The functional maturation of spermatozoa that is necessary to achieve fertilization occurs as these cells transit through the epididymis, a highly specialized region of the male reproductive tract. A defining feature of this maturation process is that it occurs in the complete absence of nuclear gene transcription or de novo, protein translation in the spermatozoa. Rather, it is driven by sequential interactions between spermatozoa and the complex external milieu in which they are bathed within lumen of the epididymal tubule. A feature of this dynamic microenvironment are epididymosomes, small membrane encapsulated vesicles that are secreted from the epididymal soma. Herein, we report comparative proteomic profiling of epididymosomes isolated from different segments of the mouse epididymis using multiplexed tandem mass tag (TMT) based quantification coupled with high resolution LC-MS/MS. A total of 1640 epididymosome proteins were identified and quantified via this proteomic method. Notably, this analysis revealed pronounced segment-to-segment variation in the encapsulated epididymosome proteome. Thus, 146 proteins were identified as being differentially accumulated between caput and corpus epididymosomes, and a further 344 were differentially accumulated between corpus and cauda epididymosomes (i.e., fold change of ≤ −1.5 or ≥ 1.5; p, < 0.05). Application of gene ontology annotation revealed a substantial portion of the epididymosome proteins mapped to the cellular component of extracellular exosome and to the biological processes of transport, oxidation-reduction, and metabolism. Additional annotation of the subset of epididymosome proteins that have not previously been identified in exosomes revealed enrichment of categories associated with the acquisition of sperm function (e.g., fertilization and binding to the zona pellucida). In tandem with our demonstration that epididymosomes are able to convey protein cargo to the head of maturing spermatozoa, these data emphasize the fundamental importance of epididymosomes as key elements of the epididymal microenvironment responsible for coordinating post-testicular sperm maturation.
Graphical Abstract
Graphical Abstract
Mammalian spermatozoa acquire the potential to fertilize an ovum as they navigate the epididymis, an exceptionally long convoluted tubule that connects the testis to the vas deferens. This maturation process encompasses a suite of cellular modifications that endow spermatozoa with the potential to sustain forward progressive motility, capacitate and subsequently participate in the cellular interactions required to achieve conception (
1.
). Among the singular features that discriminate epididymal maturation from that of the preceding phases of gamete development (2.
) is that it is driven entirely by extrinsic factors in the absence of nuclear gene transcription and de novo, protein translation in the spermatozoa (3.
, 4.
). Indeed, it is widely held that the complex intraluminal microenvironment created by the epididymal epithelium serves as the key determinant in the functional transformation of spermatozoa (5.
, 6.
). Accordingly, the epididymal soma is characterized by a marked division of labor such that the proximal segments (initial segment, caput and corpus epididymis) promote sperm maturation, whereas the distal caudal segment supports sperm storage (1.
). Such functions are reflected in distinctive gene expression profiles (7.
, 8.
, 9.
) that, in turn, dictate segment-specific secretion of proteins and a range of additional biomolecules into the luminal fluid and thus establish the unique physiological compartments that affect sperm maturation and prolonged sperm survival (3.
, 10.
, 11.
, 12.
).In recognition of the importance of epididymal function in governing sperm quality, this tissue has long been of interest as a potential site for contraceptive intervention (
13.
, 14.
, 15.
, 16.
). Conversely, the epididymis has also generated interest from the standpoint of therapeutic treatment strategies to combat sperm dysfunction associated with male factor infertility (17.
, 18.
, 19.
, 20.
). The realization of both goals is predicated on resolution of the mechanistic basis by which the sperm proteome is so dramatically altered during the key developmental window of epididymal maturation. Among the potential mechanisms capable of mediating the bulk exchange of proteomic information to maturing spermatozoa, epididymosomes have emerged as attractive candidates (21.
, 22.
, 23.
, 24.
, 25.
, 26.
). Epididymosomes represent a heterogeneous population of small membrane bound extracellular vesicles (EVs) 1
The abbreviations used are: EV, extracellular vesicle; ADAM3, a disintegrin and metallopeptidase domain 3 (cyritestin); ADAM7, a disintegrin and metallopeptidase domain 7; B4GALT1, beta-1,4-galactosyltransferase 1; BWW, Biggers, Whitten, and Whittingham medium; CD, complement dependent; CUZD1, CUB and zona pellucida-like domain-containing protein; CLU, clusterin; DAVID, database for annotation, visualization and integrated discovery; DNM2, dynamin 2; DTYMK, deoxythymidylate kinase; FDR, false discovery rate; FLOT1, flotillin 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GLUL, glutamate-ammonia ligase (glutamine synthetase); GO, gene ontology; HILIC, hydrophilic interaction chromatography; HSPA2, heat shock protein 2; HSP90B1, heat shock protein 90, beta (Grp94), member 1; IZUMO1, izumo sperm-egg fusion 1; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MFGE8, milk fat globule-EGF factor 8 protein; MPC2, mitochondrial pyruvate carrier 2; NOLC1, nucleolar and coiled-body phosphoprotein 1; NUCB1, nucleobindin-1; ODF2, outer dense fiber protein 2; PDIA6, protein disulfide isomerase associated 6; PROM2, prominin 2; PSM, peptide spectrum matches; PSMD7, proteasome (prosome, macropain) 26S subunit non-ATPase, 7; SNARE, soluble NSF attachment protein receptor; TEAB, triethylammonium bicarbonate; TMT, tandem mass tag; ZP3R, zona pellucida 3 receptor; ZPBP2, zona pellucida binding protein 2.
1The abbreviations used are: EV, extracellular vesicle; ADAM3, a disintegrin and metallopeptidase domain 3 (cyritestin); ADAM7, a disintegrin and metallopeptidase domain 7; B4GALT1, beta-1,4-galactosyltransferase 1; BWW, Biggers, Whitten, and Whittingham medium; CD, complement dependent; CUZD1, CUB and zona pellucida-like domain-containing protein; CLU, clusterin; DAVID, database for annotation, visualization and integrated discovery; DNM2, dynamin 2; DTYMK, deoxythymidylate kinase; FDR, false discovery rate; FLOT1, flotillin 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GLUL, glutamate-ammonia ligase (glutamine synthetase); GO, gene ontology; HILIC, hydrophilic interaction chromatography; HSPA2, heat shock protein 2; HSP90B1, heat shock protein 90, beta (Grp94), member 1; IZUMO1, izumo sperm-egg fusion 1; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MFGE8, milk fat globule-EGF factor 8 protein; MPC2, mitochondrial pyruvate carrier 2; NOLC1, nucleolar and coiled-body phosphoprotein 1; NUCB1, nucleobindin-1; ODF2, outer dense fiber protein 2; PDIA6, protein disulfide isomerase associated 6; PROM2, prominin 2; PSM, peptide spectrum matches; PSMD7, proteasome (prosome, macropain) 26S subunit non-ATPase, 7; SNARE, soluble NSF attachment protein receptor; TEAB, triethylammonium bicarbonate; TMT, tandem mass tag; ZP3R, zona pellucida 3 receptor; ZPBP2, zona pellucida binding protein 2.
(27.
, 28.
, 29.
) that are released from the epididymal epithelium via an apocrine secretory mechanism (30.
, 31.
, 32.
). This pathway is characterized by the formation of cytoplasmic protrusions along the apical margin of the principal epithelial cells (30.
). Following detachment, these “apical blebs” break down to release their contents, including epididymosomes, into the luminal environment (30.
) where they have the potential to interact with spermatozoa and mediate the transfer of a complex proteinaceous cargo to these cells (29.
).The participation of epididymosomes in the alteration of the sperm proteome draws on a wealth of evidence that EVs, released from virtually all somatic tissues, can facilitate the delivery of a diverse macromolecular payload (comprising proteins, lipids, and nucleic acids) to recipient cells (
33.
). It is also consistent with pioneering studies of Sullivan and colleagues who have demonstrated that bovine epididymosomes have the capacity to mediate the selective transfer of epididymal secretory proteins to homologous spermatozoa (26.
). At present however, the conservation of this form of intercellular communication for the en masse, delivery of proteins has yet to be substantiated in common laboratory models such as the rodents. To begin to address this challenge, we have surveyed the proteomic composition of epididymosomes isolated from different segments of the mouse epididymis using multiplexed tandem mass tag based relative quantification coupled with offline HPLC and LC-MS/MS. Further, we have exploited a co-culture system to demonstrate the uptake of biotinylated protein cargo from mouse epididymosomes primarily into the sperm head.DISCUSSION
A salient feature of the mammalian epididymis is its tremendous capacity for protein synthesis and secretion (
12.
). Such activity underpins the primary roles of this tissue in promoting the functional maturation of the male gamete as well as their prolonged storage in a viable state (10.
, 20.
). Both roles necessitate efficient mechanism(s) of delivering protein, and presumably other regulatory cargo (50.
), to the developing sperm cells. Among the potential mechanisms that could facilitate such bulk transfer, there is now compelling evidence supporting at least two; namely, via nonpathological amyloid matrices (61.
) and/or epididymosomes (21.
). The former of these, which may also equate to epididymal dense bodies (62.
, 63.
), have been proposed to coordinate interactions between the epididymal luminal contents and spermatozoa, although the extent and biological significance of such interactions remain to be fully resolved. By contrast, the constitutive shedding of epididymosomes appears pivotal in terms of modulating sperm function (29.
). Indeed, our study adds to a growing body of evidence that, despite the relatively simple structure of these nano-sized membranous vesicles, they encapsulate an extremely rich and diverse proteomic cargo; and one that is commensurate with their putative role as key intermediaries in soma-spermatozoa communication (21.
).Although the preparation of epididymosomes has been reported in several species, to date the comprehensive molecular profiling of their cargo has predominantly been restricted to that of large domestic species (e.g., bull). Such species hold obvious advantages in terms of permitting the collection of large volumes of uncontaminated intraluminal fluids from along the length of the epididymal tract (
21.
, 26.
). Regrettably, the application of equivalent collection protocols in smaller laboratory animals such as the mouse is technically very challenging, particularly in the context of recovering enough material to permit detailed end point characterization of the epididymosome proteome. Through necessity, we have instead pursued the isolation of epididymosomes from samples of luminal fluid obtained by puncture of the epididymis and processing by successive centrifugations to purify these vesicles. Although our previous studies have reported the utility of this approach in effectively eliminating cellular debris and sperm fragments (34.
), we readily acknowledge that we cannot entirely mitigate against the possibility of some epithelial and/or sperm cell contamination. One potential source of contamination is that of the cytoplasmic droplet, a nascent structure formed as a legacy of spermiogenesis during which spermatozoa are remodeled to remove most of their cytoplasm. This residual body is subsequently shed from the maturing sperm cell as they are conveyed through the epididymis. Of relevance to our study, the cytoplasmic droplet does contain numerous vesicles of roughly equivalent size to epididymosomes and could thus be co-isolated alongside this population of exosomes. Adding to this concern is that recognition that the cytoplasmic droplet serves to compartmentalize proteins implicated in membrane trafficking pathways, glucose transport, glycolysis, actin, tubulin and the proteasomal complex (- Reilly J.N.
- McLaughlin E.A.
- Stanger S.J.
- Anderson A.L.
- Hutcheon K.
- Church K.
- Mihalas B.P.
- Tyagi S.
- Holt J.E.
- Eamens A.L.
- Nixon B.
Characterisation of mouse epididymosomes reveals a complex profile of microRNAs and a potential mechanism for modification of the sperm epigenome.
Sci. Rep. 2016; 6: 31794
64.
). Given this possibility for contamination, it was particularly reassuring that “extracellular exosome” featured among the top ranked cellular component categories identified in the epididymosome reported herein. Indeed, ∼82% of the total epididymosome protein cargo identified herein have previously been identified as genuine exosome-borne cargo in other cellular models. Similarly, “transport” and the ancillary categories of “protein transport” and “vesicle-mediated transport” were identified among the dominant biological processes annotated from the complete mouse epididymosome proteome. Moreover, immunoblotting confirmed that the epididymosome fractions studied herein were devoid of several sperm-specific markers, including well characterized proteins of testicular origin (i.e., IZUMO1, ADAM3 and ODF2).- Au C.E.
- Hermo L.
- Byrne E.
- Smirle J.
- Fazel A.
- Kearney R.E.
- Smith C.E.
- Vali H.
- Fernandez-Rodriguez J.
- Simon P.H.
- Mandato C.
- Nilsson T.
- Bergeron J.J.
Compartmentalization of membrane trafficking, glucose transport, glycolysis, actin, tubulin and the proteasome in the cytoplasmic droplet/Hermes body of epididymal sperm.
Open. Biol. 2015; 5
Although such evidence affirms our enrichment of epididymosomes, our compiled proteomic inventory did contain several categories of protein that one may not reasonably expect to be associated with an extracellular vesicle destined to be delivered to spermatozoa, or downstream segments of the male/female reproductive tracts. We did not anticipate the presence of proteins mapping to the broad functional categories of nucleotide binding and processing; with relatively large numbers of histone variants and ribonucleoproteins serving as cases in point. Although it is difficult to envisage the functional significance of such findings, they are certainly not without precedent. In this context, equivalent proteins have been documented in populations of exosomes originating from cell types as diverse as fibroblasts, mast cells, neural stem cells, dendritic cells, and oligodendrocytes (
65.
, 66.
, 67.
, - Cossetti C.
- Iraci N.
- Mercer T.R.
- Leonardi T.
- Alpi E.
- Drago D.
- Alfaro-Cervello C.
- Saini H.K.
- Davis M.P.
- Schaeffer J.
- Vega B.
- Stefanini M.
- Zhao C.
- Muller W.
- Garcia-Verdugo J.M.
- Mathivanan S.
- Bachi A.
- Enright A.J.
- Mattick J.S.
- Pluchino S.
Extracellular vesicles from neural stem cells transfer IFN-gamma via Ifngr1 to activate Stat1 signaling in target cells.
Mol. Cell. 2014; 56: 193-204
68.
). A subset of these proteins have also been recorded among the constituents of bovine epididymosomes (24.
). Further, it is acknowledged that mature spermatozoa do harbor the basic, and presumably obsolete, machinery to synthesize proteins, including numerous cytoplasmic and mitochondrial ribosomal proteins (69.
). It is widely held that such proteins simply represent remnants of the spermatogenic process. However, our study raises the intriguing prospect that the complement of these proteins may also be supplemented during post-testicular sperm development via interaction with epididymosomes. In the absence of evidence substantiating the synthesis of proteins from nuclear-encoded genes in sperm, such proteins may be subverted for alternative non-canonical functions or may be transmitted to the oocyte upon fertilization to participate in early embryogenesis. Further work is clearly required to substantiate these possibilities and thus refine our understanding of the biological implications of such enrichment.Notably, functional annotation of the subset of the ∼18% of epididymosome proteins that were identified as not having not been reported in previous exosome proteomic catalogues, revealed an abundance of candidates linked to sperm maturation and/or fertilization; characteristics that one may logically expect to be associated with the functional transformation of the male gamete. Notable examples include the functional subunits of the chaperonin containing TCP1 complex (CCT/TRiC) as well as the putative interacting partners of ZP3R and ZPBP2, which have been implicated in the mediation of sperm-oocyte interactions (
70.
, 71.
). Such findings suggest that this “non-conserved” subset of epididymosome-borne proteins may be of interest in helping to decipher the mechanisms driving the functional maturation of spermatozoa; potentially extending to a directed analysis of species-specific elements of these pathways. In a similar context, the abundance of these proteins assigned to the broad categories of “transport” and “oxidation-reduction” may hold important information in terms of dissecting the mechanistic basis of epididymosome-biogenesis/trafficking and protection of the mature gamete from free radical injury, respectively.Our collective data also support the notion that epididymosome-sperm interaction is selective, with biotinylated epididymosome proteins being preferentially sequestered into a discrete physiological domain known as the post-acrosomal sheath, which is located within the posterior of the sperm head. Although such selectivity raises the prospect that unique compositional characteristics of the sperm plasma membrane directly influence the efficacy of epididymosome uptake, regrettably the mechanistic basis of this process has yet to be completely resolved. Notably, it has been argued that endocytic uptake, one of the principal routes of exosome internalization in somatic cells (
33.
), is severely compromised in mature spermatozoa (72.
). Indeed, cytochemical investigations have reported that spermatozoa lack the machinery needed to internalize exogenous molecules via endocytosis and are also devoid of the lysosomal organelles that serve as the typical targets for endocytosed cargo (- Jones S.
- Lukanowska M.
- Suhorutsenko J.
- Oxenham S.
- Barratt C.
- Publicover S.
- Copolovici D.M.
- Langel U.
- Howl J.
Intracellular translocation and differential accumulation of cell-penetrating peptides in bovine spermatozoa: evaluation of efficient delivery vectors that do not compromise human sperm motility.
Hum. Reprod. 2013; 28: 1874-1889
72.
). Further, mature spermatozoa are apparently also incapable of the active lipid recycling necessitated by endocytosis (- Jones S.
- Lukanowska M.
- Suhorutsenko J.
- Oxenham S.
- Barratt C.
- Publicover S.
- Copolovici D.M.
- Langel U.
- Howl J.
Intracellular translocation and differential accumulation of cell-penetrating peptides in bovine spermatozoa: evaluation of efficient delivery vectors that do not compromise human sperm motility.
Hum. Reprod. 2013; 28: 1874-1889
73.
). In view of this evidence, it is possible that spermatozoa employ non-canonical pathways for uptake of epididymosomes, such as direct fusion occurring at the interface of the respective membranes (33.
). In this context, several complementary protein families implicated as key regulators of membrane/vesicle fusion-based pathways have been identified in spermatozoa (69.
) and in the epididymosome proteome reported herein. Examples of the latter proteins include those of the soluble NSF attachment protein receptor (SNARE) (e.g., VAT1, STX3, STX4, STX5, STX6, STX7, STX8, STX12, STX16, STX17, STXBP2, STXBP3), RAB small GTPase (RAB1A, RAB1B, RAB2A, RAB2B, RAB3A, RAB5A, RAB5B, RAB5C, RAB6B, RAB7A, RAB8A, RAB8B, RAB9A, RAB11B, RAB13, RAB14, RAB18, RAB22A, RAB23, RAB24, RAB25, RAB35), and SEC (SEC11A, SEC13, SEC22B, SEC23A, SEC23B, SEC23IP, SEC24A, SEC31A) related families. Alternatively, it has been postulated that selective trafficking of epididymosome cargo to recipient sperm cells may be coordinated by the lipid raft-like properties of the vesicular membranes (74.
). In this regard, it is known that the lipid composition of mouse epididymosomes is dynamically remodeled in different epididymal segments such that these vesicles become progressively more rigid in the distal segments of the duct (75.
). At present it is not known what implications this has in terms of epididymosome-sperm and/or epididymosome-soma interactions. Clearly, additional work is needed to distinguish the relative contribution of the putative route(s) of sequestration of epididymosome contents into recipient cells. In guiding this work however, it is notable that previous studies have described the fusogenic properties of bovine epididymosomes and provided compelling evidence that such interactions lead to significant changes in the lipid and protein composition of epididymal sperm (22.
). These findings are consistent with our own immunoelectron microscopy analyses, which confirmed the presence of stalk-like projections forming at the interface of epididymosome-spermatozoa contact within the lumen of the epididymis. Such ultrastructural features have previously been recorded and taken as evidence of vesicle fusion between spermatozoa and oviductosomes (extracellular membrane vesicles released into the oviductal fluid) (76.
), raising the prospect of conserved mechanisms for facilitating cargo delivery between spermatozoa and the different populations of extracellular vesicles they encounter en route, to the site of fertilization. Accordingly, epididymosome protein transfer was significantly inhibited by antibody masking of MFGE8, a protein that possesses an RGD recognition motif implicated in integrin/ligand interactions that proceed cellular fusion (59.
).In summary, the data obtained in the present study provides novel insights into the diversity of the proteomic landscape encapsulated within mouse epididymosomes. In accordance with previous work, our findings emphasize the fundamental importance of epididymosomes as key elements of the epididymal microenvironment necessary for coordinating post-testicular sperm maturation and storage. This work encourages further studies aimed at deciphering the biogenesis and cargo-sorting mechanisms responsible for epididymosome formation as well as more detailed examination of the mechanism(s) by which they can coordinate the delivery of proteinaceous cargo to recipient cells.
DATA AVAILABILITY
The dataset (Dataset S1) analyzed here has been deposited in the Mass Spectrometry Interactive Virtual Environment (MassIVE) database with the dataset identifier MSV000082497 and is publicly accessible at: https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=5cf36b642ed54e318f3b5dbb0d1db830. The FTP download is accessible via the link: ftp://massive.ucsd.edu/MSV000082497.
Acknowledgments
This work was supported by a National Health and Medical Research Council of Australia Project Grant (APP1147932) awarded to B.N. and M.D.D. B.N. is supported by an Australian Research Council Future Fellowship. M.D.D. is supported by a Cancer Institute NSW ECR Fellowship. This work was supported by Mr Nathan Smith from The University of Newcastle Analytical and Biomolecular Research Facility (ABRF) and The Academic and Research Computing Support (ARCS) team, within IT Services at the University of Newcastle, provided high performance computing (HPC) infrastructure for supporting the bioinformatics.
Supplementary Material
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Article Info
Publication History
Published online: September 13, 2018
Received in revised form:
August 28,
2018
Received:
July 25,
2018
Footnotes
Author contributions: B.N., G.N.D.I., M.R.L., and M.D.D. designed research; B.N., G.N.D.I., H.M.H., W.Z., A.M., I.B., A.L.A., S.J.S., J.G.A., E.G.B., M.R.L., and M.D.D. analyzed data; B.N., M.R.L., and M.D.D. wrote the paper; H.M.H., W.Z., A.M., I.B., A.L.A., S.J.S., D.A.S.-B., M.F.B.J., J.G.A., E.G.B., M.R.L., and M.D.D. performed research.
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© 2019 Nixon et al.
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