The current study was designed to investigate the potential ability of various environmental compounds and mixtures to promote epigenetic transgenerational disease with a focus on pubertal and gonadal abnormalities. The alterations in the sperm epigenome were investigated to determine the similarities and differences between the different exposures on differential DNA methylation. The experimental design used pharmacologic doses, Table S1A
, based on approximately 1% of the lethal oral dose 50% (LD50) for most of the compounds that previously have been shown in vivo
to not cause direct toxicological effects. Gestating female outbred Harlan Sprague Dawley rats were given intraperitoneal (IP) injections daily between embryonic days 8–14 of fetal development correlating with gonadal sex determination. No consistent effects were observed on litter size, sex ratios or weaning weights, Figures S1
. The number of litters and male and female animals obtained for each generation is presented in Table S1B and S1C
. The F0 generation gestating female was the only animal injected IP. The F1 generation animals at 90 days of age were mated to the same lineage to generate the F2 generation and the F2 generation were mated to generate the F3 generation progeny as previously described 
. No sibling or cousin breedings were used to avoid any inbreeding artifacts. No major overt toxicity was observed in F1, F2 or F3 generations, Figure S1
. The only treatment that promotes some toxicity in the F1 generation was the high dose plastics, Table S1A
, so a lower dose at 50% that shown in Table S1A
was also used and termed “Low Dose Plastic” that had no toxicological effects, Figure S1
. Anogenital distance was measured as an indicator of exposure to androgenic compounds that promote masculinization during the perinatal period 
. Analysis of anogenital index (AGI) demonstrated some effects of the treatments on the F2 and F3 generation animals, but no effects at the F1 generation animals, Figure S3
. These actions on the AGI in the F2 generation are possibly due to the direct exposure of fetal germ cells to the endocrine disruptor activities of several of the exposure compounds (e.g. BPA, DEHP, DBP) 
, while the increased AGI in the F3 generation appears to be transgenerational. Therefore, classic endocrine disruptor actions 
are likely not involved in the F2 and F3 generation, but only in the F1 generation. In considering the actions of environmental exposure the direct versus indirect (e.g. epigenetic) actions are critical. The exposure of the F0 generation gestating female directly affects the F0 generation female, the F1 generation embryo and the germ line inside the F1 embryo that will be generating the F2 generation animal 
. Therefore, phenotypes in the F0, F1 and F2 generations may be due to direct exposures and are not transgenerational effects or phenotypes observed by definition. A transgenerational phenotype or phenomenon requires the lack of direct exposure to promote a generational effect 
. The actions on F0, F1 and F2 are due to a direct multi-generational exposure and only the F3 generation phenotype can be considered a transgenerational effect. Since the mechanisms promoting the F1 or F3 generation effects differ, the phenotypes can be distinct between the generations.
Puberty is a developmental process involving the hypothalamic – pituitary – gonadal axis which initiates during fetal development and matures in adolescence 
. The onset of puberty was investigated with the different exposure lineages of control (DMSO vehicle), pesticide, low and high dose plastics, dioxin, or hydrocarbons in the F1–F3 generation rats. The analysis was initiated for females at postnatal day 30 and males at postnatal day 35 until puberty (vaginal opening or balano-preputial separation) 
. In the F1 generation plastics promoted delayed female pubertal onset, while in the F2 generation plastics, dioxin and jet fuel promoted early onset of puberty for females, with plastics and dioxin promoting early onset of puberty in males, Figure S4
. In the transgenerational F3 generation it was demonstrated that plastics, low dose plastics, dioxin and jet fuel promote early onset of puberty in females, while having no effect on males, . Therefore, several of the exposures were found to promote early onset of puberty in females transgenerationally.
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.
Gonadal function for both testis and ovary were investigated in the F3 generation at postnatal 120 days of age. Previously vinclozolin was shown to promote a transgenerational phenotype of spermatogenic cell apoptosis 
, so potential germ cell apoptosis in the testis was investigated. The jet fuel exposure was found to transgenerationally increase spermatogenetic cell apoptosis in the F3 generation male testis, . Epididymal sperm concentration and motility for the F3 generation were also investigated and did not provide consistent alterations transgenerationally, as previously seen with vinclozolin exposure. The F3 generation ovaries were examined for total follicle number and the individual types of primordial follicles, primary follicles and developing follicles were categorized, . All the exposures were found to promote a transgenerational effect on the F3 generation ovary with a significant reduction in total follicle number, , and the follicle class primarily affected was the primordial follicle, . Therefore, all the exposures promoted the transgenerational phenotype of a reduction in the primordial follicle pool size. The large developing antral follicles were counted to determine potential effects on later stage follicle development and no differences were observed between the exposures when compared to control, . The transgenerational action of the various exposures on the ovary was a reduction in the primordial follicle pool size. This may promote premature ovarian failure as the animals age. The testis and ovary are hormone regulated and both produce endocrine steroids. Hormone levels were analyzed to determine how the endocrine system was responding transgenerationally. The F3 generation males had a reduction in testosterone levels in the plastics, dioxin and jet fuel exposure lineages, Figure S5A
, while the females had no change in progesterone levels, Figure S5B
. No change in luteinizing (LH) hormone levels was detected in either male or female F3 generation animals, Figure S5C
& D. Therefore, the endocrine system was altered transgenerationally in the males.
The mechanism involved in these transgenerational phenotypes is the reprogramming of the germ line (sperm) during male sex determination 
. This altered sperm epigenome appears to be permanently reprogrammed and escapes the DNA methylation programming at fertilization to allow transgenerational transmission of the altered sperm epigenome, that then promotes all tissues developed from that sperm to have altered cell and tissue transcriptomes that can promote transgenerational disease 
. Previously, vinclozolin was shown to promote a transgenerational (F3 generation) alteration in DNA methylation 
and a transgenerational transcriptome alteration in tissues like the testis 
. The F3 generation rat sperm from the control and all exposure groups were collected for genome wide promoter DNA methylation analysis 
. The procedure involved the use of an antibody to methylcytosine to immunoprecipitate methylated DNA (MeDIP) and then a competitive hybridization tiling array (Chip) for a MeDIP-Chip analysis 
. Differentially methylated sites were identified for all the different exposure lineages in the F3 generation sperm when compared with vehicle control F3 generation sperm. The complete lists of differential methylation regions (DMR) for each exposure in the F3 generation sperm are provided in Table S2
(A–D). The overlap of the DMR sets for each exposure is shown in a Venn diagram in . The number of DMR for hydrocarbons (jet fuel) was 33, dioxin 50, plastics (BPA, DEHP, DBP) 197 and pesticide (permethrin and DEET) 363 with a statistically significant difference (p<10−5
). Interestingly, the majority of each DMR set was specific to an exposure group and not common with the other exposure DMRs. The only exception was an overlap between plastics and pesticide of 113 DMRs, . Therefore, each exposure had a unique signature of epigenetic alterations in the F3 generation sperm. The chromosomal localizations of these sites are shown in . The DMRs are seen on all autosomes and the X chromosome. Clustering analysis of the DMRs when over represented in specific chromosomal locations identified 35 different clusters (2–5 megabase each) of DMR between the exposures that with z-score analysis have a statistically significant difference (p<0.05), . These DMR clusters may represent “epigenetic control regions” where different exposure DMRs may commonly regulate genome activity. The functional significance of these DMR clusters remains to be elucidated and are identified for individual DMR in Table S2
. In considering the combined DMR and associated gene promoters for all exposures, the potential cellular signaling processes affected demonstrated similar pathways are predominant, as shown in Table S3
. A gene network analysis for direct connections within the total gene set associated with the DMR is shown in and demonstrates extracellular, membrane, cytoplasmic and nuclear associated genes are all associated with the DMR identified. Common cellular signaling pathways and processes appear to be involved from the gene network identified. Therefore, common cellular pathways and gene networks may be influenced by the different exposures and transgenerational sperm epigenomes. Although exposure specific transgenerational differential DNA methylation regions (DMR) are predominant, the common cellular processes and gene networks effected may explain the similar disease phenotypes observed.
The transgenerational DMR associated with each exposure group identified.
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.
The identification of epigenetic alterations in specific regions of the F3 generation sperm support a role for epigenetic transgenerational inheritance of the disease phenotypes observed. Several of the top exposure specific DMRs for each exposure with the highest statistical significance were selected for confirmation with quantitative PCR of the MeDIP samples. A list of the confirmed exposure specific signatures are presented in . In addition, several of the top overlapped (common) DMR were also selected and shown. The MeDIP qPCR analysis demonstrated both increases and decreases for the exposure specific and common DMR, . These exposure specific DMR are considered potential epigenetic biomarkers for exposure and the transmission of transgenerational phenotypes. Further analysis of the epigenetic sites identified considered two genomic features associated with the DMRs. The first one was a DNA sequence motif termed “Environmentally Induced DNA Methylation Region 1” (EDM1) that was previously identified and shown to be associated with a high percentage of the vinclozolin induced sperm DMRs 
. This motif may not be at the specific altered DNA methylation site, but is within the 400–500 bp region. A DNA sequence motif such as EDM1 may promote a region of sensitivity for these DMR's to be programmed transgenerationally. The potential presence of this EDM1 motif in the epigenetic sites (DMR) identified in the current study for all the exposures was determined. An evaluation of the presence of EDM1 using the MCAST online software revealed a statistically significant higher EDM1 presence in promoter regions of the jet fuel and dioxin exposure groups (74.19% and 57.63%, respectively) compared to a computer generated random set of 144 promoters (20.83%). The presence of EDM1 in the promoter regions of the plastics (20.47%) and pesticides (7.36%) was similar or below its presence in the random set of promoters. This suggests that the molecular mechanisms involved in the targeting of these regions to produce a transgenerational change in DNA methylation may differ among the exposure groups. Another genomic feature investigated was the CpG density within the DMR identified. The frequency of CpG number per 100 bp for the DMR demonstrates the DMR identified for all exposures have an average CpG content of 4.9 CpG/100 bp with none above 15 CpG/100 bp, . A small CpG cluster in a CpG desert appears to be a primary feature of the transgenerational DMR identified, and not shores or islands of CpG. Therefore, specific genomic features such as low CpG density, isolated CpG clusters, and the presence of a unique DNA sequence motif may be involved in facilitating the programming of these epigenetic sites (DMR) in the male germ line.
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.
Differential DNA methylated region (DMR) CPG density distribution.