Analysis of the small non-protein-coding RNA profile of mouse spermatozoa reveals specific enrichment of piRNAs within mature spermatozoa

Abstract

Post-testicular sperm maturation and storage within the epididymis is a key determinant of gamete quality and fertilization competence. Here we demonstrate that mouse spermatozoa possess a complex small non-protein-coding RNA (sRNA) profile, the composition of which is markedly influenced by their epididymal transit. Thus, although microRNAs (miRNAs) are highly represented in the spermatozoa of the proximal epididymis, this sRNA class is largely diminished in mature spermatozoa of the distal epididymis. Coincident with this, a substantial enrichment in Piwi-interacting RNA (piRNA) abundance in cauda spermatozoa was detected. Further, features of cauda piRNAs, including; predominantly 29-31 nts in length; preference for uracil at their 5' terminus; no adenine enrichment at piRNA nt 10, and; predominantly mapping to intergenic regions of the mouse genome, indicate that these piRNAs are generated by the PIWIL1-directed primary piRNA production pathway. Accordingly, PIWIL1 was detected via immunoblotting and mass spectrometry in epididymal spermatozoa. These data provide insight into the complexity and dynamic nature of the sRNA profile of spermatozoa and raise the intriguing prospect that piRNAs are generated in situ in maturing spermatozoa. Such information is of particular interest in view of the potential role for paternal sRNAs in influencing conception, embryo development and intergenerational inheritance.

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

“…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). On the other hand, recent study suggests that the acquisition of piRNA sequences by cauda epididymal spermatozoa may be via an epididymosome-independent mechanism, since not like other sncRNAs, piRNAs are largely absent in the epididymosomes (Hutcheon et al, 2017). 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).…”

“…The miRNA family is the first characterized sncRNAs in the epididymal tissue and spermatozoa. The sequences of piRNAs (Yan et al, 2011;Kawano et al, 2012;Li et al, 2012;Hutcheon et al, 2017), tsRNAs (Peng et al, 2012), and rsRNA-28S (Chu et al, 2017) were revealed by RNA-seq studies and validated by qPCR and northern blot. The sequences of piRNAs (Yan et al, 2011;Kawano et al, 2012;Li et al, 2012;Hutcheon et al, 2017), tsRNAs (Peng et al, 2012), and rsRNA-28S (Chu et al, 2017) were revealed by RNA-seq studies and validated by qPCR and northern blot.…”

“…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)]. Both tsRNAs and rsRNA-28S gain their abundance in the spermatozoa during caput to cauda transit, and recently it was reported of a similar dynamics of mouse epididymal sperm-borne piRNAs (Hutcheon et al, 2017), although the mechanism of their biogenesis could be different from each other. 1A).…”

“…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.…”

“…Individual miRNA (mouse miR-7578) or microRNA-like RNA (rat mil-Hon-grES2) (Ni et al, 2011) sequences were cloned and obtained from the epididymal small RNA library, and later the expression patterns of whole miRNAs in the epididymal epithelial cells and spermatozoa were profiled using microarrays and RNA-seq. The sequences of piRNAs (Yan et al, 2011;Kawano et al, 2012;Li et al, 2012;Hutcheon et al, 2017), tsRNAs (Peng et al, 2012), and rsRNA-28S (Chu et al, 2017) were revealed by RNA-seq studies and validated by qPCR and northern blot. There are other potential sncRNA classes in the mouse epididymal spermatozoa that are similar to those reported elsewhere, including the sequences of mitochondrial RNA-derived small RNAs (mito-sRNAs) (Ro et al, 2013) and snoRNA-derived small RNAs (sno-sRNAs) (Taft et al, 2009) (Table 1).…”