Abstract
While the risks of maternal alcohol abuse during pregnancy are well-established, several preclinical studies suggest that chronic preconception alcohol consumption by either parent may also have significance consequences for offspring health and development. Notably, since isogenic male mice used in these studies are not involved in gestation or rearing of offspring, the cross-generational effects of paternal alcohol exposure suggest a germline-based epigenetic mechanism. Many recent studies have demonstrated that the effects of paternal environmental exposures such as stress or malnutrition can be transmitted to the next generation via alterations to small noncoding RNAs in sperm. Therefore, we used high throughput sequencing to examine the effect of preconception ethanol on small noncoding RNAs in sperm. We found that chronic intermittent ethanol exposure altered several small noncoding RNAs from three of the major small RNA classes in sperm, tRNA-derived small RNA (tDR), mitochondrial small RNA, and microRNA. Six of the ethanol-responsive small noncoding RNAs were evaluated with RT-qPCR on a separate cohort of mice and five of the six were confirmed to be altered by chronic ethanol exposure, supporting the validity of the sequencing results. In addition to altered sperm RNA abundance, chronic ethanol exposure affected post-transcriptional modifications to sperm small noncoding RNAs, increasing two nucleoside modifications previously identified in mitochondrial tRNA. Furthermore, we found that chronic ethanol reduced epididymal expression of a tRNA methyltransferase, Nsun2, known to directly regulate tDR biogenesis. Finally, ethanol-responsive sperm tDR are similarly altered in extracellular vesicles of the epididymis (i.e., epididymosomes), supporting the hypothesis that alterations to sperm tDR emerge in the epididymis and that epididymosomes are the primary source of small noncoding RNAs in sperm. These results add chronic ethanol to the growing list of paternal exposures that can affect small noncoding RNA abundance and nucleoside modifications in sperm. As small noncoding RNAs in sperm have been shown to causally induce heritable phenotypes in offspring, additional research is warranted to understand the potential effects of ethanol-responsive sperm small noncoding RNAs on offspring health and development.
Highlights
Studies examining the cross generational effects of alcohol have primarily focused on maternal alcohol abuse during pregnancy given the severe risk of inducing developmental deficits that typify fetal alcohol syndrome in offspring
Consistent with other studies in mice (Peng et al, 2012; Sharma et al, 2016), we found the majority (>60%) of 15–45 nucleotide sequencing reads were transfer RNA-derived small RNAs in sperm from both control and ethanol-treated mice while the remaining reads were classified as mitochondrial small RNA, piRNA, microRNA, ribosomal RNA, small nucleolar RNA and small nuclear RNA (Figure 1B)
When we examined the effect of chronic ethanol exposure on the four major small noncoding RNA types in sperm, small RNA sequencing revealed 15 transfer RNA (tRNA)-derived small RNA (tDR) (Figure 2A), 8 miRNAs (Figure 2B), 5 mitosRNAs (Figure 2C), and 0 piRNA (Supplementary Figure 2) that were significantly affected by ethanol after false discovery rate adjustment (q < 0.1, Figure 2D, Supplementary Table 1)
Summary
Studies examining the cross generational effects of alcohol have primarily focused on maternal alcohol abuse during pregnancy given the severe risk of inducing developmental deficits that typify fetal alcohol syndrome in offspring. We added to this evidence, showing that males exposed intermittently to vapor ethanol over 5 weeks produce male offspring with reduced ethanol drinking behavior, increased ethanol sensitivity and attenuated stress responsivity (Finegersh and Homanics, 2014; Rompala et al, 2016, 2017). Since these studies were performed using isogenic sires that played no role in offspring rearing and development, paternal preconception ethanol may be driving unique changes in offspring behavior through nongenomic mechanisms in sperm. Greater emphasis should be put on understanding the consequences of paternal alcohol abuse prior to conception and identifying potential epigenetic mechanisms in the germline
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