| While the importance of maternal health during pregnancy for proper offspring development is well-acknowledged, a potential role for preconception health -- especially that of the father -- is rarely considered. However, a recent surge of studies strongly implicates paternal experiences and environmental exposures prior to conception as causal drivers of complex neurobehavioral offspring phenotypes. Considering this work, my dissertation laboratory previously discovered that paternal chronic ethanol exposure increased ethanol sensitivity and decreased ethanol drinking preference selectively in male offspring. This dissertation builds off those preliminary studies with the hypothesis that paternal preconception environment alters ethanol- and stress-related phenotypes via epigenetic mechanisms in sperm. Initial experiments revealed that paternal chronic ethanol blunted the corticosterone response to acute stress, prevented stress-induced polydipsia, and altered genetic and epigenetic regulation of corticotropin-releasing factor in the hypothalamus in male offspring. In addition, paternal chronic stress reduced ethanol drinking behavior in male offspring and this phenotype was dependent on the vendor/shipping history of the sire. Subsequent experiments examined the effects of chronic ethanol on small noncoding RNA in sperm, an epigenetic mechanism causally implicated in the cross-generational effects of paternal preconception environment. The unique small noncoding RNA signature of sperm is shaped during epididymal transit by extracellular vesicles (i.e., epididymosomes). Small RNA sequencing revealed several ethanol-responsive small noncoding RNAs in sperm and some species were similarly affected in epididymosomes. Finally, the effects of epididymosomes on intergenerational ethanol- and stress-related behaviors were directly tested. Normal sperm was incubated with epididymal extracellular vesicles from chronic ethanol (Ethanol EV-donor) or control-treated (Control EV-donor) mice prior to in vitro fertilization (IVF) and embryo transfer to produce adult progeny. While Ethanol EV-donor treatment did not recapitulate the ethanol- or stress-related intergenerational effects of paternal ethanol, Ethanol EV-donors did impart increased anxiety-like behavior to IVF-derived females and modestly increased limited access ethanol intake in IVFderived males. In summary, paternal preconception environment impacts ethanol- and stressrelated behavior in offspring, possibly via small noncoding RNAs in the germline and epididymal extracellular vesicles. |
