This study extends on the previous work that has identified the miRNA class of sRNA as an additional and potentially developmentally important tier of regulation in the male reproductive tract11,22,26,28,29,30. Through development of a tractable protocol for the isolation of mouse epididymosomes, here we provide novel insight into the complexity of the segment specific miRNA profiles of these extracellular vesicles as well as exploring their capacity to deliver this important regulatory cargo to maturing spermatozoa. Our data indicate that, in addition to the more widely reported role as transporters of protein7,31,32 and lipid cargo9, epididymosomes are also carriers of miRNA, a developmentally important class of regulatory RNA. Further, many of the profiled miRNA were determined to be at considerably enriched levels compared to parent cells (the epididymal epithelial cells). Indeed, almost a third of the miRNAs detected in the epididymosome fraction were not present in our equivalent profiling of epididymal epithelial cell miRNAs26. While we cannot entirely discount the possibility that such differences may, in part, reflect either: (i) greater depth of sequence coverage achieved in our current analysis, (ii) profiling of a subset of epididymosomes originating from a non-surveyed epithelial cell population upstream of the caput epididymis, or (iii) contamination of our samples with vesicles released from ruptured cytoplasmic droplets; the data presented here does nevertheless accord with independent evidence that the epididymosome miRNA signature diverges from that of the epithelial cells from which they originate11. Taken together, the observations made to date present strong evidence that the packaging of the molecular cargo into epididymosomes is a highly selective, rather than stochastic process. Such a model draws support from a wealth of extracellular vesicle based studies33,34,35, which have shown that abundant RNA species in extracellular vesicles can remain virtually undetectable in the parent cell36. However, the precise sorting mechanism responsible for discriminating the molecular payload of these vesicles from that of their parent cells remains poorly understood. While the presence of consensus exomotifs (e.g. GGAG and CCCU)37 have been reported within the 3′ half of the mature sRNA sequence of miRNAs selectively incorporated into some exosome populations, similar motifs were only detected in the 3′ half of a small portion (25/358; ~7%) of the epididymosome-borne miRNAs identified here (Supplementary Figure S2). Such findings do not preclude the possibility that alternative exomotifs may be present among these miRNAs or that non canonical pathways38 may be adopted for the extracellular export of miRNA in the epididymis.

The epididymis may represent an interesting model to address the question of selective vesicle packaging considering the substantive segment-segment variation in epididymosome miRNA profiles reported here. Indeed, we identified marked changes in epididymosome miRNA profiles between epididymal segments, including an apparent gradient of increasing profile complexity between the proximal and distal epididymal segments. This finding mirrors that of the sperm miRNA profile, that is; progressive modification of the miRNA profile as the sperm descend through the proximal epididymal segments (caput and corpus) before undergoing extensive changes coincident with their prolonged residence in the distal (cauda) epididymis22. These miRNA profiles do however, contrast with well established paradigms of epididymal sperm maturation that attribute the majority of functional changes to the proximal segments (distal caput/proximal corpus)39. From these data we infer that the modification of the sperm miRNA profile is not strictly tied to the functional maturation of these cells. Irrespective, they identify the epididymis as an important site in establishment of the sperm epigenome and since these cells are incapable of de novo transcription, they also firmly implicate epididymosomes as a conduit for the transfer of such developmentally important regulatory information. Consistent with this hypothesis, we identified substantial overlap in the miRNA signature of both spermatozoa and epididymosomes with as many as 82% of sperm-borne miRNAs also being detected in epididymosomes. Prominent among these were members of the let7, miR-30, miR-465, miR-466, miR-467 and miR-669 clusters.

We further exploited a co-incubation strategy originally developed in the bovine model27 to provide proof-of-concept that mouse epididymosomes can directly interact with homologous spermatozoa. Moreover, we demonstrate that this is a productive interaction leading to an apparent uptake and significant accumulation, of several prominent epididymosome miRNAs. While we have yet to explore the full extent of miRNA transfer facilitated during this interaction, we did make the striking observation that it was highly selective. In this context, an encapsulated tracer dye (CFSE) was exclusively delivered from epididymosomes to the sperm head and mid-piece of the flagellum. Given the transcriptionally inert state of the mature spermatozoon, it is considered unlikely the uptake of miRNA into these intracellular domains would have any direct impact on the functional profile of these cells. Such restricted deposition would however, ideally position the miRNAs for entry into the oocyte cytosol at the time of fertilisation, thus enhancing the prospect that these miRNAs could serve as mediators of the epigenetic regulation of the resultant embryo. However, proof of an epigenetic regulatory role for epididymosome delivered miRNAs requires further evidence demonstrating that sperm-borne miRNAs control transcription homeostasis in fertilised oocytes, zygotes and two-cell embryos40. It is also notable that the sperm domains labelled with CFSE here perfectly align with the distribution of proteins trafficked to bovine spermatozoa via epididymosomes, which are also found to preferentially localise to the acrosomal and mid-piece domains27. The mechanism(s) by which such selective recognition and uptake of epididymosome cargo may be conferred have yet to be fully elucidated, but could conceivably involve complementary ligands/receptors furnished on the surface of the epididymosomes and the recipient spermatozoa41. In this context, previous work has shown that epididymosomes contain a variety of candidate adhesion molecules, including tetraspanins (CD9), integrins and milk fat globule-epidermal growth factor 8 protein42. A similar repertoire of ligands have been documented on a variety of extracellular vesicles suggesting that they may be a universal feature to help target these entities and ensure selectivity in their uptake among the hundreds of cell types that they may encounter35.

As an additional tier of specificity, it was also noted that epididymosomes appeared to exclusively interact with live cells; we consistently failed to detect any fluorescent dye labelling of dead or moribund cells. Importantly, such selectivity was not artefactual as illustrated by the strong fluorescent signal generated throughout the entire spermatozoon (both live and dead cells) upon direct incubation with free CSFC. An important precedent for these findings has been provided by the work of Sullivan and colleagues who have shown that epididymosomes constitute a heterogeneous pool that can be subdivided into at least two populations on the basis of size and the presence/absence of CD932,43. The smaller of these (~10 to 100 nm) bear the CD9 antigen and bind preferentially to live spermatozoa, whereas the larger CD9 negative sub-population possess higher affinity for dead cells6. While the scale of epididymosome recovery from mice precluded the possibility of exploring such heterogeneity, the characteristics of the epididymosomes isolated here (i.e. CD9 positive, diameter of 50 to 150 nm) suggest that our isolation protocol may have been biased toward the former population. In any case, the ability of epididymosomes to actively bind live cells supports the concept that this interaction is tightly regulated and raises the intriguing possibility that the epididymis is able to discriminate cell quality and restrict its investment to the processing of viable cells6.

Taken together, this work substantiates the growing consensus that the epididymis serves as a key staging point for establishment of the sperm epigenome14. Importantly, this epigenome may be altered by a range of environmental insults44. The relatively high degree of overlap documented among reported epididymosome miRNA profiles (Supplementary Tables S4 and S5)11,45 strongly suggests that this mode of intercellular communication may be highly conserved across mammalian species. Indeed, notwithstanding limitations imposed by the use of different methodology for miRNA identification (next generation sequencing versus microarray approaches), our mouse epididymosome dataset comprised as many as 88% of the miRNAs that have previously been documented in bovine epididymosomes11 (Supplementary Table S5). Further and while beyond the scope of the present study, it is likely that epididymosome function may also extend to the horizontal transfer of additional species of regulatory non-protein-coding RNA, including (but not limited to) transfer RNA fragments (tRFs), piwi-interacting RNAs (piRNAs) and other subclasses of small-interfering RNA (siRNA), thus potentially contributing to pronounced epigenetic alterations (such as metabolic/reproductive disruption and adverse behavioural symptoms) on subsequent generations15,16,46. Indeed, the recent studies of Rando and colleagues (2016) present compelling evidence that dietary perturbations can alter the profile of tRFs delivered to sperm via epididymosomes12. Further exploration of this pathway for information transfer is thus likely to prove a productive avenue for future research, particularly in the context of addressing pertinent questions, such as: how do epididymal soma respond to environmental cues to alter the molecular cargo of epididymosomes14? This is particularly perplexing in view of the fact that many of the stressors linked to changes in the sperm epigenome15,17,19 occur at sites distal from the male reproductive tract and that this tissue apparently lacks the innervation3 required for conveying extrinsic stress-induced neuronal factors directly to the sperm. This has encouraged speculation that the heterogeneous population of epididymosomes that sperm encounter may include contributions, albeit minor, from somatic cells that lay beyond that of the male reproductive tract13. While the validity of such a model awaits further scrutiny, it is notable that genetic markers originating from distal somatic cells have been detected in epididymal mouse spermatozoa and crude preparations of plasma extracellular vesicles47.

Notably, although a focus for our work has been epididymosome-sperm interactions, this does not discount the possibility that these extracellular vesicles hold a fundamental role in relaying regulatory information to enforce the strict control of epididymal epithelial cell function. Certainly, extracellular vesicles are replete in most biological fluids and have been conclusively shown to convey miRNA cargo to recipient cells where they act to initiate RNA regulatory pathways48. Further support for this form of paracrine regulation has been afforded by the elegant study of Sullivan and colleagues who have shown that epididymosomes can bind and subsequently transfer miRNAs, directly to cultured epididymal epithelial cells11. Such a mechanism could underpin the control of at least a portion of the >17,000 genes that are known to be expressed in the mouse epididymis49 and conceivably account (at least partially) for the segment-specific patterns of gene expression and/or protein abundance that characterise this ductal system3. Indeed, comprehensive transcriptomic analyses have led to the demarcation of 6 unique transcriptional units within the mouse epididymis49. Thus, in dividing the epididymis into three broad anatomical segments, we may have inadvertently overlooked some of the subtlety associated with epididymosome miRNA profiles. Despite this, an analysis of the key biological pathways potentially targeted by differentially accumulating miRNAs revealed a majority centred on regulation of cellular growth and proliferation, cellular development and cell death and survival, as might be expected of molecules involved in the maintenance of epididymal homeostasis. Given that epididymosomes also feature among the constituents of seminal fluid that are delivered to the female tract at the time of ejaculation, it must also be considered that they could exert similar regulatory control within the female reproductive with potential implications for conditioning of the periconceptual environment50,51.

In summary, this study reports the comprehensive mapping of the miRNA profile of mouse epididymosomes under normal physiological conditions. In so doing, we have revealed a complex profile that is discrete from that of their parent cells. These data support the selective processing and packaging of the macromolecular cargo of epididymosomes and demonstrate that this selective packaging further extends to their downstream interactions with spermatozoa. The significance of such findings lie in their validation of a widely promulgated model of intercellular communication between the epididymal soma and maturing germ cells. In addition to potential implications for epigenetic mechanisms of inheritance, these data identify epididymosomes as a potential conduit for modulating the environments of both the male and female reproductive tracts through the delivery of RNA silencing substrates. Further research is now warranted to explore the extent of the role epididymosomes play in such phenomena.