(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 …
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.
(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 …
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.
(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 …
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.
(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: …
Analytical codes of reduced representation bisulfite sequencing for Figure 3.
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 …
(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 …
Numerical source data for relative gene expression (Figure 4A) and DNA methylation status (Figure 4D), and data for Kolmogorov–Smirnov test for Figure 4A.
(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: …
Analytical codes of RNA-seq for Figure 4—figure supplement 1.
(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 …
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.
(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, …
Numerical source data of DNA methylation level for Figure 5A–C.
SPG: spermatogonia; SPC: spermatocytes; RS: round spermatids.
(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) …
Numerical source data for relative luciferase activity and data for Kolmogorov–Smirnov test for Figure 6B–D.
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 |
Methylation levels (%) of various genomic regions.
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.
List of hyper- (DEHP-oil [%] > 5) and hypo- (DEHP-oil [%] < −5) methylated genes in spermatogonia, spermatocyte, and round spermatid for Figure 3.
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.
Primers used in this study (for Figure 2, Figure 4, Figure 6, and Figure 4—figure supplement 2).