Developmental Dioxin Exposure Alters the Methylome of Adult Male Zebrafish Gonads

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.

TCDD-induced multi- and transgenerational changes in the methylome of male zebrafish gonads

“…Five percent methylation change was used as a cutoff based on previous ( 39–41 ). These cutoff values for significant methylation change are consistent with our previous publication examining changes in DNA methylation in the F0 generation ( 26 ) and other DNA methylation studies ( 40 , 42 ). Raw data and processed files were uploaded to the NCBI GEO database (record GSE149919).…”

“…This was not the case for both the F0 and F2 generations in which the majority of DMSs are in the intergenic region ( Fig. 2B ) ( 26 ).…”

“…We previously reported 397 DMSs in the F0 generation with 113 sites hypermethylated and 284 hypomethylated ( 26 ). No changes in global methylation levels were discovered in either generation, similar to the F0 generation findings ( 26 ). In the F1 generation, most DMSs are within the gene body, relatively equally divided between introns and exons ( Fig.…”

“…Previous work in our lab revealed significant differential methylation of specific genes in the testes of zebrafish exposed to TCDD during sexual differentiation and maturation ( 26 ). Testes were the target organ of this previous study because of earlier work in which we discovered transgenerational reproductive effects, primarily male-mediated, in TCDD-exposed fish ( 11 , 25 ).…”

Changes of DNA Methylation Pattern in Metabolic Pathways Induced by High-Carbohydrate Diet Contribute to Hyperglycemia and Fat Deposition in Grass Carp (Ctenopharyngodon idellus)

“…Approximately 9% of all total cytosines and 80% of CG were methylated. In zebrafish, about 12% of total cytosines were methylated; 86-87% of cytosines were methylated in CG context (28). The methylation patterns of hybrid tilapias (O. niloticus × Oreochromis mossambicus) were different from those of grass carp.…”