Small RNAs are trafficked from the epididymis to developing mammalian sperm

Abstract

SummaryRNAs 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 spermatogenesis, 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. CC-BY-NC 4.0 International license peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/194522 doi: bioRxiv preprint first posted online Sep. 27, 2017; INTRODUCTIONInheritance of phenotypic changes in the absence of any change in DNA is known as epigenetic inheritance, and is essential for cell state inheritance during mitosis. Epigenetic information is not only inherited during cell division, but at least some epigenetic information can be transmitted from one generation to the next in multicellular organisms, a mechanistically complex process that requires epigenetic information to be maintained throughout the disruptive processes of gametogenesis, fertilization, and early embryonic development. Although decades of study of cell state inheritance and other cases of epigenetic inheritance have identified several major molecular conduits of epigenetic information -chromatin structure, small RNAs, DNA modifications, prions, and transcription factors -much remains to be understood regarding the establishment and erasure of epigenetic information during gametogenesis and early development.Small (<40 nt) RNAs are one of the best-established carriers of epigenetic information, and play key roles in epigenetic inheritance paradigms in C. elegans (Fire et al

Post‐ejaculatory modifications to sperm (PEMS)

“…Temple-Smith & Bedford, 1980) and eutherian mammals (e.g. Bedford, 1979;Baker et al, 2005;Sullivan & Saez, 2013;Aalberts, Stout, & Stoorvogel, 2014;Skerget et al, 2015;Machtinger, Laurent, & Baccarelli, 2016;Sharma et al, 2018;Nixon et al, 2019a). Although less thoroughly studied, similar changes to sperm may occur within the Wolffian duct of reptiles (e.g.…”

Epididymal small non‐coding RNA studies: progress over the past decade

“…At the expense of 'resolution', this strategy maximized the affordability and provided a useful overview for following studies. Given the increasing demands for better resolutions, the progress in the sample preparation for high-throughput studies include the isolation of different epididymal regions (Belleannee et al, 2012;Chu et al, 2015) and the purification of particular cellular components such as epithelial cells (Nixon et al, 2015a), epididymosomes (Hutcheon et al, 2017), spermatozoa (Nixon et al, 2015a(Nixon et al, ,2015bSharma et al, 2016;Chu et al, 2017), and sperm cell parts (Sharma et al, 2018). Among them, spermatozoa can ooze out from the incisions and needle holes made on the epididymis.…”

“…Additionally, the existence of piRNA sequences in the mature spermatozoa in the cauda epididymis is also quite intriguing. It is gradually revealed that the substantial modifications in the sncRNA content in the spermatozoa is one of the major biological events during their epididymal transit (Nixon et al, 2015b;Chen et al, 2016a;Chu et al, 2017;Sharma et al, 2018). To present a global view of this dramatic sncRNA dynamics during spermatogenesis and sperm maturation in mice, we integrated the small RNA profiles of caput and cauda spermatozoa with those of the testicular spermatogenic cells [data collected from GEO Datasets: GSE24822 (Gan et al, 2011), GSE40397 (Peng et al, 2012) and our own RNA-seq data (GSE122006 and GSE122025)].…”

“…Apart from the well-known function of facilitating the secretion of proteins that do not have a signal peptide (Cornwall, 2009), the epididymosomes are largely involved the exchange of sncRNAs between the epithelium and the spermatozoa. The existence of different sncRNAs, including miRNAs (Belleannee et al, 2013a;Reilly et al, 2016) and tsRNAs (Sharma et al, 2018) in the epididymosomes sheds light on the previously underappreciated function of epididymosome-mediated sperm sncRNA modification, through the mechanism similar to what was described for exosomal trafficking of miRNAs in nematodes, plants (Sarkies & Miska, 2014), and somatic cell contexts (Valadi et al, 2007;Gibbings et al, 2009;Zhang et al, 2015). Recently, by using a 'thiouracil tagging (TU)' mouse model (Gay et al, 2013) to trace the newly synthesized RNAs in the epididymis, which are captured and sequenced by the metabolic sequencing of RNA (SLAM-Seq) method (Herzog et al, 2017), it was further validated that the sncRNAs transferred to the spermatozoa via epididymosomes are indeed originally produced in the epididymal epithelium (Sharma et al, 2018).…”

“…The existence of different sncRNAs, including miRNAs (Belleannee et al, 2013a;Reilly et al, 2016) and tsRNAs (Sharma et al, 2018) in the epididymosomes sheds light on the previously underappreciated function of epididymosome-mediated sperm sncRNA modification, through the mechanism similar to what was described for exosomal trafficking of miRNAs in nematodes, plants (Sarkies & Miska, 2014), and somatic cell contexts (Valadi et al, 2007;Gibbings et al, 2009;Zhang et al, 2015). Recently, by using a 'thiouracil tagging (TU)' mouse model (Gay et al, 2013) to trace the newly synthesized RNAs in the epididymis, which are captured and sequenced by the metabolic sequencing of RNA (SLAM-Seq) method (Herzog et al, 2017), it was further validated that the sncRNAs transferred to the spermatozoa via epididymosomes are indeed originally produced in the epididymal epithelium (Sharma et al, 2018). Among them, tsRNAs are likely to be generated by RNase A family members such as Angiogenin (Yamasaki et al, 2009) and RNase T family members (Andersen & Collins, 2012), as the epididymis-specific paralogs of Angiogenin-Rnase 9-12 are highly expressed in the epithelium (Castella et al, 2004;Zhu et al, 2007).…”