Mechanisms of EDC Effects via Small-RNA Cargo in Sperm Epididymosomes

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ABSTRACT Exposures to environmental endocrine-disrupting chemicals (EDCs), especially during early life, are strongly linked to adverse health outcomes. Furthermore, EDC exposures to the fetus, which also expose the germ cells (precursors to sperm and egg) within the fetus, can cause heritable epigenetic changes that are passed to future generations. Previous work has implicated epimutations of DNA CpG methylation as one molecular mechanism by which EDCs program the germline. Proposed work will explore this mechanism, which is under-studied, and in addition, seek to understand how external factors from the male reproductive tract involved in sperm maturation may themselves contribute to epimutations. More specifically, the proximal portion (caput) of the epididymis has a substantial influence on sperm during their final steps of maturation. Extracellular vesicles (EVs) from the epididymis – epididymosomes - released from the epithelium fuse and transfer their diverse molecular contents to sperm, including small noncoding RNAs (sncRNA), thereby contributing to the functional competency of sperm and to the developmental trajectory of a fertilized embryo. These sncRNAs have the capacity to regulate gene expression and direct DNA methylation in sperm. In the current proposal, we will establish whether and how two different EDC classes that signal via differing hormonal mechanisms cause direct CpG methylation changes to DNA, and/or change the transfer of sncRNA content of epididymosomes to sperm to indirectly cause epimutations. The two EDC classes studied here are the polychlorinated biphenyl mix Aroclor 1221 (A1221), an industrial mixture that has weakly estrogenic properties; and vinclozolin (VIN), an anti- androgenic fungicide. Both EDCs are still environmentally relevant. Rats are exposed to human relevant dosages of A1221 or VIN, or vehicle, through feeding the dams during gestation and lactation. In adult male rats, sncRNA cargo of EVs from caput epididymis and mature sperm from the cauda epididymis will be extracted and used for deep sequencing and bioinformatics. DNA from sperm in the same samples will be subjected to reduced representation bisulfite sequencing to identify epimutations in CpG islands. These data will establish a definitive epigenetic profile that will allow us to pinpoint the origin of EDC induced epimutations. This proposal is biomedically relevant, as all humans are exposed to EDCs, with mechanisms of sperm maturation highly conserved across mammals.