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BOR - Papers in Press, published online ahead of print January 5, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.036426
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BIOLOGY OF REPRODUCTION 72, 1452–1465 (2005)
DOI: 10.1095/biolreprod.104.036426
© 2005 by the Society for the Study of Reproduction, Inc.

Identification, Proteomic Profiling, and Origin of Ram Epididymal Fluid Exosome-Like Vesicles1

Jean-Luc Gatti 2 3, Sonia Métayer 3, Maya Belghazi 4, Françoise Dacheux 3, and Jean-Louis Dacheux 3

Equipe Gamètes Males et Fertilité3 and Service de Spectrométrie de Masse pour la Protéomique,4 URM 6175 INRA-CNRS-Université de TOURS-Haras Nationaux, Physiologie de la Reproduction et des Comportements, Institut National de la Recherche Agronomique de Nouzilly, Monnaie 37380, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Small membranous vesicles, between 25- and 75-nm diameter, were collected by high-speed centrifugation from the ram cauda epididymal fluid and were found to be normal constituents of this fluid and of the seminal plasma. The SDS-PAGE protein pattern of these vesicles was specific and very different from that of the caudal fluid, seminal plasma, sperm extract, and cytoplasmic droplets. After two-dimensional electrophoresis separation and mass spectrometry analysis, several proteins were identified and grouped into i) membrane-linked enzymes, such as dipeptidyl peptidase IV (DPP-IV), neprilysin (NEP), phosphodiesterase-I (E-NPP3), and protein G-beta; ii) vesicle-associated proteins, such as lactadherin (MFEG8-PAS6/7) and vacuolar ATPase; iii) several cytoskeleton-associated proteins, such as actin, ezrin and annexin; and iv) metabolic enzymes. The presence of some of these proteins as well as several different hydrophobic proteins secreted by the epididymis was further confirmed by immunoblotting. These markers showed that the majority of the vesicles originated from the cauda epididymal region. The physical and biochemical characteristics of these vesicles suggest they are the equivalent of the exosomes secreted by several cell types and epithelium. The main membrane-linked proteins of the vesicles were not retrieved in the extract from cauda or ejaculated sperm, suggesting that these vesicles did not fuse with sperm in vivo.

epididymis, epididymosomes, exosomes, gamete biology, male reproductive tract, mass spectrometry, protein, proteomic, sperm, spermatozoa, sperm maturation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mammalian sperm collected from the testis are immobile and unable to bind to and then fertilize the oocyte. They acquire these properties gradually while they travel through the epididymal tubule. During this transit, the sperm lose their cytoplasmic droplets and their plasma membrane undergoes profound changes, including addition, removal, or transformation of proteins and lipids [1–3]. These gradual surface modifications occur in response to variations in the luminal fluid composition and are thought to be responsible for their final maturation [4].

One of the most striking changes is the transfer to the sperm membrane of very hydrophobic proteins found in the epididymal fluid and of proteins that normally bear glycosyl phosphatidyl inositol (GPI) tails [5–7]. Because such proteins normally exist in a membrane environment and are not free in the fluid, it has been suggested that a transport system, for example, vesicles (named epididymosomes) or lipid micelles, might exist in the epididymis [2, 3, 8]. The presence of membrane vesicles in seminal plasma of human and mammals (named prostasomes) has been well documented, and it has been suggested that these vesicles are derived mainly from the accessory glands, such as the prostate or the seminal vesicles (for reviews, see [8, 9]).

We have recently provided evidence that such vesicles exist in the cauda epididymal fluid of rams [10], and in vitro experiments have shown that such vesicles, which are also present in the bovine cauda epididymis and seminal plasma, can transfer some proteins to the sperm in zinc-and pH-dependent manners (for reviews, see [8, 9]).

In this study, we definitively establish that an intraluminal system of membrane vesicles occurs naturally in the ram cauda epididymis. We have isolated, purified, and biochemically characterized these vesicles from the epididymal fluid and the seminal plasma, and we defined biochemical markers that help us to determine their origin. Their relationship to the vesicles present in the seminal plasma and to exosome vesicles secreted by other types of epithelium [11] is discussed, as well as their role in the transport and transfer of proteins to the sperm membrane.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Fluids and Sperm Collection

Experiments on animals were conducted according to the International Guiding Principles for Biomedical Research Involving Animals as promulgated by the Society for the Study of Reproduction. Testis and epididymis were obtained from adult Ile de France and Romanov rams (at least 10 different animals). Testicular fluid was obtained after puncture of the rete testis. Epididymal fluids were collected by microperfusion with PBS from the 10 different zones (0–9) defined in the three main regions (caput, corpus, cauda) and the caudal fluid (CEP) by retroperfusion of zones 8 and 9 from the deferent duct (see Fig. 1). Ejaculates were obtained from five intact adult animals or two vasectomized animals using an artificial vagina.



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  		FIG. 1. Photograph of an epididymis from adult ram with the different zones of microperfusion indicated. Caput, corpus, and cauda indicate the gross morphological regions. (Real size, 15 cm)

The spermatozoa were separated from the fluid or seminal plasma by two successive centrifugations (15 min, 15 000 x g, 4°C). Where indicated, the sperm from different zones were diluted in PBS and layered on a discontinuous percoll gradient (40% and 90%) and centrifuged at 3000 x g for 10 min at 4°C. The sperm pellets were carefully collected, diluted with PBS, and centrifuged again to remove the percoll. Under both conditions (directly washed or after percoll gradient), the last pellet was directly extracted with an equal volume of nonreducing Laemmli sample buffer and centrifuged again; the supernatant was boiled after addition of beta-mercaptoethanol (2%) and is referred to as the sperm extract.

Cytoplasmic droplets were purified from the top layer of the discontinuous percoll gradient from the sperm samples of zones 8 and 9. This suspension was diluted in PBS, centrifuged at 15 000 x g, and the pellet resuspended and loaded on a 10–90% percoll gradient. The top white band separated after centrifugation was recovered. The purity of the droplets was assessed by microscopy after two successive washes in PBS (15 000 x g, 10 min, 4°C) and also by electron microscopy. This preparation was highly enriched in droplets, with very few contaminating sperm.

Each experiment was repeated at least twice with samples from two different animals. The most representative results are shown.

Vesicle Preparation

The 15 000 x g supernatant from the caudal fluid was ultracentrifuged at 48 000 x g (JA18; Beckman, Villepinte, France) or 100 000 x g (SW21; Beckman) for 2 h at 4°C (no major differences in the protein pattern of vesicles were observed between the two procedures by SDS-PAGE; see also [10]). After the centrifugation, a yellowish pellet was found at the bottom of the tube. This pellet was suspended in PBS and submitted to a second high-speed centrifugation. The final pellet gave a cloudy, refracting solution in PBS, which was divided into aliquots to be processed for electron microscopy or prepared for one- and two-dimensional gel separation.

Electron Microscopy

The washed pellet was fixed at room temperature with 2.5% glutaraldehyde in 0.175 M cacodylate at pH 7.2. After 24 h, the pellet was postfixed with osmium tetroxide (2% final) and potassium ferricyanide (1.5%), washed with deionized water, and dehydrated with ethanol before embedding in epoxy-propan glycidether 100. The fixed pellet was cut with an ultramicrotome (80–90 nm). Contrast was enhanced by uranyl-acetate and lead citrate. Samples were observed on a Philips CM 10 microscope (60 KV). Measurement of the vesicle diameters and of the thickness of the vesicle membranes was performed either manually or using digital imaging system (Soft Imaging System, Munster, Germany). Both techniques gave very similar results.

Gel Electrophoresis

SDS 6–16% gradient polyacrylamide gels were used for protein separation, and iso-electrofocalization was performed as previously described [12]. The gels were Coomassie blue-stained before spot cutting and mass-spectrometry analysis or silver stained when better visualization of the protein spots was needed. For protein quantification, the Coomassie blue-stained gels were scanned and the total protein content of each lane analyzed using the 1D-Elite software package (Amersham-Pharmacia Biotech AB, Uppsala, Sweden).

Western Blotting

Semidry transfer of proteins was performed over 2 h at 0.8 mA/cm2. The western blots were blocked with TBS-Tween 20 (0.5%, w/v) and, depending on the antibodies used, supplemented with lyophilized low-fat milk (5% [w/v]), gelatin (0.5% [w/v]), or BSA (1% [w/v]).

The first antibodies were either rabbit polyclonals or mouse monoclonals. The anti-HE1, anti-RABP, anti-PGDS, and anti-acrosin are rabbit polyclonal antibodies obtained in our laboratory against the ram epididymal cholesterol transfer protein (CTP/HE1/NP-C2), retinoic acid binding protein (E-RABP), prostaglandin D2 synthase (PGDS) [13], and sperm acrosin [14], respectively. The anti-lactadherin (P47/PAS-6/7/MFG-E8) rabbit polyclonal antibody was a gift from Dr. J.T. Rasmussen [15, 16]. The anti-phosphodiesterase I rabbit polyclonal (B10; nucleotide pyrophosphatase 3; E-NPP3) was a gift of Dr. M. Maurice [17]. Anti-CD10 (neprilysin/CALLA/neutral endopeptidase) and anti-protein G-beta (against a peptide mapping at the carboxy-terminus of human G-beta 1 and recognizing G-beta 1, 2, 3, and 4) were rabbit polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), and anti-actin was a monoclonal antibody (Sigma, Saint Quentin Fallavier, France, and Prolabo, Fontenay-sous-Bois, France).

Membranes were incubated with the antisera under mild agitation overnight at 4°C or for 2 h at 37°C. The second antibody was a goat anti-rabbit or a goat anti-mouse antibody conjugated with peroxidase (dilution 1:5000). The peroxidase was revealed with chemoluminescent substrates and the images recorded on film or digitized with a cooled CCD camera. No reaction was observed with the secondary antibodies alone or the preimmune sera when available (for anti-CTP/HE1, -PGDS, -ERABP, and -acrosin).

Mass Spectrometry

The identification of the major protein spots from two-dimensional gels was obtained by mass spectrometry. Coomassie blue-stained spots were obtained from at least two different bidimensional gels from two different separately treated preparations. The spots were cut into small blocks with a sterile scalpel blade. The blocks were rinsed, then reduced and alkylated with iodoacetamide, and incubated overnight at 37°C in a microtube with 12.5 ng/µl trypsin (sequencing grade; Roche, Meillan, France) in 25 mM NH4HCO3 as previously described [18]. The tryptic fragments were extracted, dried, reconstituted with 0.1% formic acid, and sonicated for 10 min.

Tryptic peptides were analyzed either directly by MALDI (M@LDI-L/R; Waters Micromass, Manchester, UK) or sequenced by liquid chromatography coupled to tandem Mass Spectrometry (nano-LC-MS/MS) (Q-TOF-Global equipped with a nano-ESI source; Waters Micromass) in automatic mode. The peptides were loaded on a C18 column (Atlantis dC18, 3 µm x 75 µm x 150 mm, Nano Ease; Waters Micromass) and eluted with 5–60% linear gradient at a flow rate of 180 nl/min for 30 min (buffer A, water:acetonitrile 98:2 [v/v] 0.1% formic acid; and buffer B, water: acetonitrile 20:80 [v/v], 0.1% formic acid). The peptide masses and sequences obtained were either matched automatically to proteins in a nonredundant database (NCBI) using the Mascot program (http://www.matrixscience.com) or de novo sequenced using the ProteinLynx Global Server program (Waters Micromass) and blasted manually against the current databases.

Chemicals

All reagents were of the best available grade (Sigma). Low molecular weight standards for electrophoresis were from Amersham-Pharmacia Biotech AB and Biorad (Ivry sur Seine, France).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Electron Microscopy of the Vesicle Pellet

The white-yellowish pellet obtained after ultracentrifugation of the cauda epididymal fluid was analyzed morphologically by electron microscopy (Fig. 2). This pellet was composed of vesicles with a distribution size between 25- and 75-nm diameter (64 ± 22 nm; mean ± SD, n = 292). Some of these vesicles contained electron-dense material and a large majority showed a typical membrane bilayer of an average size of 7.9 ± 1.4 nm (mean ± SD, n = 200), suggesting a homogeneous population.



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  		FIG. 2. Electron micrograph and analysis of the high-speed pellet from the cauda epididymal fluid. Electron microscropy of the high-speed pellet showed small vesicles with a typical bilayer membrane. The diameters of the vesicles from two different preparations (10 different microscopic fields, 292 vesicles) were measured to establish the size distribution of these vesicles

Characterization of the Vesicle Proteins by 1-D SDS-PAGE

The proteins from these vesicles were analyzed by SDS-PAGE (Fig. 3A). The protein pattern obtained was very specific and different from that of the cauda fluid, particularly bands at 140, 110, and 45 kDa that were strongly enriched in the pellet. To estimate the protein quantity represented by these vesicles, the pellet was resuspended to concentrate the vesicles from the starting caudal fluid 20, 10, 5, 2.5, and 1 times (1 = no concentration). The total protein quantity of the different lanes analyzed by a digital system indicated that these proteins were less than 3% of the total protein of the caudal fluid. This value was similar to that obtained by the Bradford assay using BSA as the standard and was also demonstrated by the lack of difference in the pattern of proteins of the cauda fluid before (CEF) or after ultracentrifugation (CEF-hs).



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  		FIG. 3. Protein pattern of the vesicles. A) SDS-PAGE comparison of the proteins from caudal fluid (CEF), caudal fluid after high-speed centrifugation (CEF-hs), and from epididymal vesicles (vesicles). The same volume of CEF and CEF-hs were loaded while vesicles were resuspended in the initial volume of fluid (1) or concentrated 2.5, 5, 10, or 20 times. B) Protein separation of the vesicle pellet obtained from the seminal plasma (SP Intact) of an intact and a vasectomized (SP Vas.) ram. The same volume of 10-fold concentrated pellet was loaded per lane. 6–16% SDS-PAGE; Coomassie blue-stained gel

Small Membraneous Vesicles Are Normal Constituents of the Epididymal Fluid

To ascertain that these vesicles had a physiological significance and were not artifacts induced by the collection method, we compared the pellet obtained with the seminal fluid of normal and vasectomized rams collected 3 mo after the surgery (no sperm could be observed in the ejaculate at this time). After high-speed centrifugation, seminal plasma from both types of animals yielded a pellet, but the pellet obtained from the vasectomized rams was much smaller than from the controls. After separation by SDS-PAGE (Fig. 3B), the protein pattern of vesicles obtained from the vasectomized animal was very different from that of the normal ram and that of the cauda epididymis. All the characteristic proteins of these latter pellets were absent from the pellet of the vasectomized animals. In contrast, the pattern obtained from the epididymal vesicles and the seminal plasma vesicles from the normal animals differed only by two protein bands at 14 and 55 kDa.

Identification of the Vesicle Proteins by Two-Dimensional SDS-PAGE, Mass Spectrometry, and Western Blotting

The vesicle proteins from the caudal fluid (Fig. 4) and from the seminal plasma (not shown) were separated by two-dimensional gel electrophoresis to allow mass spectrometry identification. Both preparations gave identical protein profiles except for two series of spots at about 14 and 55 kDa present only in the seminal plasma (see also Fig. 3B). The major protein spots from the vesicles observed by Coomassie-blue staining (Fig. 4) were processed for identification by mass spectrometry. Two different approaches were used: Maldi-TOF, which allowed comparison of the tryptic pattern of a protein with the theoretical tryptic pattern in databases; and nano-HPLC-MS-MS, which allowed sequencing of specific tryptic fragments and search for homology in data bases (see Tables 1 and 2).



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  		FIG. 4. Two-dimensional gel electrophoresis of the vesicle proteins. The vesicles were treated and separated by two-dimensional gel electrophoresis under denaturing conditions. The spots were cut and processed by mass spectrometry to identify the proteins (see also Table 1). 6–16% SDS-PAGE; silver-stained gel


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  		TABLE 1. Names, accession numbers, size, sequence of the peptides obtained by LC-MS-MS, and number of peptides and percentage of the protein coverage obtained by Maldi for each protein described in Figure 3


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  		TABLE 1. Continued


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  		TABLE 1. Continued


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  		TABLE 2. Properties of identified proteins


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  		TABLE 2. Continued

Fragments matching several proteins were obtained in the major spots at about 140 and 120 kDa, ie: dipeptidyl peptidase IV (DPPIV) and neprilysin (NEP) (140 kDa), and phosphodiesterase-I (E-NPP3) and beta-mannosidase (120 kDa). These proteins were also found when the 140 kDa and 120 kDa bands from a one-dimensional gel were processed in the same way. Because of their abundance and the vertical smear they produced in the gel, dipeptidyl peptidase IV and neprilysin signatures were found in a large number of other spots with lower molecular weights. The cytoskeleton protein actin (45 kDa) was also one of the major proteins of the vesicles; this protein was separated as a series of at least four different spots composed of alpha-and beta-actin isoforms. The actin-associated protein Ezrin/Cytovillin was found in two major trains of spots: one at 75–80 kDa, which is its normal molecular weight, and one at 50 kDa, which could represent a degradation product of this protein. Tubulin and annexin II were two other cell-membrane cytoskeleton components identified. Several other membrane-linked proteins were identified such as lactadherin-PAS6/7 protein, chaperone heat shock protein-71 (HSC71), vacuolar ATPase (V-ATPase), protein G-beta (G-beta) and NAP22/BASP1. One protein expressed in testis (HPAST1-testilin-EHD1) at the germ cell level was also identified. The metabolic enzymes creatine kinase (CreaKin), pyruvate kinase (PyrKin), glutamate carboxypeptidase-like-1 (named carnosinase 1; CARN1), alpha-enolase (Enolase), aldehyde and isocitrate dehydrogenase (Ald-dh and Sorb-dh, respectively) and aldose reductase (Ald-red) were also present. Varying amounts of lactotransferin and serum albumin (OSA) were also found depending upon the preparation variable. Because these proteins were present in the caudal fluid, they may be adsorbed to the vesicle membrane. More information on these proteins is available in Table 2.

Western blots were performed using specific antibodies to confirm the identities of some of the major vesicle proteins (Fig. 5). Immunoreactions with neprilysin, ENPP3, protein G-beta, lactadherin-PAS6/7and actin were observed in raw cauda epididymal fluid (CEF) and purified vesicles (Fig. 5), but their intensity levels decreased significantly after removal of the vesicles from the caudal fluid by centrifugation (Fig. 5, CEF-hs). This result thus confirmed that these proteins are mainly associated with the vesicles.



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  		FIG. 5. Immunological identification of some of the vesicle proteins. Caudal fluid (CEF), caudal fluid after high-speed centrifugation (CEF-hS), and epididymal vesicles (10 times concentrated) were separated on a 6– 16% SDS-PAGE and transferred to nitrocellulose and probed with specific antibodies to confirm the identity of some of the proteins obtained by mass spectrometry. Similar Western blots were probed with antibodies directed against hydrophobic or lipophilic proteins known to be in the ram cauda epididymal fluid and/or on sperm membrane

Presence of Epididymal Hydrophobic Proteins Within the Vesicles

To investigate whether hydrophobic proteins cosediment with the vesicles, we used a similar immunoblotting approach (Fig. 5). The first protein sought was the 17-kDa hydrophobic protein that has previously been described in rams [5]. This protein is secreted exclusively by the epithelium of the cauda region (zones 7–9) and transferred to the sperm membrane. The Western blot showed that this protein was linked to the vesicles but that a large part also remained in the fluid after centrifugation.

Because PEBP (phosphatidyl-ethanolamine binding protein) was found by mass spectrometry to be associated with the vesicles, we analyzed whether other proteins that bind and transport hydrophobic compounds in epididymal fluid were also present. One isoform of the retinol acid binding protein (E-RABP) was found to be present in the vesicles as well as prostaglandin D2. In contrast, the cholesterol transport protein (HE1/NP-C2/CTP) was not found to be associated with the vesicles.

Epididymal Distribution of the Vesicles

Using these immunological tools, the origin of the vesicles along the epididymis was investigated. We probed the epididymal fluids from different zones with antibodies against the proteins linked to the vesicles, i.e., phosphodiesterase E-NPP3, neprylisin, lactadherin-PAS 6/7, and protein G-beta (Fig. 6A). We found that E-NPP3, NEP, and the 35-kDa G-beta protein were restricted to the cauda epididymal fluid (zones 6/7 to 9). We observed that one form (30 kDa) of PAS6/7-lactadherin was mainly associated with the caudal fluid and another (50–55 kDa) was present throughout the epididymis.



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  		FIG. 6. Regional distribution of the vesicle in the epididymal fluid. A) Equivalent amounts of protein from the rete testis and the different epididymal zones were run on 6–16% SDS-PAGE and transferred to nitrocellulose and probed with antibodies reactive against vesicle proteins. B) An equivalent Western blot, actin was present throughout the epididymal fluid (control); high-speed centrifugation (high speed) decreased the quantity of actin in zone 7 and removed it from the fluid in zones 8/9. C) The fluid of zones 7 and 9 were probed with antibodies reactive against vesicle proteins before (–) or after (+) high-speed centrifugation. After this treatment, the immunoreactive proteins were significantly reduced or removed, indicating they are linked to vesicles

The Western blot probed with the anti-actin antibody (Fig. 6B, control) showed the presence of this protein in the fluid throughout the epididymis. Because this might suggest that some of the vesicles were transported from the upper epididymal tract, we submitted these different fluids to high-speed centrifugation. After this treatment, actin was slightly decreased in the high-speed supernatant from zone 7 (Fig. 6B, high speed) as compare with the control (Fig. 6B, control) and was completely removed from the fluid in zones 8 and 9. It was also in these zones that actin was retrieved associated with the pellet (see Fig. 5).

We therefore analyzed the behavior of other vesicle proteins. We observed that the amounts of E-NNP3, neprilysin, lactadherin-PAS 6/7, 35-kDa G-beta in zone 7 were strongly decreased after high-speed centrifugation (Fig. 6C), confirming that these proteins are associated with the vesicles as soon as they are present in the fluid.

Origin(s) of the Vesicles

Because we have previously observed that 5–10% of sperm died during epididymal transit [14] and because sperm lose their cytoplamic droplet during the corpus transit, we checked whether some of the vesicles were derived from the sperm cells or their droplets. For this, we used different antibodies to investigate whether the vesicle proteins were also present on the sperm and/or the cytoplasmic droplet.

Sperm obtained from the caput region (zone 2) that retain their cytoplasmic droplets; from the corpus region (zone 6), where sperm with or without droplets coexist; from the cauda epididymis (zones 8–9), where sperm have lost their droplets were washed by percoll gradient centrifugation before extraction. This also purified intact cytoplasmic droplets released in the caudal fluid. As a control, an equal volume of fluid from the cauda containing both the sperm and the droplets was centrifuged and washed only with PBS.

The PBS- and percoll-washed cauda sperm, the cytoplasmic droplets, the caudal fluid and the vesicles pellet were run on SDS-PAGE (Fig 7A). Almost no difference was observed in the protein pattern between the two caudal sperm preparations (first and second lanes) and the sperm extracts from zones 2 and 6 (not shown). The cytoplasmic droplet protein pattern was very different from those of the sperm extracts, the caudal fluid, and the vesicles. We have previously shown that acrosin could be released in the fluid by dying sperm [14], and thus, if some of the vesicles were derived from the acrosomal membranes of these damaged sperm, some of the acrosin should remain associated with it. With our ram anti-acrosin antibody, three different acrosin forms were observed in the sperm extracts between 43 and 50 kDa and a 43-kDa band was slightly visible in the caudal fluid (Fig. 7B). No reactive bands were found in cytoplasmic droplets or in the vesicle preparation, suggesting that the vesicles did not derive from the degraded acrosomal sperm membranes and indicating also that the cytoplasmic droplet preparation was not contaminated with sperm.



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  		FIG. 7. Origin of the protein vesicles. Equivalent amounts of epididymal cauda sperm before (–) and after percoll wash (+), cytoplasmic droplets, cauda epididymal fluid (CEF), and purified vesicles were separated by 6– 16% SDS-PAGE. After Coomassie blue staining, except for sperm before and after percoll washing, a very different pattern of proteins was observed for each type of sample. An equivalent gel was transferred to nitrocellulose and probed with a ram anti-acrosin antibody. Sperm were strongly reactive and a slight reaction was observed in the cauda fluid

The previous samples and sperm extracts from zones 2 and 6 were probed with the different antibodies reacting with the vesicle proteins (Fig. 8). The anti-phosphodiesterase and anti-neprilysin antibodies showed reactive bands in the cauda fluid containing the vesicles, but no reaction was obtained with the cauda sperm extract or cytoplasmic droplet. The neprilysin antibody reacted with the caput sperm extract but not with corpus and cauda sperm extracts, but the neprilysin form observed in the fluid showed a lower molecular weight (less than 120 kDa; see Fig. 8, compare lanes Z2 and CEF). Moreover, this neprilysin fluid form appeared only in zones 6–7, suggesting that it was a different form than the sperm neprilysin. The anti-actin antibody reacted with the sperm extracts but the decrease in the reaction observed in the percoll-washed sperm and the substantial reaction with the cytoplasmic droplets indicated that actin was mainly present in these later (Fig. 8). As expected, the total fluid was reactive because actin was present in the vesicles.



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  		FIG. 8. Common proteins between sperm and vesicles. Equivalent amounts of epididymal percoll-washed sperm from zones 2 and 6, cauda sperm before (–) and after percoll washing (+), purified cytoplasmic droplets, and cauda epididymal fluid (CEF) were separated, transferred, and probed with antibodies reactive to vesicle proteins. The major proteins ENPP3 and neprilysin were not found on sperm from zones 6 and 9 nor on cytoplasmic droplets, while actin, G-beta, and the 43-kDa PAS6/ 7 were mainly associated with cytoplasmic droplets but not with cauda epididymal percoll-washed sperm

The anti-G beta antibody recognized different bands on sperm extracts, the most reactive at 43 and 35 kDa and a strong doublet at about 20 kDa. The 43-kDa band showed varying intensity depending on the samples, and a less reactive band at 55 kDa could also be observed. The 35-kDa band was decreased on the percoll-washed sperm extract but was highly enriched in cytoplasmic droplets while the other bands remained at the same level (Fig. 7). This 35-kDa band was also found in the CEF and in the vesicles. Anti-lactadherin-PAS6/7 reacted slightly with the 50-kDa and 35-kDa bands in sperm, and a 43-kDa band was also visible in PBS-washed sperm but decreased after washing with Percoll. An equivalent 43-kDa band and the 50-kDa band were retrieved in cytoplasmic droplets and in the caudal fluid (Fig. 8). Similar results were obtained when ejaculated sperm extracts were used instead of cauda epididymal sperm extracts (not shown).

All these findings indicated that none of the immunoreactive proteins found in the vesicles were present on mature sperm extracts and that only three of the five antibodies cross-reacted between the cytoplasmic droplets and the vesicles, suggesting no direct relationships between the sperm and the vesicles.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results demonstrated clearly that membranous vesicles are normal constituents of the cauda epididymal fluid and that, although a small number of vesicles originate from the other accessory glands, this fluid is the main source of the vesicles retrieved in the ram seminal plasma. This is demonstrated by the very similar one-dimensional and two-dimensional SDS-Page protein profiles and the protein compositions found for the epididymal and seminal plasma vesicles of normal rams and the absence of these vesicles in the seminal plasma of vasectomized rams.

Several groups have suggested that small membranous vesicles could be formed by apocrine or bleb secretions from the testis, efferent ducts, and the epididymal epithelium [53, 58, 59]. It has also previously been shown that the seminal plasma of different mammals, including the humans, contains neurosecretory-like vesicles, and some findings have indicated that these vesicles originated from the prostate and seminal vesicles [60, 62]. The epididymal membrane vesicles described here are quite homogeneous in size and delimited by a single bilayer membrane. Although their mean diameter is smaller than the average diameter reported for seminal plasma vesicles, it can be concluded that the seminal-fluid vesicles are composed of a mixture of vesicles originating from the different accessory glands, to which the epididymis largely contributes (see also [63]). Moreover, almost all proteins identified in ram epididymal vesicles have also been found in a recent proteomic profiling study of human seminal plasma vesicles [26], and one protein, aldose reductase, has also been described in bull epididymal vesicles [27] (see also Table 2). This suggests that, as in the ram, a large proportion of the vesicles found in seminal plasma in humans (and certainly in bulls) are derived from the epididymis. Alternatively, these different types of vesicles may be shed from the cell membrane by a very similar mechanism, including the same membrane domains containing the same proteins.

Both the epididymal and seminal plasma vesicles also show some physical and biochemical similarities to vesicles secreted by different types of epithelia and cells called exosomes [11]. They are in the same size range, they are not retained by 0.22-µm filtration, and they can be purified by size exclusion on gel filtration. Indeed, the ram cauda fluid size-exclusion peak obtained by gel filtration was found to have a similar protein pattern as the vesicle pellet, and this peak was considerably decreased when the fluid was centrifuged at high speed before separation (unpublished data). All this suggests that epididymal and seminal plasma vesicles may be members of an exosome-vesicle family. This hypothesis is also supported by the fact that the different proteome of the exosomes published to date also contain similar proteins to epididymal and seminal plasma vesicles (for review, see [11, 64]). It has been suggested that exosomal vesicles are secreted from cells upon fusion of multivesicular endosomes with the cell membrane by an unknown mechanism [64]. However, we still do not have enough information on epididymal vesicles to know whether they are a true homogeneous population of exosomes. For example, no V-ATPase subunits (a marker of intracellular acidic vesicles) were found in exosomes, while one is present in the epididymal vesicles. Alternatively, this is in agreement with the suggestion that the composition of exosomes may vary in function with the tissue from which they are derived [11, 64].

Several types of membrane secretion have been described in the efferent ducts and epididymis, including bleb secretions, which involved a large surface of the apical membrane and then form larger vesicles in the fluid. Destruction of these large vesicles could give rise to smaller vesicles similar to exosomes. Other possible origins include the confluence of smaller vesicles coming from the upper tract (which may explain the actin results we have obtained). However, our findings that these vesicles have a specific protein composition, which is different from the mature sperm and to a large extent from cytoplasmic droplets, and contain proteins that are only present in the cauda region strongly suggest that they are mainly the result of a specific secretion from this region.

Different roles have been suggested for seminal plasma vesicles (prostasomes), like immunosuppression, oxidative protection, and bacterial defense (for reviews, see [8, 9, 63, 65]). Similar properties have been also suggested for exosomes, and these diverse roles may reflect the different biological activity of the composing proteins (see Table 2). Both seminal plasma and epididymal vesicles have been suggested to be involved in sperm membrane maturation by transferring proteins, particularly hydrophobic proteins, to the cell under very specific conditions (acidic pH, presence of zinc [48, 66]). Indeed, these vesicles are rich in GPI anchored proteins such as CDw52, CD55, CD59, prion protein, and p25b, and it has been shown that, in vitro, the vesicles can fuse with sperm [8, 61, 65, 67, 68]. Immunoblotting revealed that several new hydrophobic and lipophilic compounds were bound with the epididymal vesicles. We observed the presence of the very hydrophobic protein 17 kDa secreted by the cauda epididymis [5] and of PGDS secreted in the caput epididymis [13]. One form of E-RABP is also associated with the vesicles, although several different isoforms are secreted in the epididymis [4]. The 17-kDa protein has been immunolocalized on cauda sperm flagellum, but PGDS and E-RABP were not found in the sperm membrane ([13] and unpublished results). We have also observed that the main prion protein isoform, a GPI-anchored protein present on epididymal exosomes, was not transferred to the cauda or ejaculated sperm membrane [10]. Exchange of other seminal plasma vesicle membrane-linked proteins have also been described, particularly for dipeptidyl peptidase IV [68]; transfer has been suggested after ejaculation in horse [52, 68, 69].

However, our data demonstrated that none of the epididymosomes major membrane-bound proteins was present in vivo on cauda epididymal or ejaculated sperm extracts. Thus, if these vesicles are involved in sperm membrane transformations, it is via very subtle exchange mechanisms that remain to be explained but not by a vesicle-fusion mechanism. Moreover, the epididymal exososomes represent only a small percent of the total cauda protein, and the most hydrophobic proteins were mainly present in the caudal fluid bulk phase under a soluble form. It is possible that these soluble proteins exist in micelles or complexes, which could explain the mechanism of their transfer to the cell membrane. Alternatively, these vesicles could play a role in the female genital tract, where their immunosuppressive and anti-oxidant properties might protect the sperm from degradation.

In conclusion, we have clearly demonstrated that vesicles with a specific protein content exist in the caudal fluid and form the majority of the vesicles retrieved in the seminal plasma of the ram. Using different markers, we have also provided evidence that the majority of these vesicles are mainly secreted in the cauda/corpus regions of the epididymis and accumulated in the caudal fluid. These vesicles, which are similar to the previously described vesicles from the seminal plasma, are physically and biochemically related to exosomes. They represented only a small fraction of the caudal-fluid proteins and do not contain all the hydrophobic compounds of this fluid. We found no evidence for the exchange of the major proteins between vesicles and sperm within the epididymis or during ejaculation. The role of these vesicles in male reproductive physiology requires further investigations.


   ACKNOWLEDGMENTS
 
The authors thank Dr J Rossier and the staff of the laboratory of Neurobiology (Ecole Supérieure de Physique et de Chimie Industrielles, Paris, France) for their help with the mass spectrometry. We are grateful to Dr C. Thery (U520, Institut Curie, Paris, France), for valuable advice and providing us with antibodies known to react with exosomes. We also thank different colleagues that provided specific antibodies used in this study, particularly Dr M. Maurice (U538 INSERM, CHU St-Antoine, Paris, France) and Dr J. T. Rasmussen (Dept of Molecular and Structural Biology, University of Aarhus, Denmark). We thank A. Collet and G. Tsikis for technical help and J.-C. Nicolle and B. Delaleu for their expertise in electron microscopy.


   FOOTNOTES
 
1 Thesis support was given to S.M. by Région Centre. Back

2 Correspondence: Dr. Jean-Luc Gatti, UMR 6175 INRA-CNRS-Université de TOURS-Haras Nationaux, Physiologie de la Reproduction et des Comportements, Institut National de la Recherche Agronomique de Nouzilly, Monnaie 37380, France. FAX: 33 247 427 743; gatti{at}tours.inra.fr Back

Received: 20 September 2004.

First decision: 11 October 2004.

Accepted: 20 December 2004.


   REFERENCES
TOP
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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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