Histological and Transcriptomic Changes in Male Zebrafish Testes Due to Early Life Exposure to Low Level 2,3,7,8-Tetrachlorodibenzo-p-Dioxin

Abstract

We have shown that zebrafish (Danio rerio) are an excellent model for evaluating the link between early life stage exposure to environmental chemicals and disease in adulthood and subsequent unexposed generations. Previously, we used this model to identify transgenerational effects of dioxin (2,3,7,) on skeletal development, sex ratio, and reproductive capacity. Transgenerational inheritance of TCDD toxicity, notably decreased reproductive capacity, appears to be mediated through the male germ line. Thus, we examine testicular tissue for structural and gene expression changes using histology, microarray, and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Histological analysis revealed decreased spermatozoa with concurrent increase in spermatogonia, and decreased germinal epithelium thickness in TCDD-exposed males compared with controls. We also identified altered expression of genes associated with testis development, steroidogenesis, spermatogenesis, hormone metabolism, and xenobiotic response. Altered genes are in pathways involving lipid metabolism, molecular transport, small molecule biochemistry, cell morphology, and metabolism of vitamins and minerals. These data will inform future investigations to elucidate the mechanism of adult-onset and transgenerational infertility due to TCDD exposure in zebrafish.

Developmental Dioxin Exposure Alters the Methylome of Adult Male Zebrafish Gonads

“…The purpose of this study is to utilize 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 also interpret our findings of changes in the testicular methylome within the context of this data (Baker et al, 2016). To our knowledge, we have performed the first WGBS on zebrafish in response to TCDD exposure.…”

“…All results are derived from three independent TCDD exposure experiments performed in successive blocks. The fish in the current experiment were collected and stored from the same blocks of the previously published microarray and histology studies (Baker et al, 2013, 2016).…”

“…Our earlier research demonstrated impaired fertility in adult zebrafish in response to TCDD exposure during sexual differentiation (two separate hour-long exposures to 50 pg/mL TCDD at 3 and 7 weeks post fertilization), modeling fetal basis of adult-onset disease (Baker et al, 2013). Several reproductive outcomes (altered sex ratio, decreased egg release, and transcriptional changes in reproductive genes) persisted through two subsequent generations (Baker et al, 2014a,b, 2016; Meyer et al, 2018). 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 (F 0 –F 2 ).…”

“…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). Subsequent microarray and histologic analyses revealed differentially expressed genes involved in reproduction and defects in spermatogenesis, indicating a possible mechanism for the male-mediated decreased fertility (Baker et al, 2016; Meyer et al, 2018).…”

“…Parental exposures to environmental chemicals and other stressors, including paternal exposures, can contribute to adverse outcomes in offspring and subsequent generations (Jirtle and Skinner, 2007; Rodgers et al, 2013; Robledo et al, 2015). 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). Because the DNA methylation pattern in zebrafish is passed down paternally through the sperm (Potok et al, 2013; Jiang et al, 2014), inheritance of epimutations in the DNA methylome is a promising mechanism of transgenerational male-mediated reproductive defects resulting from TCDD exposure.…”