Small Noncoding RNA Contents Of Rat Epididymal Extracellular Vesicles Including A Putative Novel Small RNA

Extracellular vesicles (EVs) released from the epididymal epithelium, referred to as epididymosomes, impart functional competence on sperm as they transit the epididymis by merging with sperm and releasing a complex repertoire of molecules. The cargo of epididymal EVs includes small noncoding RNAs (sncRNAs) that are modified by external factors such as stress, nutrition, and drug use, which are delivered to sperm and affect offspring development and outcomes. The rat is an important translational model system for many fields including reproduction and transgenerational epigenetics, but there is little work on rat epididymosomes and their sncRNA cargo. To fill this gap in knowledge, in the current study we performed a comprehensive characterization of an epididymal EV preparation that we refer to as extracellular particles (EPs) because of the potential presence of other non-vesicular extracellular particles along with the EVs. EPs were collected from the caput epididymis, isolated, subjected to verification of the presence of EVs, and sncRNAs were sequenced. We considered nearly all known categories of small RNA and their fragment sub-types and in the process identified a subset that did not strictly fit the definition of any known category. These unique small RNAs are expressed strictly from within the boundaries of CpG islands and have a distinct 19-nt length. Their functional significance remains unknown, but they have characteristics of RNA fragments that can associate with the Argonaute/PIWI family of proteins and therefore could have regulatory function via RNA induced silencing or de novo DNA methylation.