Extracellular vesicles (EVs) regulate multiple physiological processes. Seminal plasma contains numerous EVs which may deliver functional molecules such as small RNAs (sRNAs) to the sperm. However, the RNA profiles in the boar seminal plasma extracellular vesicles (SP-EVs) and its function has not been characterized. The aim of this study was to establish an effective method for isolation of boar SP-EVs, and characterize the functions and sRNAs profiles in the boar SP-EVs using deep sequencing technology. Briefly, boar SP-EVs were isolated by differential ultracentrifugation, and confirmed with the transmission electron microscope (TEM), nanoparticle tracking analysis (NTA) and western blot. The isolated boar SP-EVs contained numerous and diverse sRNAs families, including microRNAs (miRNAs, 9.45% of the total reads), PIWI-interacting RNAs (piRNAs, 15.25% of the total reads), messenger RNA fragments (mRNA, 25.30% of the total reads) and tRNA-derived small RNAs (tsRNA, 0.01% of the total reads). A total of 288 known miRNAs, 37 novel miRNA and 19749 piRNAs were identified in boar SP-EVs. The identified miRNAs may confer negative effects on sperm fertility based on dual-luciferase reporter experiment. This study therefore provides an effective method to isolate SP-EVs which is likely to spur research on SP-EVs in relation to sperm function and male fertility.
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