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Prenatal Exposure to DEHP Affects Spermatogenesis and Sperm DNA Methylation in a Strain-Dependent Manner.

Prados J, Stenz L, Somm E, Stouder C, Dayer A, Paoloni-Giacobino A - PLoS ONE (2015)

Bottom Line: Di-(2-ethylhexyl)phtalate (DEHP) is a plasticizer with endocrine disrupting properties found ubiquitously in the environment and altering reproduction in rodents.The number of differentially methylated regions (DMRs) by DEHP-exposure across the entire genome showed increased hyper- and decreased hypo-methylation in C57BL/6J compared to FVB/N.In contrast, a large set of micro-RNAs were hypo-methylated, with a trend more pronounced in the FVB/N strain.

View Article: PubMed Central - PubMed

Affiliation: Department of Mental Health and Psychiatry, Division of Psychiatric Specialties, University Hospitals of Geneva, Geneva, Switzerland; Department of Microbiology and Molecular Medicine, University of Geneva, Geneva, Switzerland.

ABSTRACT
Di-(2-ethylhexyl)phtalate (DEHP) is a plasticizer with endocrine disrupting properties found ubiquitously in the environment and altering reproduction in rodents. Here we investigated the impact of prenatal exposure to DEHP on spermatogenesis and DNA sperm methylation in two distinct, selected, and sequenced mice strains. FVB/N and C57BL/6J mice were orally exposed to 300 mg/kg/day of DEHP from gestation day 9 to 19. Prenatal DEHP exposure significantly decreased spermatogenesis in C57BL/6J (fold-change = 0.6, p-value = 8.7*10-4), but not in FVB/N (fold-change = 1, p-value = 0.9). The number of differentially methylated regions (DMRs) by DEHP-exposure across the entire genome showed increased hyper- and decreased hypo-methylation in C57BL/6J compared to FVB/N. At the promoter level, three important subsets of genes were massively affected. Promoters of vomeronasal and olfactory receptors coding genes globally followed the same trend, more pronounced in the C57BL/6J strain, of being hyper-methylated in DEHP related conditions. In contrast, a large set of micro-RNAs were hypo-methylated, with a trend more pronounced in the FVB/N strain. We additionally analyze both the presence of functional genetic variations within genes that were associated with the detected DMRs and that could be involved in spermatogenesis, and DMRs related with the DEHP exposure that affected both strains in an opposite manner. The major finding in this study indicates that prenatal exposure to DEHP can decrease spermatogenesis in a strain-dependent manner and affects sperm DNA methylation in promoters of large sets of genes putatively involved in both sperm chemotaxis and post-transcriptional regulatory mechanisms.

No MeSH data available.


Related in: MedlinePlus

Confirmatory bisulfite pyrosequencing experiment in Tmem125 & Smim8.(A, B, C) Images related with Tmem125. (D, E, F) Images related with Smim8. (A, D) Full scan derived from MBDseq data reflecting methylation levels in the entire genes and with the promoter regions as defined in the manuscript for the four tested conditions. The densities of CpG throughout the entire gene are shown as well as SNP between strains. The legend is located at the bottom of Fig 5 and the colors encoding the four conditions are respected all over the Fig 5. (B, E) Promoter methylation according to MBDseq experiments probing 2.2 kb promoters delimited and localized with vertical lines on the full scan in (A) and (D). (C, F) Promoter methylation according to pyrosequencing experiments probing the regions defined by dashed line on the full scan shown in (A) and (D) with an arrow indicating the sense of the pyrosequencing experiment. P-values resulting from T-Tests performed between conditions smaller than 0.05 and coordinates of the probed regions according to the mm10 mouse genome are shown (B, E, C, F).
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pone.0132136.g005: Confirmatory bisulfite pyrosequencing experiment in Tmem125 & Smim8.(A, B, C) Images related with Tmem125. (D, E, F) Images related with Smim8. (A, D) Full scan derived from MBDseq data reflecting methylation levels in the entire genes and with the promoter regions as defined in the manuscript for the four tested conditions. The densities of CpG throughout the entire gene are shown as well as SNP between strains. The legend is located at the bottom of Fig 5 and the colors encoding the four conditions are respected all over the Fig 5. (B, E) Promoter methylation according to MBDseq experiments probing 2.2 kb promoters delimited and localized with vertical lines on the full scan in (A) and (D). (C, F) Promoter methylation according to pyrosequencing experiments probing the regions defined by dashed line on the full scan shown in (A) and (D) with an arrow indicating the sense of the pyrosequencing experiment. P-values resulting from T-Tests performed between conditions smaller than 0.05 and coordinates of the probed regions according to the mm10 mouse genome are shown (B, E, C, F).

Mentions: Result of the confirmatory pyrosequencing experiments that were performed for the two unknown targets Tmem125 and Smim8 in order to validate the opposite promoter methylation impacts between strains upon DEHP exposure is summarized on (Fig 5). Pyrosequencing derived methylation percentages obtained for CpG1 in Tmem125 promoter were statistically significantly higher (p = 8*10−3) in C57BL/6J controls (69±4%) compared to FVB/N controls (58±7%), (Fig 5C), which is consistent with MBDseq results in C57BL/6J controls (19.8±3.9) compared to FVB/N controls (7.6±2.5), although that time not statistically significant (p = 0.11), (Fig 5B). Inversely, a statistically highly significant DEHP-induced decreased reads numbers (p = 9*10−15) was observed in Tmem125 promoter region in MBDseq experiment in C57BL/6J controls (19.8±3.9) compared with DEHP300 (0.9±3.9%), (Fig 5B), consistent with a DEHP-induced decreased methylation levels recorded in pyrosequencing experiment in CpG1 although not statistically significant (p = 0.7) in C57BL/6J controls (69±4%) compared to DEHP300 (65±10%), (Fig 5C). Note that in the Tmem125 assay probing 6 sites, we do not consider CpG2–6, because they go away form the MBDseq pic detected (Fig 5A) and because pyrosequencing is prone to inaccuracy in distant site. In Smim8, a statistically significantly lower methylation level was recorded by pyrosequencing experiment in CpG1 (p = 3*10−2) in C57BL/6J controls (73±8%) when compared with FVB/N controls (83±5%), Fig 5F, which is consistent with an also statistically significant lower average of reads (p = 2*10−3) that was observed in C57BL/6J controls (0±0) when compared with FVB/N controls (1.18±0.8), Fig 5E. Inversely, both statistically significant and opposite impacts between strains of the DEHP treatments on methylation levels observed in MBDseq experiments (Fig 5E) are concordant with same trends although not statistically significant observed in pyrosequencing experiments (Fig 5F). Indeed, MBDseq resulted in an increased reads averages recorded in DEHP 300 (1.8±1) compared with those recorded in controls (0±0) in C57BL/6J background (p = 6.4*10−5), and a decrease reads in DEHP 300 (0±0) compared with controls (1.1±0.8) in the other FVB/N background (p = 3*10−3), (Fig 5E), consistent with pyrosequencing derived increased methylation levels recorded in DEHP300 condition (78±9%) compared with controls (73±8%) in C57BL/6J background and a decrease methylation level in DEHP 300 (77±7%) compared with controls (83±5%) in FVB/N background, (Fig 5F).


Prenatal Exposure to DEHP Affects Spermatogenesis and Sperm DNA Methylation in a Strain-Dependent Manner.

Prados J, Stenz L, Somm E, Stouder C, Dayer A, Paoloni-Giacobino A - PLoS ONE (2015)

Confirmatory bisulfite pyrosequencing experiment in Tmem125 & Smim8.(A, B, C) Images related with Tmem125. (D, E, F) Images related with Smim8. (A, D) Full scan derived from MBDseq data reflecting methylation levels in the entire genes and with the promoter regions as defined in the manuscript for the four tested conditions. The densities of CpG throughout the entire gene are shown as well as SNP between strains. The legend is located at the bottom of Fig 5 and the colors encoding the four conditions are respected all over the Fig 5. (B, E) Promoter methylation according to MBDseq experiments probing 2.2 kb promoters delimited and localized with vertical lines on the full scan in (A) and (D). (C, F) Promoter methylation according to pyrosequencing experiments probing the regions defined by dashed line on the full scan shown in (A) and (D) with an arrow indicating the sense of the pyrosequencing experiment. P-values resulting from T-Tests performed between conditions smaller than 0.05 and coordinates of the probed regions according to the mm10 mouse genome are shown (B, E, C, F).
© Copyright Policy
Related In: Results  -  Collection

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pone.0132136.g005: Confirmatory bisulfite pyrosequencing experiment in Tmem125 & Smim8.(A, B, C) Images related with Tmem125. (D, E, F) Images related with Smim8. (A, D) Full scan derived from MBDseq data reflecting methylation levels in the entire genes and with the promoter regions as defined in the manuscript for the four tested conditions. The densities of CpG throughout the entire gene are shown as well as SNP between strains. The legend is located at the bottom of Fig 5 and the colors encoding the four conditions are respected all over the Fig 5. (B, E) Promoter methylation according to MBDseq experiments probing 2.2 kb promoters delimited and localized with vertical lines on the full scan in (A) and (D). (C, F) Promoter methylation according to pyrosequencing experiments probing the regions defined by dashed line on the full scan shown in (A) and (D) with an arrow indicating the sense of the pyrosequencing experiment. P-values resulting from T-Tests performed between conditions smaller than 0.05 and coordinates of the probed regions according to the mm10 mouse genome are shown (B, E, C, F).
Mentions: Result of the confirmatory pyrosequencing experiments that were performed for the two unknown targets Tmem125 and Smim8 in order to validate the opposite promoter methylation impacts between strains upon DEHP exposure is summarized on (Fig 5). Pyrosequencing derived methylation percentages obtained for CpG1 in Tmem125 promoter were statistically significantly higher (p = 8*10−3) in C57BL/6J controls (69±4%) compared to FVB/N controls (58±7%), (Fig 5C), which is consistent with MBDseq results in C57BL/6J controls (19.8±3.9) compared to FVB/N controls (7.6±2.5), although that time not statistically significant (p = 0.11), (Fig 5B). Inversely, a statistically highly significant DEHP-induced decreased reads numbers (p = 9*10−15) was observed in Tmem125 promoter region in MBDseq experiment in C57BL/6J controls (19.8±3.9) compared with DEHP300 (0.9±3.9%), (Fig 5B), consistent with a DEHP-induced decreased methylation levels recorded in pyrosequencing experiment in CpG1 although not statistically significant (p = 0.7) in C57BL/6J controls (69±4%) compared to DEHP300 (65±10%), (Fig 5C). Note that in the Tmem125 assay probing 6 sites, we do not consider CpG2–6, because they go away form the MBDseq pic detected (Fig 5A) and because pyrosequencing is prone to inaccuracy in distant site. In Smim8, a statistically significantly lower methylation level was recorded by pyrosequencing experiment in CpG1 (p = 3*10−2) in C57BL/6J controls (73±8%) when compared with FVB/N controls (83±5%), Fig 5F, which is consistent with an also statistically significant lower average of reads (p = 2*10−3) that was observed in C57BL/6J controls (0±0) when compared with FVB/N controls (1.18±0.8), Fig 5E. Inversely, both statistically significant and opposite impacts between strains of the DEHP treatments on methylation levels observed in MBDseq experiments (Fig 5E) are concordant with same trends although not statistically significant observed in pyrosequencing experiments (Fig 5F). Indeed, MBDseq resulted in an increased reads averages recorded in DEHP 300 (1.8±1) compared with those recorded in controls (0±0) in C57BL/6J background (p = 6.4*10−5), and a decrease reads in DEHP 300 (0±0) compared with controls (1.1±0.8) in the other FVB/N background (p = 3*10−3), (Fig 5E), consistent with pyrosequencing derived increased methylation levels recorded in DEHP300 condition (78±9%) compared with controls (73±8%) in C57BL/6J background and a decrease methylation level in DEHP 300 (77±7%) compared with controls (83±5%) in FVB/N background, (Fig 5F).

Bottom Line: Di-(2-ethylhexyl)phtalate (DEHP) is a plasticizer with endocrine disrupting properties found ubiquitously in the environment and altering reproduction in rodents.The number of differentially methylated regions (DMRs) by DEHP-exposure across the entire genome showed increased hyper- and decreased hypo-methylation in C57BL/6J compared to FVB/N.In contrast, a large set of micro-RNAs were hypo-methylated, with a trend more pronounced in the FVB/N strain.

View Article: PubMed Central - PubMed

Affiliation: Department of Mental Health and Psychiatry, Division of Psychiatric Specialties, University Hospitals of Geneva, Geneva, Switzerland; Department of Microbiology and Molecular Medicine, University of Geneva, Geneva, Switzerland.

ABSTRACT
Di-(2-ethylhexyl)phtalate (DEHP) is a plasticizer with endocrine disrupting properties found ubiquitously in the environment and altering reproduction in rodents. Here we investigated the impact of prenatal exposure to DEHP on spermatogenesis and DNA sperm methylation in two distinct, selected, and sequenced mice strains. FVB/N and C57BL/6J mice were orally exposed to 300 mg/kg/day of DEHP from gestation day 9 to 19. Prenatal DEHP exposure significantly decreased spermatogenesis in C57BL/6J (fold-change = 0.6, p-value = 8.7*10-4), but not in FVB/N (fold-change = 1, p-value = 0.9). The number of differentially methylated regions (DMRs) by DEHP-exposure across the entire genome showed increased hyper- and decreased hypo-methylation in C57BL/6J compared to FVB/N. At the promoter level, three important subsets of genes were massively affected. Promoters of vomeronasal and olfactory receptors coding genes globally followed the same trend, more pronounced in the C57BL/6J strain, of being hyper-methylated in DEHP related conditions. In contrast, a large set of micro-RNAs were hypo-methylated, with a trend more pronounced in the FVB/N strain. We additionally analyze both the presence of functional genetic variations within genes that were associated with the detected DMRs and that could be involved in spermatogenesis, and DMRs related with the DEHP exposure that affected both strains in an opposite manner. The major finding in this study indicates that prenatal exposure to DEHP can decrease spermatogenesis in a strain-dependent manner and affects sperm DNA methylation in promoters of large sets of genes putatively involved in both sperm chemotaxis and post-transcriptional regulatory mechanisms.

No MeSH data available.


Related in: MedlinePlus