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

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.

Preconception paternal alcohol exposure decreases IVF embryo survival and pregnancy success rates in a mouse model.

Increasingly, couples struggling with fertility turn to assisted reproductive techniques, including IVF, to have children. Despite the demonstrated influence of periconception male health and lifestyle choices on offspring development, studies examining IVF success rates and child health outcomes remain exclusively focused on maternal factors. Using a physiologically relevant mouse model, we tested the hypothesis that chronic paternal preconception alcohol intake adversely affects IVF success and negatively impacts IVF offspring fetoplacental growth. Using a voluntary, binge-like mouse model, we exposed sexually mature C57BL/6J males to three preconception treatments (0% (Control), 6% EtOH or 10% EtOH) for 6 weeks, isolated and cryopreserved caudal sperm from treated males, and then used these samples to fertilize oocytes before assessing IVF embryo developmental outcomes. We found that preconception paternal alcohol use reduced IVF embryo survival and pregnancy success rates in a dose-dependent manner, with the pregnancy success rate of the 10% EtOH treatment falling to half those of the Controls. Mechanistically, we found that preconception paternal alcohol exposure disrupts embryonic gene expression, including Fgf4 and Egfr, two critical regulators of trophectoderm stem cell growth and placental patterning, with lasting impacts on the histological organization of the late-term placenta. The changes in placental histoarchitecture were accompanied by altered regulation of pathways controlling mitochondrial function, oxidative phosphorylation and some imprinted genes. Our studies indicate that male alcohol use may significantly impede IVF success rates, increasing the couple's financial burden and emotional stress, and highlights the need to expand prepregnancy messaging to emphasize the reproductive dangers of alcohol use by both parents.

Paternal alcohol exposures program intergenerational hormetic effects on offspring fetoplacental growth.

Hormesis refers to graded adaptive responses to harmful environmental stimuli where low-level toxicant exposures stimulate tissue growth and responsiveness while, in contrast, higher-level exposures induce toxicity. Although the intergenerational inheritance of programmed hormetic growth responses is described in plants and insects, researchers have yet to observe this phenomenon in mammals. Using a physiologically relevant mouse model, we demonstrate that chronic preconception paternal alcohol exposures program nonlinear, dose-dependent changes in offspring fetoplacental growth. Our studies identify an inverse j-shaped curve with a threshold of 2.4 g/Kg per day; below this threshold, paternal ethanol exposures induce programmed increases in placental growth, while doses exceeding this point yield comparative decreases in placental growth. In male offspring, higher paternal exposures induce dose-dependent increases in the placental labyrinth layer but do not impact fetal growth. In contrast, the placental hypertrophy induced by low-level paternal ethanol exposures associate with increased offspring crown-rump length, particularly in male offspring. Finally, alterations in placental physiology correlate with disruptions in both mitochondrial-encoded and imprinted gene expression. Understanding the influence of ethanol on the paternally-inherited epigenetic program and downstream hormetic responses in offspring growth may help explain the enormous variation observed in fetal alcohol spectrum disorder (FASD) phenotypes and incidence.

Alcohol induced increases in sperm Histone H3 lysine 4 trimethylation correlate with increased placental CTCF occupancy and altered developmental programming.

Using a mouse model, studies by our group reveal that paternal preconception alcohol intake affects offspring fetal-placental growth, with long-lasting consequences on adult metabolism. Here, we tested the hypothesis that chronic preconception male alcohol exposure impacts histone enrichment in sperm and that these changes are associated with altered developmental programming in the placenta. Using chromatin immunoprecipitation, we find alcohol-induced increases in sperm histone H3 lysine 4 trimethylation (H3K4me3) that map to promoters and presumptive enhancer regions enriched in genes driving neurogenesis and craniofacial development. Given the colocalization of H3K4me3 with the chromatin binding factor CTCF across both sperm and embryos, we next examined CTCF localization in the placenta. We find global changes in CTCF binding within placentae derived from the male offspring of alcohol-exposed sires. Furthermore, altered CTCF localization correlates with dysregulated gene expression across multiple gene clusters; however, these transcriptional changes only occur in male offspring. Finally, we identified a correlation between genomic regions exhibiting alcohol-induced increases in sperm H3K4me3 and increased CTCF binding in male placentae. Collectively, our analysis demonstrates that the chromatin landscape of sperm is sensitive to chronic alcohol exposure and that a subset of these affected regions exhibits increased placental CTCF enrichment.

Chromatin alterations during the epididymal maturation of mouse sperm refine the paternally inherited epigenome.

BACKGROUND: Paternal lifestyle choices and male exposure history have a critical influence on the health and fitness of the next generation. Accordingly, defining the processes of germline programming is essential to resolving how the epigenetic memory of paternal experiences transmits to their offspring. Established dogma holds that all facets of chromatin organization and histone posttranslational modification are complete before sperm exits the testes. However, recent clinical and animal studies suggest that patterns of DNA methylation change during epididymal maturation. In this study, we used complementary proteomic and deep-sequencing approaches to test the hypothesis that sperm posttranslational histone modifications change during epididymal transit. RESULTS: Using proteomic analysis to contrast immature spermatozoa and mature sperm isolated from the mouse epididymis, we find progressive changes in multiple histone posttranslational modifications, including H3K4me1, H3K27ac, H3K79me2, H3K64ac, H3K122ac, H4K16ac, H3K9me2, and H4K20me3. Interestingly, some of these changes only occurred on histone variant H3.3, and most involve chromatin modifications associated with gene enhancer activity. In contrast, the bivalent chromatin modifications, H3K4me3, and H3K27me3 remained constant. Using chromatin immunoprecipitation coupled with deep sequencing, we find that changes in histone h3, lysine 27 acetylation (H3K27ac) involve sharpening broad diffuse regions into narrow peaks centered on the promoter regions of genes driving embryonic development. Significantly, many of these regions overlap with broad domains of H3K4me3 in oocytes and ATAC-seq signatures of open chromatin identified in MII oocytes and sperm. In contrast, histone h3, lysine 9 dimethylation (H3K9me2) becomes enriched within the promoters of genes driving meiosis and in the distal enhancer regions of tissue-specific genes sequestered at the nuclear lamina. Maturing sperm contain the histone deacetylase enzymes HDAC1 and HDAC3, suggesting the NuRD complex may drive some of these changes. Finally, using Western blotting, we detected changes in chromatin modifications between caput and caudal sperm isolated from rams (Ovis aries), inferring changes in histone modifications are a shared feature of mammalian epididymal maturation. CONCLUSIONS: These data extend our understanding of germline programming and reveal that, in addition to trafficking noncoding RNAs, changes in histone posttranslational modifications are a core feature of epididymal maturation.

Maternal background alters the penetrance of growth phenotypes and sex-specific placental adaptation of offspring sired by alcohol-exposed males.

Epigenetic mechanisms of paternal inheritance are an emerging area of interest in our efforts to understand fetal alcohol spectrum disorders. In rodent models examining maternal alcohol exposures, different maternal genetic backgrounds protect or sensitize offspring to alcohol-induced teratogenesis. However, whether maternal background can mitigate sperm-inherited alterations in developmental programming and modify the penetrance of growth defects induced by preconception paternal alcohol exposures remains unaddressed. In our previous studies examining pure C57Bl/6J crosses, the offspring of alcohol-exposed sires exhibited fetal growth restriction, enlarged placentas, and decreased placental efficiency. Here, we find that in contrast to our previous studies, the F1 offspring of alcohol-exposed C57Bl/6J sires and CD-1 dams do not exhibit fetal growth restriction, with male fetuses developing smaller placentas and increased placental efficiencies. However, in these hybrid offspring, preconception paternal alcohol exposure induces sex-specific changes in placental morphology. Specifically, the female offspring of alcohol-exposed sires displayed structural changes in the junctional and labyrinth zones, along with increased placental glycogen content. These changes in placental organization are accompanied by female-specific alterations in the expression of imprinted genes Cdkn1c and H19. Although male placentae do not display overt changes in placental histology, using RNA-sequencing, we identified programmed alterations in genes regulating oxidative phosphorylation, mitochondrial function, and Sirtuin signaling. Collectively, our data reveal that preconception paternal alcohol exposure transmits a stressor to developing offspring, that males and females exhibit distinct patterns of placental adaptation, and that maternal genetic background can modulate the effects of paternal alcohol exposure.

Programmed suppression of oxidative phosphorylation and mitochondrial function by gestational alcohol exposure correlate with widespread increases in H3K9me2 that do not suppress transcription.

BACKGROUND: A critical question emerging in the field of developmental toxicology is whether alterations in chromatin structure induced by toxicant exposure control patterns of gene expression or, instead, are structural changes that are part of a nuclear stress response. Previously, we used a mouse model to conduct a three-way comparison between control offspring, alcohol-exposed but phenotypically normal animals, and alcohol-exposed offspring exhibiting craniofacial and central nervous system structural defects. In the cerebral cortex of animals exhibiting alcohol-induced dysgenesis, we identified a dramatic increase in the enrichment of dimethylated histone H3, lysine 9 (H3K9me2) within the regulatory regions of key developmental factors driving histogenesis in the brain. However, whether this change in chromatin structure is causally involved in the development of structural defects remains unknown. RESULTS: Deep-sequencing analysis of the cortex transcriptome reveals that the emergence of alcohol-induced structural defects correlates with disruptions in the genetic pathways controlling oxidative phosphorylation and mitochondrial function. The majority of the affected pathways are downstream targets of the mammalian target of rapamycin complex 2 (mTORC2), indicating that this stress-responsive complex plays a role in propagating the epigenetic memory of alcohol exposure through gestation. Importantly, transcriptional disruptions of the pathways regulating oxidative homeostasis correlate with the emergence of increased H3K9me2 across genic, repetitive, and non-transcribed regions of the genome. However, although associated with gene silencing, none of the candidate genes displaying increased H3K9me2 become transcriptionally repressed, nor do they exhibit increased markers of canonical heterochromatin. Similar to studies in C. elegans, disruptions in oxidative homeostasis induce the chromatin looping factor SATB2, but in mammals, this protein does not appear to drive increased H3K9me2 or altered patterns of gene expression. CONCLUSIONS: Our studies demonstrate that changes in H3K9me2 associate with alcohol-induced congenital defects, but that this epigenetic change does not correlate with transcriptional suppression. We speculate that the mobilization of SATB2 and increased enrichment of H3K9me2 may be components of a nuclear stress response that preserve chromatin integrity and interactions under prolonged oxidative stress. Further, we postulate that while this response may stabilize chromatin structure, it compromises the nuclear plasticity required for normal differentiation.

Programmed increases in LXRα induced by paternal alcohol use enhance offspring metabolic adaptation to high-fat diet induced obesity.

OBJECTIVES: Paternally inherited alterations in epigenetic programming are emerging as relevant factors in numerous disease states, including the growth and metabolic defects observed in fetal alcohol spectrum disorders. In rodents, chronic paternal alcohol use induces fetal growth restriction, as well as sex-specific alterations in insulin signaling and lipid homeostasis in the offspring. Based on previous studies, we hypothesized that the observed metabolic irregularities are the consequence of paternally inherited alterations liver x receptor (LXR) activity. METHODS: Male offspring of alcohol-exposed sires were challenged with a high-fat diet and the molecular pathways controlling glucose and lipid homeostasis assayed for LXR-induced alterations. RESULTS: Similar to findings in studies employing LXR agonists we found that the male offspring of alcohol-exposed sires display resistance to diet-induced obesity and improved glucose homeostasis when challenged with a high-fat diet. This improved metabolic adaptation is mediated by LXRα trans-repression of inflammatory cytokines, releasing IKKß inhibition of the insulin signaling pathway. Interestingly, paternally programmed increases in LXRα expression are liver-specific and do not manifest in the pancreas or visceral fat. CONCLUSIONS: These studies identify LXRα as a key mediator of the long-term metabolic alterations induced by preconception paternal alcohol use.