 2).
- https://cdn.elifesciences.org/articles/70322/elife-70322-supp5-v1.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/70322/elife-70322-transrepform-v1.docx
