Alterations in sperm RNAs persist after alcohol cessation and correlate with epididymal mitochondrial dysfunction

Abstract

Background: Chronic preconception paternal alcohol use adversely modifies the sperm epigenome, inducing fetoplacental and craniofacial growth defects in the offspring of exposed males. A crucial outstanding question in the field of paternal epigenetic inheritance concerns the resilience of the male germline and its capacity to recover and correct sperm-inherited epigenetic errors after stressor withdrawal.

Objectives: We set out to determine if measures of the sperm-inherited epigenetic program revert to match the control treatment 1 month after withdrawing the daily alcohol treatments.

Materials and methods: Using a voluntary access model, we exposed C57BL/6J males to 6% or 10% alcohol for 10 weeks, withdrew the alcohol treatments for 4 weeks, and used RNA sequencing to examine gene expression patterns in the caput section of the epididymis. We then compared the abundance of sperm small RNA species between treatments.

Results: In the caput section of the epididymis, chronic alcohol exposure induced changes in the transcriptional control of genetic pathways related to the mitochondrial function, oxidative phosphorylation, and the generalized stress response (EIF2 signaling). Subsequent analysis identified region-specific, alcohol-induced changes in mitochondrial DNA copy number across the epididymis, which correlated with increases in the mitochondrial DNA content of alcohol-exposed sperm. Notably, in the corpus section of the epididymis, increases in mitochondrial DNA copy number persisted 1 month after alcohol cessation. Analysis of sperm noncoding RNAs between control and alcohol-exposed males 1 month after alcohol withdrawal revealed a ∼100-fold increase in mir-196a, a microRNA induced as part of the nuclear factor erythroid 2-related factor 2 (Nrf2)-driven cellular antioxidant response.

Discussion and conclusion: Our data reveal that alcohol-induced epididymal mitochondrial dysfunction and differences in sperm noncoding RNA content persist after alcohol withdrawal. Further, differences in mir-196a and sperm mitochondrial DNA copy number may serve as viable biomarkers of adverse alterations in the sperm-inherited epigenetic program.

Keywords: alcohol; epididymis; epigenetics; mitochondrial dysfunction; paternal epigenetic inheritance; sperm.

Figure 1.. A mouse model to determine the capacity of the sperm epigenome to recover one month after the cessation of alcohol exposures.

A) Experimental design: We exposed C57BL6/J males to 6% and 10% alcohol for ten weeks, then collected tissues from a cohort of active drinkers (Cohort 1). We then ceased the alcohol exposures, allowed males to recover for four weeks, collected tissues and sperm (Cohort 2), then used RNA-sequencing to compare RNA profiles between treatments. Comparison of male B) average weekly weight gain between treatment groups, C) average weekly fluid consumption, and D) average daily dose of ethanol between treatment groups (n = 8). We compared treatments using either a two-way ANOVA followed by Tukey’s post hoc analysis or a Kruskal-Wallis One-Way ANOVA followed by Dunn’s multiple comparisons test. For C, Asterix denote significant differences compared to the Control treatment. Error bars represent the standard error of the mean, *P < 0.05, **P < 0.01, ****P < 0.0001.

Figure 2.. Chronic alcohol exposure induces altered gene expression patterns in the caput section of the epididymis.

A) Principal Component Analysis of gene expression between the caput epididymis isolated from Control, 6% EtOH, and 10% EtOH-treated males (Left Cohort 1 Active Drinkers, Right Cohort 2 EtOH-Cessation Males). Volcano plot contrasting down- and upregulated differentially expressed genes between B) Control vs. 6%EtOH-treated males and C) Control vs. 10%EtOH-treated males (log2FC, q-value <0.05). D) Ingenuity Pathway Analysis of differentially expressed genes identified in the 10% EtOH treatment group (log2FC, p-value <0.05). n = 3 males per treatment for each cohort.

Figure 3.. Chronic alcohol exposure induces lasting changes in mitochondrial DNA copy number within the epididymis.

A) Quantitative PCR analysis of mitochondrial DNA copy number (mtDNAcn) across the caput, corpus, and cauda sections of the epididymis isolated from Control males (n=8). For ease of comparison, we normalized qPCR ratios to the caput section. Comparison of mtDNAcn in the B) liver and C) testis isolated from Control and 10% EtOH males in the cohort of active drinkers (Cohort 1; n=8). Alcohol-induced alterations in mtDNAcn across the D) caput, E) corpus, and F) cauda sections of the epididymis isolated from Control and actively drinking 10% EtOH males (Cohort 1; n=8). Segment-specific differences in mtDNAcn across the G) caput, H) corpus, and I) cauda sections of the epididymis four weeks after the cessation of alcohol (Cohort 2; n=8). We compared the impacts of alcohol treatment on mtDNAcn using either a Kruskal-Wallis One-Way ANOVA, followed by Dunn’s multiple comparisons test, a student’s t-test with Welch’s Correction, a Mann-Whitney test, or a one-way ANOVA followed by a Fisher’s exact test, depending on treatment and the normality of the dataset. Error bars represent the standard error of the mean, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4.. Analysis of mitochondrial copy number in alcohol-exposed sperm.

A) Quantitative PCR analysis of mitochondrial DNA copy number (mtDNAcn) in cryopreserved sperm used in our previously published IVF experiments (n=6). Analysis of mtDNAcn in fresh sperm isolated from Control and 10% EtOH-treated actively drinking males (Cohort 1; n=8). We used a student’s t-test to compare treatments. Error bars represent the standard error of the mean, *P < 0.05, **P < 0.01.

Figure 5.. Sperm isolated from alcohol-exposed EtOH-Cessation males retain a distinct small RNA signature.

Comparison of total small RNA species in sperm isolated from A) Cessation-Control and B) EtOH-exposed males in the EtOH-Cessation cohort after four weeks of abstinence from alcohol (n=4). Differential enrichment of sequenced C) microRNAs (miRNAs) and D) exonic sequences between EtOH-Cessation-Control and alcohol-exposed males (n=4). Principal Component Analysis comparing enrichment of sperm E) miRNA, F) exonic, and G) transposable element (TE)-derived sequences between Cessation-Control and alcohol-exposed males (n=4). Volcano plot comparing differentially enriched H) miRNA-, I) Structural RNA-, and J) exonic-derived sequences between Cessation-Control and EtOH-exposed males after four weeks of abstinence from alcohol (log2FC, q-value <0.05; n=4). We used a Mann-Whitney test to compare the percentages of miRNA and exonic-derived sequences between treatments. Error bars represent the standard error of the mean, *P < 0.05, **P < 0.01.