Transgenerational actions of environmental compounds on reproductive disease and identification of epigenetic biomarkers of ancestral exposures

Abstract

Environmental factors during fetal development can induce a permanent epigenetic change in the germ line (sperm) that then transmits epigenetic transgenerational inheritance of adult-onset disease in the absence of any subsequent exposure. The epigenetic transgenerational actions of various environmental compounds and relevant mixtures were investigated with the use of a pesticide mixture (permethrin and insect repellant DEET), a plastic mixture (bisphenol A and phthalates), dioxin (TCDD) and a hydrocarbon mixture (jet fuel, JP8). After transient exposure of F0 gestating female rats during the period of embryonic gonadal sex determination, the subsequent F1-F3 generations were obtained in the absence of any environmental exposure. The effects on the F1, F2 and F3 generations pubertal onset and gonadal function were assessed. The plastics, dioxin and jet fuel were found to promote early-onset female puberty transgenerationally (F3 generation). Spermatogenic cell apoptosis was affected transgenerationally. Ovarian primordial follicle pool size was significantly decreased with all treatments transgenerationally. Differential DNA methylation of the F3 generation sperm promoter epigenome was examined. Differential DNA methylation regions (DMR) were identified in the sperm of all exposure lineage males and found to be consistent within a specific exposure lineage, but different between the exposures. Several genomic features of the DMR, such as low density CpG content, were identified. Exposure-specific epigenetic biomarkers were identified that may allow for the assessment of ancestral environmental exposures associated with adult onset disease.

Figure 1. Ancestral (F0 generation female) exposures to environmental compounds promote transgenerational diseases, altering onset of puberty, testicular spermatogenic function and ovarian follicular development in F3 generation rat progeny.

(A) Onset of female puberty was advanced from exposures to plastics, dioxin and jet fuel. (B) Onset of male puberty was unaffected from these exposures. (C) Increased apoptotic spermatogenic cells per testis section were observed from jet fuel exposure. (D) Total numbers of ovarian follicles per section were reduced in individuals from all exposures, (E) Total numbers of primordial follicles per section declined. (F) Total numbers of large ovarian antral follicles were unaffected. The animal n value is presented in Table S1C (*p<0.05; **p<0.01, ***p<0.001).

Figure 2. The transgenerational DMR associated with each exposure group identified.

(A) Venn diagram of exposure DMR lists of F3 generation rat genes with differential DNA methylation due to in vivo exposure of F0-generation gestating female with Dioxin, Pesticide, Plastics or Hydrocarbons/Jet fuel. (B) Chromosomal location of each exposure group DMR are indicated with red arrow (plastics), green arrow (dioxin), blue arrow (hydrocarbon) and black arrow (pesticide). The chromosome number and size are indicated. The box below the line indicates DMR cluster in 2–5 megabase regions with statistical significance (p<0.05).

Figure 3. Direct connection gene sub-network for combined genes with transgenerational DMR associated exposures for Dioxin (red shapes), Pesticide (light blue shapes), Plastics (pink shapes) or Hydrocarbons/Jet fuel (dark blue shapes) indicated.

Only 140 directly connected genes out of 499 unique genes associated with the combined lists are shown. Node shapes code: oval and circle – protein; diamond – ligand; circle/oval on tripod platform – transcription factor; ice cream cone – receptor; crescent – kinase or protein kinase; irregular polygon – phosphatase. Arrows with plus sign show positive regulation/activation, arrows with minus sign – negative regulation/inhibition; grey arrows represent regulation, lilac – expression, purple – binding, green – promoter binding, and yellow/olive – protein modification.

Figure 4. The MeDIP-qPCR analysis of (A) selected DMR for each exposure was used to confirm MeDIP-Chip analysis and (B) relative change (exposure/control) ratio presented for each DMR.

All changes shown are statistically significant between control and exposure (p<0.05).

Figure 5. Differential DNA methylated region (DMR) CPG density distribution.

The CpG density (CpG/100 bp) associated with all exposure DMR are presented with number of DMR on y axis and density (CpG per 100 bp) on x axis.