Environmental toxicant induced epigenetic transgenerational inheritance of ovarian pathology and granulosa cell epigenome and transcriptome alterations: ancestral origins of polycystic ovarian syndrome and primary ovarian insufiency

Abstract

Two of the most prevalent ovarian diseases affecting women's fertility and health are Primary Ovarian Insufficiency (POI) and Polycystic Ovarian Syndrome (PCOS). Previous studies have shown that exposure to a number of environmental toxicants can promote the epigenetic transgenerational inheritance of ovarian disease. In the current study, transgenerational changes to the transcriptome and epigenome of ovarian granulosa cells are characterized in F3 generation rats after ancestral vinclozolin or DDT exposures. In purified granulosa cells from 20-day-old F3 generation females, 164 differentially methylated regions (DMRs) (P < 1 x 10^-6) were found in the F3 generation vinclozolin lineage and 293 DMRs (P < 1 x 10^-6) in the DDT lineage, compared to controls. Long noncoding RNAs (lncRNAs) and small noncoding RNAs (sncRNAs) were found to be differentially expressed in both the vinclozolin and DDT lineage granulosa cells. There were 492 sncRNAs (P < 1 x 10^-4) in the vinclozolin lineage and 1,085 sncRNAs (P < 1 x 10^-4) in the DDT lineage. There were 123 lncRNAs and 51 lncRNAs in the vinclozolin and DDT lineages, respectively (P < 1 x 10^-4). Differentially expressed mRNAs were also found in the vinclozolin lineage (174 mRNAs at P < 1 x 10^-4) and the DDT lineage (212 mRNAs at P < 1 x 10^-4) granulosa cells. Comparisons with known ovarian disease associated genes were made. These transgenerational epigenetic changes appear to contribute to the dysregulation of the ovary and disease susceptibility that can occur in later life. Observations suggest that ancestral exposure to toxicants is a risk factor that must be considered in the molecular etiology of ovarian disease.

Keywords: DNA methylation; Ovary; PCO disease; POI; epigenetic; granulosa; ncRNA; sperm; transcriptome; transgenerational.

Figure 1.

Ovarian pathology frequency. Transgenerational ovarian disease in F3 generation control, vinclozolin and DDT lineage rats at 1 y of age. Numbers for diseased individuals versus the total number of individuals analyzed is shown and (***) indicates statistical significance of P < 7 x 10⁻³ for vinclozolin and P < 1 x 10⁻⁵ for DDT by Fisher’s Exact Test. Transgenerational ovarian disease frequency (i.e., presence of ovarian cysts) from control, vinclozolin, and DDT lineage rats at 1 y of age.

Figure 2.

DMR identification. The number of DMRs found using different P value cutoff thresholds. The all window column shows all DMRs. The multiple window column shows the number of DMRs containing at least two significant windows. Lower table of each set shows the number of DMR having each specific number of significant windows at a P < 1x10⁻⁶. (a) Granulosa cell vinclozolin F3 generation DMRs P < 1x10⁻⁶. (b) Granulosa cell DDT F3 generation DMRs P < 1x10^−6.

Figure 3.

DMR chromosomal locations. The DMR locations on the individual chromosomes for all DMRs at a P value threshold of < 1x10⁻⁶. (a) Vinclozolin F3 generation. (b) DDT F3 generation. Red arrowheads indicate positions of DMRs and black boxes indicate clusters of DMRs.

Figure 4.

DMR genomic features. (a & c) The number of DMRs at different CpG densities for all DMRs at a p-value threshold of P < 1x10⁻⁶. (b & d) The DMR lengths for all DMRs are at a P value threshold of < 1x10⁻⁶. (a & b) Vinclozolin F3 generation. (c & d) DDT F3 generation.

Figure 5.

Differential RNA expression at different p-value thresholds for vinclozolin (a) and DDT (b). P value < 1x10⁻⁴ was used for subsequent analysis. Venn diagrams show overlap of RNA categories between the two lineages for (c) sncRNA, (d) lncRNA, and (e) mRNA.

Figure 6.

Differentially expressed small, noncoding RNA broken down by class and lineage. P value < 1 x 10⁻⁴ was used for analysis.

Figure 7.

Differentially expressed RNAs from the vinclozolin lineage. Chromosomal locations of differentially expressed sncRNA (a), lncRNA (b), or mRNA (c). Individual RNAs are shown as red arrows and clusters are shown as black boxes. RNAs with unknown locations are not shown. (d) Venn diagram showing overlap of all differentially expressed epigenetic modifications from the vinclozolin lineage.

Figure 8.

Differentially expressed RNAs from the DDT lineage. Chromosomal locations of differentially expressed sncRNA (a), lncRNA (b), or mRNA (c). Individual RNAs are shown as red arrows and clusters are shown as black boxes. RNAs with unknown locations are not shown. (d) Venn diagram showing overlap of all differentially expressed epigenetic modifications from the DDT lineage.

Figure 9.

Differentially expressed epigenetic modifications were broken down by predicted associated gene category. Top row: mRNAs from vinclozolin (a) and DDT (b) lineages. Bottom row: DMRs from DDT (c) and vinclozolin (d). Genes or DMRs that could not be assigned a category are not shown.

Figure 10.

Associated gene pathways for DMRs (a) DDT and (b) vinclozolin lineages and for mRNA (c) DDT and (d) vinclozolin lineages.

Figure 11.

Ovarian disease gene associations. (a) DMR associated gene overlap with published ovarian disease genes. (b) mRNA gene overlaps with published ovarian disease genes. (c) Specific ovarian disease associated gene overlap with DMR associated genes and mRNA genes.