Abstract
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental toxicant and endocrine disrupting compound with reproductive and developmental effects in humans and model organisms, including zebrafish. Our previous microarray and histological studies found defects in spermatogenesis and fertility of zebrafish in response to acute developmental TCDD exposure. These effects are apparent following exposure during reproductive development, modeling fetal basis of adult-onset disease. Some outcomes of these previous studies (reduced fertility, changes in sex ratio, transcriptomic alterations) are also transgenerational – persisting to unexposed generations – through the male germline. We hypothesized that DNA methylation could be a possible mechanism for these reproductive effects and performed whole genome bisulfite sequencing (WGBS), which identifies whole genome DNA methylation status at the base pair level, on testes of adult zebrafish exposed to TCDD (two separate hour-long exposures to 50 pg/mL TCDD at 3 and 7 weeks post fertilization). In response to TCDD exposure, multiple genes were differentially methylated; many of which are involved in reproductive processes or epigenetic modifications, suggesting a role of DNA methylation in later-life health outcomes. Additionally, several differentially methylated genes corresponded with gene expression changes identified in TCDD-exposed zebrafish testes, indicating a potential link between DNA methylation and gene expression. Ingenuity pathway analysis of WGBS and microarray data revealed genes involved in reproductive processes and development, RNA regulation, the cell cycle, and cellular morphology and development. We conclude that site-specific changes in DNA methylation of adult zebrafish testes occur in response to acute developmental TCDD exposure.
Highlights
Manikkam et al, 2012; Warner et al, 2013)
The purpose of this study is to utilize whole genome bisulfite sequencing (WGBS) to expand the current knowledge of TCDD-induced epigenetic effects, demonstrating that sublethal TCDD exposure during zebrafish gonadal differentiation and maturation causes changes in DNA methylation at specific genome loci which persist to adulthood
As our previous work revealed transcriptomic alterations in zebrafish testes induced by TCDD exposure, we interpret our findings of changes in the testicular methylome within the context of this data (Baker et al, 2016)
Summary
Manikkam et al, 2012; Warner et al, 2013). People exposed to TCDD suffer decreased semen quality, have longer menstrual cycles, and produce more female than male offspring (i.e., altered sex ratios) (Signorini et al, 2000; Eskenazi et al, 2002; Mocarelli et al, 2008). Reduced reproductive capacity was characterized by decreases in both egg release (a product of courtship between male and female zebrafish) and percentage of fertilized eggs, which presented across three generations (F0–F2) We determined these effects were male-mediated by outcrossing control and exposed lineage fish from each generation; only males from the exposed lineage demonstrated transgenerational reproductive effects (Baker et al, 2014b). The mechanisms of adult-onset and transgenerational TCDD effects are unknown but thought to be epigenetic as studies have demonstrated changes in common epigenetic mechanisms such as DNA and histone methylation, as well as differential expression of DNA methyltransferases in response to TCDD exposure (Manikkam et al, 2012; Olsvik et al, 2014; Aluru et al, 2015; Ma et al, 2015; Baker et al, 2016; Sanabria et al, 2016). These data will provide valuable insight into the epigenetic effects of environmental endocrinedisrupting compounds as potential mechanisms of adult-onset and multigenerational disease
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