Transgenerational epigenetic programming of the brain transcriptome and anxiety behavior

Abstract

Embryonic exposure to the endocrine disruptor vinclozolin during gonadal sex determination promotes an epigenetic reprogramming of the male germ-line that is associated with transgenerational adult onset disease states. Further analysis of this transgenerational phenotype on the brain demonstrated reproducible changes in the brain transcriptome three generations (F3) removed from the exposure. The transgenerational alterations in the male and female brain transcriptomes were distinct. In the males, the expression of 92 genes in the hippocampus and 276 genes in the amygdala were transgenerationally altered. In the females, the expression of 1,301 genes in the hippocampus and 172 genes in the amygdala were transgenerationally altered. Analysis of specific gene sets demonstrated that several brain signaling pathways were influenced including those involved in axon guidance and long-term potentiation. An investigation of behavior demonstrated that the vinclozolin F3 generation males had a decrease in anxiety-like behavior, while the females had an increase in anxiety-like behavior. These observations demonstrate that an embryonic exposure to an environmental compound appears to promote a reprogramming of brain development that correlates with transgenerational sex-specific alterations in the brain transcriptomes and behavior. Observations are discussed in regards to environmental and transgenerational influences on the etiology of brain disease.

Figure 1. Brain transcriptome microarray analysis from F3 generation control (con) and vinclozolin (vin) animals.

(A) Dendrogram for male whole brain, male hippocampus and male amygdala for statistically significant regulated genes with signal above 75. (B) Dendrogram for female hippocampus or female amygdala for statistically significant regulated genes. The scale at the bottom margin indicates an increase (red) and decrease (green) in expression. The number of regulated genes is listed at the right of each gene set. The whole male brain was from 6 month old animals, and amygdala and hippocampus from 12–15 month old animals.

Figure 2. Comparison of the F3 generation regulated gene sets (i.e. transcriptomes).

Venn diagram with total regulated (>1.5 fold-change between control and vinclozolin) genes listed and the overlap, (A) brain nucleus differences within a sex; (B) sex differences within a brain nucleus.

Figure 3. Categorization of genes into functionally related gene groups with the number of genes up-regulated or down-regulated.

(A) male hippocampus regulated gene list; (B) male amygdala regulated gene list; (C) female hippocampus regulated gene list; and (D) female amygdala regulated gene list.

Figure 4. Performance in the light:dark box in young and aged third-generation female and male rats.

Data are mean±SEM of latency to enter dark (A, G), time spent in the light side of the box (B, H) and the total number of transitions made between the light and dark sides of the box (C, I) in young (left panel) and aged (right panel) control and vinclozolin generation rats. For young and aged female rats N = 8–9/group. For B aged: t₁₅ = 2.65, p<0.018. * P<0.05, compared with control. Performance in the elevated plus-maze in young and aged third-generation female and male rats. Data are mean±SEM of percent open arm time (D, J), percent open arm entries (E, K) and total entries (F, L) in young (left panel) and aged (right panel) control and vinclozolin generation rats. For young rats, all animals from above are shown; for aged rats, N = 5 (control) and 7 (vinclozolin). The aged group represents a subset of those run in the light:dark box shown because of technical (unstable lighting) conditions during testing for the first 5 rats; these rats were omitted from the analysis. For D young: t₁₅ = 2.30, p<0.036. For E young: t₁₅ = 2.51, p<0.024. For D aged: t₁₀ = 2.40, p<0.037. For E aged: t₁₀ = 2.82, p<0.018. *P<0.05, compared with control. For young male rats, N = 9–12/group. For aged male rats, N = 9–10/group. For H young: t₁₉ = −2.65, p<0.016. For I young: t₁₉ = −2.46, p<0.024. * P<0.05, compared with control. All young male animals L: t₁₉ = −3.32, p<0.004. *P<0.05, compared with control.