Heavy Chronic Intermittent Ethanol Exposure Alters Small Noncoding RNAs in Mouse Sperm and Epididymosomes

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.

Keywords: epididymosomes; epigenetics; ethanol; noncoding RNA; sperm.

Figure 1

Chronic ethanol shifts the tDR profile of sperm small noncoding RNA. (A) Chronic intermittent ethanol vapor exposure (left panel) induced an average blood ethanol concentration of 159.2 ± 9.2 mg/dl [mean (μ) ± standard error of the mean (SEM)] over its 5 week duration. There was no effect of chronic ethanol on body weight (right panel) compared to the control group (p > 0.05). (B) Pie charts displaying the percentage of each small RNA class represented in sperm from control and ethanol treatment groups. (C) Most tDR are 30–35 nt 5′-derived tRNA halves (5′-tRH) (see insert) and chronic ethanol significantly altered the percentage of 30, 31, and 35 nt tDR reads. (D) Most 30–36 nt tDR reads map to Glu-CTC and Gly-GCC relative to all other tDR species. Data in bar graphs presented as μ ± SEM. N = 9/treatment in all panels. ^**p < 0.01. ^***p < 0.001.

Figure 2

Chronic ethanol alters abundance of several tDR, miRNA, and mitosRNA species in sperm. Volcano plots depicting fold change and log-transformed p-value for sperm (A) tDR, (B) miRNA, and (C) mitosRNA. Red dots indicate significance (q ≤ 0.1). (D) Heat map of differentially expressed sperm small noncoding RNAs representing fold change in normalized counts for each small RNA sequencing sample represented by each column. (E) RT-qPCR validation of sequencing results revealed a significant effect of chronic ethanol on sperm tDRs Glu-CTC (p < 0.05), His-GTG (p < 0.01), Ser-AGA (p < 0.05), with no change in Pro-AGG (p > 0.05) and significantly increased miR-10a (p < 0.05) and miR-99b (p < 0.05), N = 7–11/treatment. RT-qPCR data presented as μ ± SEM with black dots representing biological replicates (one mouse/replicate). ^*p < 0.05, ^**p < 0.01.

Figure 3

Analyzing predicted gene targets of ethanol-responsive sperm miRNA and tDR Glu-CTC. (A) Genes with 3′-UTRs targeted by three or more miRNAs. (B) Gene ontology analysis of predicted target genes of ≥ 3 miRNA. (C) Number of genes with predicted 5′-UTR, coding, or 3′-UTR targets of ethanol-responsive sperm tDR. (D) Gene ontology analysis for genes with predicted 5′-UTR targets of tDR Glu-CTC.

Figure 4

Chronic ethanol alters select RNA modifications in sperm small noncoding RNA. UHPLC-MS/MS was performed on the ~30–40 nt fraction of sperm RNA from chronic ethanol and control exposed groups. Post-transcriptional modifications were examined for each of the parent nucleosides, (A) uridine, (B) cytidine, (C) adenosine, and (D) guanosine. Chronic ethanol increased the uridine modification, 5-methylaminomethyl-2-thiouridine (mnm⁵s²U) (q < 0.1) and the cytidine modification formylcytidine (f⁵C) (q < 0.01). Data presented as μ ± SEM bars. N = 3 pooled samples/group. ^*q < 0.1, ^**q < 0.01.

Figure 5

Effects of chronic ethanol on sperm tDR are reflected in epididymosomes. (A) RT-qPCR showing no effect of chronic ethanol on tDR Glu-CTC, His-GTG, and Ser-AGA in caput epididymal sperm (p > 0.05). (B) Transmission electron microscopy image of epididymosomes (arrows) isolated from adult mouse cauda epididymis. (C) RT-qPCR reveled a significant effect of chronic ethanol on tDR Glu-CTC with no change in His-GTG or Ser-AGA with 2 weeks of ethanol exposure. (D) RT-qPCR showing increased tDR His-GTG with no change in Glu-CTC or Ser-AGA with 5 weeks ethanol exposure. N = 4–11/treatment. Data presented as μ ± SEM with black dots representing biological replicates (one mouse/replicate). ^*p < 0.05.

Figure 6

Effect of chronic ethanol on epididymal expression of genes regulating tDR biogenesis. RT-qPCR in cauda epididymal tissue revealed a significant effect of chronic ethanol on the tRNA methyltransferase Nsun2 (p < 0.05) with no change in Dnmt2 (p > 0.5). N = 7–8/treatment. Data presented as μ ± SEM with black dots representing biological replicates (one mouse/replicate). ^*p < 0.05.