RNAs present in mature mammalian sperm are delivered to the zygote at fertilization, where they have the potential to affect early development. The biogenesis of the small RNA payload of mature sperm is therefore of great interest, as it may be a target of signaling pathways linking paternal conditions to offspring phenotype. Recent studies have suggested the surprising hypothesis that the small RNA payload carried by mature sperm may include RNAs that were not synthesized during testicular

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... matogenesis, but that are instead delivered to sperm during the process of post-testicular maturation in the epididymis. To further test this hypothesis, we characterized small RNA dynamics during testicular and post-testicular germ cell maturation in mice. We show that purified testicular germ cell populations, including mature testicular spermatozoa, carry extremely low levels of tRNA fragments (tRFs), and that tRFs become highly abundant only after sperm have entered the epididiymis. The process of small RNA delivery to sperm can be recapitulated in vitro, as caput epididymosomes deliver small RNAs including tRFs and microRNAs to mature testicular spermatozoa. Finally, to definitively identify the tissue of origin for small RNAs in sperm, we carried out tissue-specific metabolic labeling of RNAs in intact mice, finding that mature sperm carry small RNAs that were originally synthesized in the somatic cells of the epididymis. Taken together, our data demonstrates that soma-germline small RNA transfer occurs in male mammals, most likely via vesicular transport from the epididymis to maturing sperm.