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.

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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.

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.

Pathologic evaluation is crucial to the study of medical devices and integral to the Food and Drug Administration and other regulatory entities' assessment of device safety and efficacy. While pathologic analysis is tailored to the type of device, it generally involves at a minimum gross and microscopic evaluation of the medical device and associated tissues. Due to the complex nature of some implanted devices and specific questions posed by sponsors, pathologic evaluation inherently presents many challenges in accurately assessing medical device safety and efficacy. This laboratory's experience in numerous collaborative projects involving veterinary pathologists, biomedical engineers, physicians, and other scientists has led to a set of interrelated assessments to determine pathologic end points as a means to address these challenges and achieve study outcomes. Thorough device evaluation is often accomplished by utilizing traditional paraffin histology, plastic embedding and microground sections, and advanced imaging modalities. Combining these advanced techniques provides an integrative, comprehensive approach to medical device pathology and enhances medical device safety and efficacy assessment.