Mercury-induced epigenetic transgenerational inheritance of abnormal neurobehavior is correlated with sperm epimutations in zebrafish

Abstract

Methylmercury (MeHg) is a ubiquitous environmental neurotoxicant, with human exposures predominantly resulting from fish consumption. Developmental exposure of zebrafish to MeHg is known to alter their neurobehavior. The current study investigated the direct exposure and transgenerational effects of MeHg, at tissue doses similar to those detected in exposed human populations, on sperm epimutations (i.e., differential DNA methylation regions [DMRs]) and neurobehavior (i.e., visual startle and spontaneous locomotion) in zebrafish, an established human health model. F0 generation embryos were exposed to MeHg (0, 1, 3, 10, 30, and 100 nM) for 24 hours ex vivo. F0 generation control and MeHg-exposed lineages were reared to adults and bred to yield the F1 generation, which was subsequently bred to the F2 generation. Direct exposure (F0 generation) and transgenerational actions (F2 generation) were then evaluated. Hyperactivity and visual deficit were observed in the unexposed descendants (F2 generation) of the MeHg-exposed lineage compared to control. An increase in F2 generation sperm epimutations was observed relative to the F0 generation. Investigation of the DMRs in the F2 generation MeHg-exposed lineage sperm revealed associated genes in the neuroactive ligand-receptor interaction and actin-cytoskeleton pathways being effected, which correlate to the observed neurobehavioral phenotypes. Developmental MeHg-induced epigenetic transgenerational inheritance of abnormal neurobehavior is correlated with sperm epimutations in F2 generation adult zebrafish. Therefore, mercury can promote the epigenetic transgenerational inheritance of disease in zebrafish, which significantly impacts its environmental health considerations in all species including humans.

Fig 1. Schematic of the experimental design presented in comparison to a human exposure scenario.

Breeding/exposure scheme and summary of analyses are presented. *In our paradigm, F0 generation zebrafish embryos are developmentally exposed to MeHg ex vivo with the intention of emulating a human maternal exposure.

Fig 2. Transgenerational phenotypes.

(A) Visual deficit and (B) hyperactivity observed in F2 generation zebrafish due to ancestral MeHg exposure (*p<0.001). (A) Analysis of variance (ANOVA) on Ranks and post-hoc analysis via Dunn’s Method revealed a statistically significant decrease in the visual startle response of the MeHg lineages compared to control (df = 5, H = 34.596, p<0.001). (B) One-way ANOVA and post-hoc Holm-Sidak analyses showed a statistically significant increase in the spontaneous locomotion in all MeHg lineages compared to control (df = 102, F = 3.498, p = 0.006). n = number of individual adult zebrafish. Solid horizontal lines represent the median, dashed horizontal lines represent the mean, the box represents the 25th and 75th percentiles, the whiskers show the 5th and 95th percentiles and outliers are represented by dots. (C) Proportion of the F2 generation lineages showing transgenerational phenotypes.

Fig 3. DMR chromosomal locations.

(A) The F0 generation sperm DMR locations on the individual chromosomes. Only multiple window DMRs at a p-value threshold of 1e-07 are shown here. (B) The F2 generation sperm DMR locations on the individual chromosomes. Only multiple window DMRs at a p-value threshold of 1e-07 are shown here. The red arrowhead identifies the location of the DMR and black box identifies the DMR cluster site.

Fig 4. DMR CpG density and length.

(A) The F0 generation sperm DMR CpG density. (B) The F0 generation sperm DMR lengths. (C) The F2 generation DMR CpG density. (D) The F2 generation DMR lengths. Only DMRs containing at least two significant sites/windows at a p-value threshold of <10⁻⁷.

Fig 5. DMR association gene categories.

(A) F0 generation and (B) F2 generation sperm DMR associated gene categories separated by gene classification for the sperm and number of DMR presented.

Fig 6. DMR associated neuroactive ligand-receptor gene pathway.

The pathway with the DMR associated genes identified (circled).