Extracellular Vesicles as Mediators of EDC-Induced Epigenetic Changes in Male Reproductive Health - PROJECT SUMMARY/ ABSTRACT Exposures to environmental endocrine-disrupting chemicals (EDCs), especially during early life, are strongly linked to adverse health outcomes including neurobehavioral, reproductive, and other endocrine dysfunctions. EDC exposures to a fetus (F1) also exposes the germline (F2) and causes heritable epigenetic changes that are passed on to future generations. The male reproductive tract is of primary interest, particularly the epididymis, where spermatozoa undergo essential maturational processes. Epididymosomes, extracellular vesicles of the epididymal epithelium, transfer bioactive cargo like small non-coding RNAs (sncRNAs) to sperm, influencing their molecular composition. SncRNAs, including micro-RNA and transfer RNA fragments (tRFs), are emerging as significant regulators of epigenetics effects. The epigenome is environmentally responsive; thus, EDC exposure can modify the selection and packing of sncRNAs into epididymosomes, which are then transferred to sperm, thus affecting reproductive outcomes. There are several limitations to prior work that I will overcome in the current F31 application. Most EDC research is limited to a single tissue type or a single mechanism with a limited number of targets. The field is also limited by a surprisingly small number of studies that address how epigenetic programming propagates from somatic cells to gametes and causes reproductive disorders. Lastly, the ability of sncRNA from epididymosomes to respond to environmental insults and communicate such changes to sperm is a fundamental question, one that (to my knowledge) has never been addressed using an EDC mixture. Therefore, this F31 application has three overarching areas of inquiry. 1) How does EDC exposure modify epididymosome cargo in the F1 and F2 generations, specially the sncRNA profile? 2) Are sperm sncRNA profiles influenced by changes to epididymosomes? 3) What are the effects of EDC exposure on reprogramming of sperm DNA methylation in the F1 and F2 generations? To address these questions, we will use our established rat EDC exposure model with human-relevant chemicals, dosages, and route, in which direct (F1) and intergenerational (F2) work will be performed in the epididymis—specifically epididymosomes and sperm. We will profile the epididymosomes and sperm at the DNA and/or sRNA level, enabling us to pinpoint sncRNAs influenced by EDCs, and how phenotypes are propagated from gametes to individuals and across generations. Novel bioinformatic pipelines established by our laboratory will inform on these mechanisms individually, as well as their relationships. Crucially, the lines of work in sRNA and DNA will be connected by relating sncRNA reads to sperm DNA methylation status, thereby determining how epigenomic marks in gametes relate to epididymosome and sperm sncRNA cargo. These data will establish definitive epigenetic profiles that will allow us to identify the origin of EDC induced epigenetic modifications and provide potential targets for therapeutics in humans, with which the mechanisms studied in rats are highly conserved. The support provided by the F31 NRSA fellowship will enable me to fully realize the promise of this likely paradigm-shifting research.
