Hum. Reprod. Advance Access originally published online on May 14, 2008
Human Reproduction 2008 23(8):1698-1707; doi:10.1093/humrep/den181
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Protein composition of human epididymosomes collected during surgical vasectomy reversal: a proteomic and genomic approach
1 Centre de Recherche en Biologie de la Reproduction and Département d'Obstétrique-Gynécologie, Université Laval, Quebec City, Quebec, Canada 2 Laboratoire épididyme et maturation des gamètes. CNRS UMR 6547, Université Blaise Pascal, Clermont-Ferrand Cedex 1, France 3 Urologie, Faculté de Médecine, Université Laval, Quebec City, Quebec, Canada
4 Correspondence address. Unité d'Ontogénie-Reproduction, Centre de Recherche du Centre Hospitalier de l'Université Laval, 2705 boulevard Laurier, T1-49, Quebec City, Quebec, Canada G1V 4G2. Tel: +1-418-656-4141; Fax: +1-418-654-2765; E-mail: robert.sullivan{at}crchul.ulaval.ca
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Abstract |
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BACKGROUND: The epididymal epithelium secretes membranous vesicles, called epididymosomes, with which a complex mixture of proteins is associated. These vesicles transfer to spermatozoa selected proteins involved in sperm maturation. Epididymosomes in the human excurrent duct have been described, but their protein composition and possible functions are unknown.
METHODS AND RESULTS: Epididymosomes were collected during vasovasostomy procedures, purified and submitted to liquid chromatography with hybrid quadrupole time-of-flight mass spectrometry. From all the mass spectra generated, 1022 peptides allowed the identification of 146 different proteins. Identification of some of these proteins was confirmed by western blots. Furthermore, western blot showed that the protein composition of epididymosomes differed from that characterizing prostasomes; membranous vesicles secreted by the prostate. Organization of the epididymosomes proteome according to common functional features suggests that epididymosomes have multiple functions. In order to understand the origin of epididymosomes collected distally, microarray databases of caput, corpus and cauda epididymidis were analysed to determine where along the excurrent duct the encoded proteins associated to epididymosomes are synthesised. Results suggest that some proteins synthesized in the caput and corpus epididymidis are associated with epididymosomes collected distally.
CONCLUSIONS: Epididymosomes thus transit along the excurrent duct, and vesicles collected distally represent a mixed population.
Key words: epididymosome/protein/vasectomy/sperm/epididymus
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Introduction |
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In vertebrate species practicing internal fertilization, spermatozoa have to transit along the epididymis. In mammals, the epididymis is a long convoluted tubule located between the vasa efferentia and the vas deferens (Jones, 1998
). Under the influence of androgens and unidentified testicular factors, the epididymis secretes into the intraluminal compartment proteins that interact with spermatozoa (Cuasnicu et al., 2002). Classically, the epididymis is anatomically divided into caput, the most proximal segment, corpus and distal cauda epididymidis (Cooper, 1998b). The gene expression pattern is highly segregated along the epididymis (Kirchhoff, 1999; Dube et al., 2007; Thimon, 2007b) resulting in variation of intraluminal protein composition along the tubule (Cuasnicu et al., 2002; Dacheux and Dacheux, 2002; Sullivan et al., 2003). The epididymis is involved in sperm transport, storage and maturation. Sperm maturation includes a series of biochemical modifications undergone by epididymal spermatozoa which are essential for the acquisition of fertilizing ability and forward motility (Cooper and Yeung, 2006).
The mechanisms of interaction between transiting spermatozoa and the intraluminal epididymal proteins have raised interest for three decades (Cuasnicu et al., 2002; Dacheux and Dacheux, 2002). Newly acquired proteins by epididymal spermatozoa are generally referred to as coating proteins since hydrophobic interactions are responsible for sperm acquisition (Cooper, 1998a). Some sperm proteins acquired during maturation do not behave as coating proteins when sperm cells are experimentally submitted to different biophysical treatments. Some of them behave as integral membrane proteins, including GPI (glycosylphosphatidylinositol) anchored-proteins (Kirchhoff and Hale 1996; Legare et al., 1999a, b). Other epididymal proteins have been shown to be integrated into sperm intracellular compartments during maturational process and proposed to be involved in sperm motility (Eickhoff et al., 2001, 2004, 2006; Frenette et al., 2005). The acquisition of epididymal proteins involved in sperm maturation is a puzzling phenomenon which cannot be explained by the classical secretory pathway targeting newly synthesized proteins into different subcellular compartments (Cooper, 1998a; Sullivan et al., 2007).
As an alternative to the classical merocrine protein secretory pathway, the apocrine mode of secretion has been described, particularly in male and female reproductive organs (Aumuller et al., 1997, 1999). This mechanism involves the formation of cytoplasmic blebs at the apical pole of secretory epithelial cells. These blebs detach from the plasma membrane and liberate their contents when they disintegrate in the intraluminal compartment of the secretory organ (Hermo and Jacks, 2002). Apocrine secretion has been described in the epididymis of many mammalian species and is responsible for the secretion in the intraluminal compartment of small membranous vesicles named epididymosomes (Yanagimachi et al., 1985; Fornes et al., 1995; Eickhoff et al., 2001; Frenette and Sullivan, 2001; Frenette et al., 2002; Saez et al., 2003; Rejraji et al., 2006). In the human, similar vesicles (prostasomes) secreted by the prostate are found in semen (Ronquist and Brody, 1985).
Using the bovine as a model, we previously showed that complex protein patterns are associated with epididymosomes (Frenette et al., 2002), and that the protein composition of epididymosomes varies along the bovine epididymis (Frenette et al., 2006). We also demonstrated that, when co-incubated with epididymal spermatozoa, epididymosomes transfer selected proteins to these cells (Frenette et al., 2002; Sullivan et al., 2001, 2005, 2007). Some of these proteins have been identified and their functions in sperm physiology hypothesized: P25b (Frenette and Sullivan, 2001), a zona pellucida binding protein, MIF (macrophage migration inhibitory factor) (Eickhoff et al., 2001; Frenette et al., 2003, 2005), enzymes of the polyol pathway (Frenette et al., 2003) and HE5/CD52 (sperm maturation-associated epididymal protein) (Kirchhoff and Hale, 1996). Some of these proteins are GPI anchored to epididymosomes (Frenette and Sullivan, 2001). The protein transfer from epididymosomes to spermatozoa is saturable, pH- and temperature-dependent, and much more efficient in presence of Zn2+ (Frenette et al., 2002). It is thus hypothesized that epididymosomes are involved in the acquisition of new proteins by spermatozoa transiting the epididymis. These vesicles thus play a major role in sperm maturation (Sullivan et al., 2001, 2007).
In human, prostasomes in semen have been exhaustively studied (Ronquist and Brody, 1985; Saez et al., 2003; Ronquist and Nilsson, 2004; Sullivan et al., 2005) and proposed to be involved in sperm motility (Carlsson et al., 1997) and capacitation (Cross, 1996; Cross and Mahasreshti, 1997). Furthermore, prostasomes are characterized by antibacterial (Carlsson et al., 2000) and immunomodulation (Rooney et al., 1993, 1996) activities and play a protective role against reactive oxygen species (Saez et al., 1998, 2000). In the human, epididymosomes prepared from fluids collected from the distal epididymis are characterized by a protein electrophoretic pattern showing major differences compared with that of prostasomes (Utleg et al., 2003; Frenette et al., 2005). It is thus thought that epididymosomes from the distal epididymis do not contribute significantly to the population of vesicles present in semen (Frenette et al., 2005). In contrast to what is known about prostasomes (Utleg et al., 2003), the protein composition and possible functions of human epididymosomes remain to be defined. In this work, epididymosomes were prepared from epididymal fluid collected during surgical vasectomy reversal and their protein composition and possible functions defined using a proteomic approach. To define the possible origin of these vesicles along the human epididymis, these results were analysed in parallel with data describing the transcriptome of the caput, corpus and cauda human epididymidis (Thimon, 2007b). Taken together, the results contribute to our understanding of the role of epididymosomes in human epididymal sperm maturation.
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The use of human tissues and body fluids and consent forms were approved by our institutional ethic committee.
Epididymosomes and prostasomes
Human epididymosomes were prepared as previously described (Frenette et al., 2005). Epididymal fluid was collected from the open end of the scrotal portion of the vas deferens during surgical vasectomy reversal. Fifty to two hundred microlitre of fluid was collected from each vas deferens. The fluids were diluted in Tris 30 mM, NaCl 130 mM, pH 7.5 (TN), pooled, and centrifuged twice at 3000g for 10 min to remove contaminating cells and cellular debris. Supernatants were resuspended in the same buffer and ultracentrifuged at 120 000g for 2 h. The pellet was suspended in TN and ultracentrifuged a second time. Collected fluid samples containing red blood cells were discarded, as were epididymosomes pellets presenting with a reddish colour. Based on these criteria, only half of the collected fluids were used in this study.
Prostasomes were prepared according toRonquist and Brody (1985). Briefly, normozoospermic semen samples obtained by masturbation were centrifuged twice at 3000g for 10 min to remove spermatozoa and cellular debris. Supernatants were ultracentrifuged at 120 000g for 2 h, the pellets resuspended in TN buffer and chromatographed on a Sephacryl S-500 HR column (Pharmacia, Baie d'Urfé, Québec, Canada). The void volume containing the prostasomes was centrifuged at 120 000g for 2 h and the resulting pellets were used as prostasomes samples.
Electrophoresis and proteomic analysis
Epididymosomes and prostasomes were resuspended in Laemmli sample buffer [2% sodium dodecyl sulphate (SDS), 3% β-mercaptoethanol, 50 mM Tris, pH 6.8] and protein concentrations estimated by dot blots on nitrocellulose (Chapdelaine et al., 2001). The proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS–PAGE) on a 12% polyacrylamide gel and stained with Coomassie blue. For peptide analysis using liquid chromatography with hybrid quadrupole time-of-flight mass spectrometry (LC-QToF analysis), the gels were cut in 0.5 cm long slices corresponding to proteins of molecular weight ranges: no 1 (>100 kDa), no 2 (60–100 kDa), no 3 (40–60kDa), no 4 (35–40 kDa), no 5 (28–35 kDa), no 6 (22–28 kDa), no 7 (16–22 kDa) and no 8 (10–16 kDa).
Proteins in polyacrylamide slices were digested with trypsin for LC-QToF at the Proteomic service platform of McGill University-Genome Quebec (Canada) (http://genomequebec.mcgill.ca/services/proteomics.php). Analyses of data generated by LC-QToF (QToF; MS/MS) were performed by Mascot search (http://www.matrixscience.com/search_form_select.html) using NCBInr (taxonomy: mammalian) database. The «Hugo» identification protein names were further validated using UnitProt Knowledge base (http://ca.expasy.org/sprot/). Protein identifications were based on an individual ion score over 100 and a minimum of two matched peptides for P < 0.05 (Working group on publication guidelines for peptide and protein identification data:Carr et al., 2004). Some protein identification was confirmed by western blot using specific antibodies.
Western blot identification of selected proteins
Electrophoretic patterns of epididymosomes and prostasomes were electrotransfered onto a nitrocellulose membrane and saturated with PBS containing 0.1% Tween-20 and 5% skimmed milk, or 3% BSA, depending on the first antibody. The antibodies used in this study were as follow: rabbit polyclonal antibodies against NPC2 (HE1) (Niemann-Pick disease type C2 and human epididymal protein 1, 1/2000: gift from Dr Jean-Luc Gatti, INRA-Tours, France) (Fouchecourt et al., 2000), PGAM2 (phosphoglycerate mutase 2, 1.5ug/mL: gift of Dr Yushi Matuo and Kohji Uchida from Oriental Yeast Co) (Uchida et al., 1995), HSP90 (Heat-shock protein 90) beta (3 µg/ml: Santa Cruz Biotechnology, Santa-Cruz, USA), mouse monoclonal antibodies against Annexin II (Annexin 2) (1/4000: BD Transduction Laboratory), alpha-tubulin (1/50 000) and β-actin (1/1000) (Sigma), goat polyclonal antibodies against CRISP 1 (acidic glycoprotein homolog or «Cysteine-rich secretory protein 1, 1ug/ml: Santa Cruz Biotechnology). Membranes were incubated with the relevant second antibody coupled to peroxidase. Immune complexes were revealed using a peroxidase chemiluminescent substrate.
Epididymal tissues, RNA extraction and microarray analysis
In order to understand if epididymosomes collected at the end of the epididymis were secreted distally or if they originated from more proximal parts followed by a transport along the organ, proteomic data were analysed in parallel with transcriptome of different epididymal segments. Considering that epididymosomes were prepared from fluids collected during surgical vasectomy reversal, microarrays were hybridized using RNA extracted from epididymal tissues collected from vasectomized men. Human epididymal tissues were obtained through collaboration with our local organ transplantation programme, ‘Quebec Transplant’. Donors were 26–50 years of age with no medical pathologies that could affect the reproductive function except vasectomy. Tissues were collected under optimal conditions, while artificial circulation was maintained to preserve organs assigned for transplantation. Vasectomy being a common practice in Canada, epididymides after vasectomy were occasionally identified by the presence of clips on the scrotal part of the vas deferens. Epididymides were dissected into caput, corpus and cauda epididymidis (Legare et al., 1999a, b), snap frozen in liquid nitrogen and stored at –80°C until used for RNA extraction.
Total RNA from the caput, corpus and cauda epididymidis from three vasectomized men was extracted by Trizol (Invitrogen, Burlington, ON, Canada) and used for microarray hybridization as previously described (Thimon, 2007b). Briefly, labelled complementary RNA fragments were hybridized to human oligonucleotide array U133 Plus 2.0 (Genechip, Affynetrix). The array comprises 55 000 oligonucleotide features covering over 47 000 transcripts and variants representing 39 000 of the best characterized human genes. Gene chips were scanned and images were extracted with the GeneChip Operating Software (Affimetrix CGOSv1.4). Gene signal intensities of β-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were used as internal quality controls. To identify differentially expressed genes, hybridization intensities were compared using a moderated t-test and a Bayes smoothing approach developed for a low number of replicates (Smyth, 2004).
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Proteomic analysis
Fluids recovered during surgical vasectomy reversal procedures were pooled in order to prepare a sufficient amount of epididymosomes to perform proteomic analysis. Coomassie blue stained one-dimensional gel of epididymosomes proteins revealed a major band around 80 kDa. This band corresponded to LTF (lactoferrin, also known as lactotransferrin). This identification was confirmed by LC-QToF performed on the trypsin digested, excised and stained-80 kDa band (data not shown). The electrophoretic pattern revealed many other bands covering all the spectrum of MWs between 100 and 10 kDa (Fig. 1).
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Using the criteria of an ion score >100 and a minimum of two matching peptides, 1022 peptides retrieved from the mass spectra allowed the identification of 146 different proteins. An average of 7.4 matching peptides (min 2; max 188) was identified per protein. The ion score was as high as 3186 with 188 peptides covering 66% of the deduced amino acid sequence of the proteins, in this case LTF. As described in Table I, the identified proteins covered a large MW spectrum. In general, the excised region of the electrophoretic pattern generating the peptides used to identify the protein was in the range of the expected MW of the identified protein. In few cases, peptides corresponding to a single protein were generated from different excised electrophoretic regions, probably due to partial proteolysis (data not shown).
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A few proteins were selected to confirm proteomic results by western blotting. Protein extracts of epididymosomes were blotted in parallel with proteins prepared from prostasomes. Prostasomes are vesicles similar to epididymosomes present in human semen (Ronquist and Brody 1985
; Saez et al., 2003). ANXA2 (annexin 2) and PGAM2 were detected at similar levels in both epididymosome and prostasome preparations. Alpha-tubulin and β-actin were also detected in both preparations but appeared to be more abundant in prostasomes when compared with epididymosomes. In contrast, HSP90AB1 was more abundant in epididymosomes while detectable to a small extent in prostasomes. CRISP1 and HE1/NPC2 are part of epididymosome signature, being undetectable in prostasomes (Fig. 2). In brief, all the proteins detected by western blots performed on epididymosomes protein extracts confirmed the LC-QToF results. While epididymosomes showed some similarities in their protein composition with prostasomes, western blots showed that these membranous vesicles from the male reproductive tract are two distinct populations.
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-tubulin), ACTB (β-actin), HSP90AB1 (heat shock protein 90), CRISP1 (acidic epididymal glycoprotein 1), NPC2 (Niemann-Pick disease type 2/HE1) on epididymosomes and prostasomes.In order to understand the potential functions of epididymosomes in human, the proteome was organized according to common functional features using Gene Ontology (GO) categories. When organized according to their molecular functions, 27% of the identified proteins fitted in the enzymes category, 14% were adhesion molecules, 13% were transporters or were involved in protein trafficking, whereas 12% were involved in signal transduction. Structural proteins, defence molecules and chaperones were represented by 9, 5 and 4% of the identified proteins, respectively. The 16% remaining proteins were in an unclassified category (Fig. 3).
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Genomic analysis
Primary microarray databases generated for this work have been registered in the public domain «Gene Expression Omnibus»:gSE7808 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=flktdmggsqkkovg&acc=GSE7808).
Analysis of the human epididymal transcriptome revealed the presence of mRNAs encoding for 134 (92%) of the 146 proteins associated to epididymosomes. Of the remaining 12 identified proteins to which no transcript was identified by the Affimetrix microarrays, 4 belong to the histone family, 2 (YWAHZ and HSP90β) are associated to prostasomes and one (PGAM2) has been previously shown to be associated to bovine epididymosomes.
In animal models it has been demonstrated that epididymosomes are present in the intraluminal fluid all along the epididymis (Yanagimachi et al., 1985; Rejraji et al., 2002, 2006; Frenette et al., 2006) and that proteins associated with these vesicles vary from one epididymal segment to another (Frenette et al., 2006). We thus asked the question whether the epididymosomes collected distally during vasovasosotomy procedures were secreted along the epididymis. For obvious reasons, it was not possible to collect fluids at different sites of the human epididymis. Microarray databases of caput, corpus and cauda epididymides from vasectomized men were thus analysed to determine if some encoded proteins present on epididymosomes collected distally were expressed in a segment-specific manner along the human epididymis. mRNAs coding for a total of 19 proteins of the epididymosomal proteome were shown to be differentially expressed along the human epididymis (Table II). Three, 13 and 3 of these transcripts were preferentially expressed in the caput, corpus and cauda epididymidis, respectively (Table II). Many other proteins associated to epididymosomes are known to be expressed by the epididymis but, according to the microarray databases, their expression was not modulated along the epididymis (Table I).
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Preparation of epididymosome suspensions is not an easy task. Using laboratory animals, epididymal tissues from whole organ or from specific segments are homogenized and then processed by successive centrifugations to purify these vesicles. These preparations are obviously contaminated by microsomal fractions. In order to avoid this artefact, we chose in previous works to use epididymides from large domestic animals that allow the collection of uncontaminated intraluminal epididymal fluids (for reviews seeSullivan et al., 2005
, 2007). Transposition of these results to human physiology is not obvious. In this work, human epididymosomes were collected during surgical vasectomy reversal as previously described (Frenette et al., 2005). To these preparations are associated many proteins known to be secreted by the human epididymis such as CRISP1, epididymal secretory protein E12 (E12), serpin A1 (SERPINA1), NPC2 and many others (Table I). Even though presence of few proteins, such as four members of the histone family, suggests contamination by lysed somatic cells, 92% of the identified proteins are encoded by mRNAs detected in the epididymal transcriptome. Furthermore, the protocols used to prepare epididymosomes are known to eliminate sperm fragments and cellular debris (Utleg et al., 2003; Ronquist and Nilsson, 2004; Frenette et al., 2005). Keratins are detected in the proteome of our samples and are known to be a common contamination when protein samples are analysed by SDS–PAGE and by western blotting (Berube et al., 1994). Epididymosomes were collected from vasectomized men. We previously showed that vasectomy affects the expression pattern of selected genes along the epididymis (Legare et al., 2001, 2004, 2006). Vas deferens obstruction may thus influence the protein composition of the collected epididymosomes. In fact, expression patterns of transcripts known to be modulated along the epididymis and encoding proteins associated with epididymosomes, show some differences in their patterns of expression in microarray databases of epididymal segments of normal versus vasectomized men. Molecular function analysis of transcriptomes suggests that vasectomy does not significantly affect the overall pattern of gene expression or the deduced functions of each epididymal segment (Table II; Thimon, 2007a, b). Preparation of epididymosomes during surgical vasectomy reversal was the only alternative to obtain this biological material and provides a general overview of the proteome of these membranous vesicles. When selected proteins identified by LC-QToF were analysed by western blotting, the association of these proteins to epididymosomes was confirmed (Fig. 2).
Proteome reveals 146 different proteins covering a large spectrum of GO categories, suggesting that epididymosomes play diversified functions. A major feature of the epididymosomes proteome is the number of GTPase proteins. Many of these proteins belong to the Ras family, especially RAB proteins: RAB1A, RAB2, RAB7, RAB8A, RAB10 and RAB11B. In mammalian cells, >60 of these small monomeric RAS-like GTPases have been identified and show a wide range of tissue distribution (Schultz et al., 2000). RABs are involved in membrane traffic, including vesicle delivery, vesicle tethering and fusion of vesicle membrane with those of the target compartment. The large number of RABs thus favours the specificity of membrane-targeting in different mammalian tissues or cell types (Grosshans et al., 2006). Considering that epididymosomes have been shown to transfer selectively epididymal proteins to specific sub-compartments of maturing spermatozoa, RABs may be involved in these processes (Grosshans et al., 2006). Other RAS-like proteins such as RAC1, RAP1A or B associated with human epididymosomes may also be involved in membrane vesicle trafficking and in the interaction between the epididymal vesicles and spermatozoa. Other proteins such as SYPL1 (synaptophysin-like1 isoforms) and TSPAN1 (tetraspan) involved in membrane vesicle exocytosis and membrane fusion, respectively, may also target epididymosomes to spermatozoa. Other cell adhesion proteins also involved in protein trafficking associated with epididymosomes such as Syntenins (SDCBP and SDCBP2), may also modulate sperm–epididymosome interactions.
Epididymosomes are resistant to boiling, freezing–thawing cycles, sonication and non-ionic detergent treatments (Frenette and Sullivan, 2001). These membranous vesicles are thus very stable, suggesting a particular ultrastructural organization. This is reflected by the presence of moesin, ezrin and radixin linking actin (ACTA1 and ACTB) to the plasma membrane, possibly playing a structural role, as well as tektin (TEKT3), which is a tubulin organizer; TUBA and TUBB (tubulin alpha and beta) being constituents of epididymosomes. CLTC (Clathrin) is another epididymosomal protein linking the membrane with the cytoskeleton that may stabilize these intraluminal membranous vesicles. Different types of annexins are associated with epididymosomes. Annexins are proteins that bind to phospholipids in a calcium-dependent manner: ANXA1, ANXA11, ANXA2, ANXA4, ANXA5, ANXA6 associated with these epididymal membranous vesicles may play different roles as they do in plant and animal cell physiology. Obviously, proteomic analysis suggests that epididymosomes play different functions. Some of these proteins support the concept that epididymosomes interact in a highly regulated manner with spermatozoa. Furthermore, many structural proteins confirm the observation that these vesicles have a complex ultrastrutural organization that contributes to the stability of these extracellular organelles (Frenette and Sullivan, 2001; Frenette et al., 2002).
Epididymosomes are secreted by epididymal principal cells in an apocrine manner (Aumuller et al., 1997; Hermo and Jacks, 2002; Sullivan et al., 2005). This peculiar mode of secretion is particularly common in reproductive organs such as mammary glands and the prostate: in the latter case, the secreted vesicles, named prostasomes, are found in seminal plasma. Protein composition of prostasomes appears to be as complicated as in epididymosomes (Utleg et al., 2003). When GO categories based on molecular functions are used to compare prostasomes to epididymosomes, these two proteomes reveal functional similarities. Some proteins are also identified in both types of vesicles, mainly those in transport and structural proteins and chaperone proteins (Table I; Utleg et al., 2003). When the association of selected proteins associated with prostasomes and epididymosomes is analysed, differences are detected between these two types of membranous vesicles; this is particularly true for proteins known to be specifically secreted by the epididymis. This confirms our previous observations showing that epididymosomes do not contribute significantly to membranous vesicles found in seminal plasma (Frenette et al., 2005). This suggests that spermatozoa interact in a sequential manner with different membranous vesicles along the epididymis (Frenette et al., 2006) and in seminal plasma (Burden et al., 2006).
Many proteins of the epididymosomes proteome have already been described as being expressed in the epididymis: LCN2 (neutrophil gelatinase-associated lipocalin), LTF, NPC2, ACE (angiotensin converting enzyme), AK1 (adenylate kinase 1), CA (carbonic anhydrase), GSTP1 (glutathion S-transferase, class Pi), LDH (lactate dehydrogenase), PRDX4' Triose-phosphate isomerase, CSTB (cystatin B), E12 (Dacheux and Dacheux, 2002; Dacheux et al., 2003, 2006; Kirchhoff, 2007; Thimon, 2007b). This confirms the enrichment of our preparation in material originating from the excurrent duct. Some of these proteins of epididymal origin become associated with spermatozoa during maturation along the epididymis. Thus, epididymosomes are involved in a docking process of specific proteins to defined sperm membrane domains or subcellular compartments. Some examples of these proteins are MIF that is transferred to dense fibres and is hypothesized to modulate sperm motility, and CRISP1, which plays a dual role in capacitation and sperm–egg interactions. Other proteins, even though they are not added to the sperm structure, are involved in the modifications undergone by sperm cells during maturation: E12 and NPC2. Some proteins play an indirect role such as clusterin or apolipoprotein J (CLU), serpins (SERPINA1 and SERPINB6) and LTF. Proteome analysis thus reveals that epididymosomes are involved in complex functions in both epididymal physiology and sperm maturation.
LTF is the major protein constituent of epididymosomes (Fig. 1) and its encoding transcript is highly expressed in the distal part of the excurrent duct (Table II). It has previously been shown to be an epididymal secretory protein in many species (Dacheux et al., 2003) including man (Dacheux et al., 2006). LTF is an iron-scavenging defence protein that plays a bacteriostatic role. Peptides derived from this protein have bactericidal properties (Ward and Conneely, 2004; Ward et al., 2005). Lipocalin family members such as PTGDS and LCN2 are also associated with epididymosomes. The association of these proteins to epididymosomes shows that these vesicles play a role in microbial defence in the distal part of the excurrent duct.
Many proteins already known to be secreted by the epididymis are associated with distally collected epididymosomes. Epididymal transcriptome analysis shows that some of these proteins are translated in a segment-specific manner (Table II). Proteins principally synthesized in the cauda epididymidis such as ANXA1 and LTF, are associated with epididymosomes collected in this portion of the excurrent duct. Analysis of microarray databases of different epididymal segments demonstrate that protein synthesized in the caput epididymidis [AK1, ANPEP (Aminopeptidase N), and SLC44A5 (Choline transporter-like protein 5)] or in the corpus segment [especially ADAM7, E12, FAM12B (Human epididymis-specific 3 beta), and LCP1 (L-plastin, or lymphocyte cytosolic protein 1)] are associated with epididymosomes collected in the most distal part of the excurrent duct. This suggests that epididymosomes can transit along the epididymis and that vesicles collected distally represent a mixed population resulting from apocrine secretion of principal cells all along the epididymis.
Some proteins associated with epididymosomes are known to be constituents of mature spermatozoa. Hexokinase (Kalab et al., 1994) and ADAM7 (Lin et al., 2001) are plasma membrane proteins, sp38 is involved in zona pellucida binding (Mori et al., 1995) and MIF is a structural protein of the dense fibres of ejaculated spermatozoa (Eickhoff et al., 2001; Frenette et al., 2005). This support the concept that in human, as it has been shown in bovine (Legare et al., 1999a, b; Frenette and Sullivan, 2001; Frenette et al., 2002, 2006), epididymosomes are involved in the docking of epididymal secreted proteins to different sub-compartments of spermatozoa.
In conclusion, epididymosomes represent a constituent of the intraluminal compartment of the human excurrent duct and their protein composition suggests that they play different functions in sperm maturation and epididymal physiology.
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This work was supported by Natural Sciences and Engineering Research Council of Canada (NSERC) and by Canadian Institutes for Health Research (CIHR) grants to R.S.
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We wish to thank Mrs Christine Légaré for technical help and stimulating discussion as well as Drs E. Calvo and O. Koukoui for helping us in microarray databases analysis. Technical support provided by the McGill University and Genome Quebec Innovation Centre (Prov Quebec, Canada) is also acknowledged.
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These two authors contributed equally to this work. 
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Submitted on November 26, 2007; resubmitted on March 26, 2008; accepted on April 12, 2008.
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