Epigenetic transgenerational inheritance of vinclozolin induced mouse adult onset disease and associated sperm epigenome biomarkers

Abstract

The endocrine disruptor vinclozolin has previously been shown to promote epigenetic transgenerational inheritance of adult onset disease in the rat. The current study was designed to investigate the transgenerational actions of vinclozolin on the mouse. Transient exposure of the F0 generation gestating female during gonadal sex determination promoted transgenerational adult onset disease in F3 generation male and female mice, including spermatogenic cell defects, testicular abnormalities, prostate abnormalities, kidney abnormalities and polycystic ovarian disease. Pathology analysis demonstrated 75% of the vinclozolin lineage animals developed disease with 34% having two or more different disease states. Interestingly, the vinclozolin induced transgenerational disease was observed in the outbred CD-1 strain, but not the inbred 129 mouse strain. Analysis of the F3 generation sperm epigenome identified differential DNA methylation regions that can potentially be utilized as epigenetic biomarkers for transgenerational exposure and disease.

Figure 1

Testicular spermatogenic cell apoptosis in (a) 129 mouse strain vinclozolin lineage, (b) 129 mouse strain flutamide lineage, and (c) CD-1 mouse strain vinclozolin lineage. The mean ± SEM for control (white bars) and treated lineage (black bars). The CD-1 mouse vinclozolin lineage treatment of V1 (black bar) and V2 (gray bar) is presented. No significant difference in tubule cross section numbers was detected between treatment lineages or treatments. The asterisks (*) indicate a statistically significant difference (P<0.05) between control and vinclozolin/flutamide lineage mice. The visible apoptotic cells (TUNEL assay) for control (d) and treated (e) testes are presented.

Figure 2

Disease frequency presented as (a) Percent of Disease F3 generation Males with the ratio of disease/total animal number presented above each bar for Control (white bar), V1 (black bar) and V2 (gray bar). Representative tissue micrographs from control (b,d,f) and V1 (c,e,g) F3 mice samples at >1 yr of age. (b) and (c) represent testis tubules cross sections at 100× magnification with 400× insets. (d) and (e) represent ventral prostate cross sections at 100X magnification with 400X insets. (f) and (g) represent kidney Bowman’s capsule at 400X magnification.

Figure 3

(a) Percentage of ovarian cysts in aged vinclozolin female CD-1 mice. White bar represents control, black bar represents V1, gray bar represents V2. The ratio of animals with cystic ovaries/total animal number is presented above each bar. Representative histology of control (b) and vinclozolin (c) lineage ovaries are presented with asterisks indicating an ovarian cyst.

Figure 4

Regions presenting vinclozolin-induced transgenerational change in F3 generation sperm DNA methylation: (a) chromosomal locations for regions detected with MeDip-Chip to have transgenerational change in DNA methylation for regions confirmed (closed arrowhead) and not able to be tested (open arrowhead) with Real Time qPCR validation are shown; (b) Real Time qPCR validation of regions showing transgenerational change in methylation with values presented as fold change of Vinclozolin/Control and normalized by DNA concentration in the MeDIP samples. The criteria for a RT-qPCR value to be considered as a change are passed t-test with p<0.05, and the trend of the change observed in the qPCR is the same as the observed in the MeDIP-Chip array.

Figure 5

Examples of tiling array data for the most dramatic changes in DNA methylation (a) Elf3 or highest increase and (b) for Mro, for highest decrease. The log signal intensity for vinclozolin lineage, blue line, and control lineage, red line, is presented for chromosomal locations and the bar represents the site for qPCR confirmation. The distribution of differential methylation sites versus CpG density is shown in (c). Black bar represent all regions significantly changed in the MeDIP-Chip array and hatched bar shows only confirmed regions by Real Time qPCR.

Figure 6

Motif analysis of the transgenerational sperm DMR. (a) The similarity of the mouse EDM1 motif created from the 40 confirmed DMR with the rat EDM1 motif. The optimal nucleotide for each nucleotide position is on top with alternations listed. (b) Transcription factor binding sequence motif comparison with mouse and rat EDM1. The transcription factor name, statistical E value, sequence alignment and motif are presented for rat on left and mouse on right.