The type and severity of ethanol-induced alterations following prenatal ethanol exposure is strongly impacted by a number of factors. A growing body of evidence suggests that sex is one of the important variables in ethanol's teratogenic effects. Further, recent data shows that acute ethanol exposure at the beginning of CNS development can have long-term sex-specific effects on behavior (Fish et al., Behav Brain Res. 338:173-184, 2018). This proposal will test the hypotheses that sex is an important variable in mediating ethanol's effects on the CNS just after neural tube closure, an early stage of CNS development. In the parent RO1, we are examining the role of genotype in ethanol's neuroteratogenesis, and sex and genotype are often interacting variables. These experiments will expand this by conducting an initial analysis of sex-by-genotype interactions by evaluating two different mouse strains. We will examine changes in DNA methylation in the telencephalon (primordial forebrain) of the developing neural tube because ethanol has been shown to alter DNA methylation and there is a wide literature on sex-specific differences in DNA methylation. Two groups of mice will be examined in each strain: ethanol-exposed given 5.9 g/kg ethanol in a binge model and controls given isocaloric maltose-dextrin. DNA methylation will be examined using an unbiased, whole-genome method, Reduced Representation Bisulfite Sequencing. Two strains will be examined and these were chosen because they exhibit differential sensitivity to prenatal alcohol exposure: 1 sensitive and 1 resistant to ethanol's effects. Data from these experiments fits the first of the stated goals of the NIH Strategic Plan for Women's Health Research from The Office of Research on Women's Health: increase sex differences research in basic science studies. These experiments will address several areas of interest under this goal including: 1) Evaluation of prenatal development, 2) Comparative study of cells, and specifically early?forming neural cells, from males and females, 3) Conduction of neuroscience research to understand vulnerability to ethanol-induced CNS damage, and 4) Evaluation of epigenetic modifications, specifically DNA methylation, using a whole-genome approach. Outcomes from this experiment will determine 1) Whether DNA methylation plays in a role in sex-specific neuroteratogenic effects following early ethanol exposure and 2) whether there is a sex-by-strain interaction suggesting that both variables are critically important in determining neuroteratogenicity. Additionally, the evaluation of sex differences in control embryos could provide information that would have a broader impact in our understanding of developmental conditions that have a sex ratio bias, such as autism. The ability to understand sex and sex-by-genotype differences in DNA methylation, particularly early in CNS development, will likely improve our understanding of how sex differences in brain function occur and potentially identify molecular pathways that mediate this difference.
It is becoming increasingly clear that sex is an important factor in mediating the type and severity of ethanol's deleterious actions in the brain, although the mechanism that controls this and whether sex interacts with other factors is currently unknown. The experiments in this proposal will evaluate one epigenetic process, DNA methylation, and its role in the sex-specific effects of alcohol exposure early in the development of the brain. Further, these experiments will examine two genotypes with differing phenotypic responses to developmental alcohol exposure to evaluate whether there is an interaction between genotype and sex in ethanol's neuroteratogenic effects.
