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.

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.

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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.

Increasingly, couples struggling with fertility turn to assisted reproductive techniques, including in vitro fertilization (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 six weeks, isolated and cryopreserved caudal sperm from treated males, and then used these samples to fertilise 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.