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a URA 1291 INRA-CNRS, Institut National de la Recherche Agronomique, Station de Physiologie de la Reproduction des Mammifères Domestiques, 37380 Monnaie, France
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The fluid and membrane 17- and 23-kDa antigens had a low isoelectric point and were glycosylated. The fluid 17- and 23-kDa proteins had hydrophobic properties: they were highly enriched in the Triton X-114 detergent phase and could be extracted from the cauda epididymal fluid by a chloroform-methanol mixture. These proteins were further purified, and their N-terminal sequences did not match any protein in current databases.
A polyclonal antibody against the fluid 17-kDa protein recognized the protein in the cauda epididymal sperm extract and immunolocalized it on the sperm flagellum membrane and at the luminal border of all cells in the cauda epididymal epithelium.
These results indicated that secreted glycoproteins with hydrophobic properties could be directly integrated in a specific domain of the sperm plasma membrane.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the ram, sperm membrane protein and glycoprotein changes during epididymal transit have been documented by SDS-PAGE studies [13–18] and by analysis of lectins and gold particle binding [19, 20]. It has been shown that most of the proteins from the testis are reabsorbed in the rete testis or in the first region of the epididymis and replaced by specific epididymal secretions [13–16, 21, 22]. Thereafter the sperm membrane changes are intimately linked with variations in the composition of the surrounding fluid due to epithelium activity, and some of the cauda epididymal components have also been shown to bind to or to be integrated to the sperm surface [23, 24].
In order to increase understanding of the relationship between ram epididymal secretions and the variations in sperm plasma membrane composition, we examined the common antigens in the sperm membrane and epididymal fluid. The further biochemical characterization of these proteins should allow study of their integration mechanism and their putative role in the sperm maturation process.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult Ile de France rams were used for this study. Epididymides were obtained by castration or from freshly killed animals at the local slaughterhouse. The epididymal fluids were obtained free of blood by microperfusion of the tubule at 10 different sites [25], i.e., zones 0–4 (caput region), zones 5–6 (corpus region), and zones 7–9 (cauda region), with PBS (137 mM NaCl, 15.4 mM KCl, 7.7 mM Na2HPO4, 1.5 mM KH2PO4, 0.5 mM MgCl2, 0.5 mM CaCl2, pH 7.4). Spermatozoa were separated from the fluid by centrifugation (1500 x g for 15 min). The supernatant was carefully removed and centrifuged twice at 15 000 x g for 10 min and was used directly or stored at -20°C.
For protein purification, zones 8 and 9 of the cauda epididymidis were retroperfused from the vas deferens with PBS, and the sperm and the fluid were separated as described above. The centrifuged fluid was supplemented with 0.5 mM PMSF (Sigma, Saint Quentin Fallavier, France) and passed through a 0.2-µm filter before overnight dialysis against 5 mM ammonium carbonate. The protein solution was then lyophilized and stored in a desiccator at 4°C (these lyophilized proteins are referred to in the text as CEP).
The extraction of proteins from Percoll-washed sperm by N-octyl-ß-D-glucopyranoside (1.5% w:v) in the presence of protease inhibitors has been previously described [10]. The sperm protein extract was used directly for gel electrophoresis or after overnight dialysis against PBS for immunization.
For the castration-supplementation experiments, orchidectomy was performed on adult rams weighing about 80 kg. After 6 mo of castration to obtain complete epididymal regression, animals were injected daily with 40 mg testosterone propionate (Fluka, Saint Quentin Fallavier, France). Epididymides were recovered after 3, 8, 20, 40, and 60 days of treatment. The luminal content was obtained directly by incubating about 50 mg of excised tissue from 10 different zones of the epididymis in 1 ml of Dulbecco's modified Eagle's medium at 35°C under 5% CO2. After 1 h under agitation, the incubation medium was removed and centrifuged at 15 000 x g for 10 min. The supernatant was centrifuged and kept at -20°C until use.
Gel Electrophoresis and Protein Blotting
Preparation of gel samples and methods for isoelectric focalization were performed as previously described [26]. For SDS-polyacrylamide gel separation, a linear 6–16% polyacrylamide gradient or 20% polyacrylamide gel was used. The apparent molecular weights of the separated proteins were obtained by comparison with standard proteins (Pharmacia, Orsay, France). The gels were silver stained.
Semidry transfer of proteins to nitrocellulose was at 0.8 mA/cm2 over 2 h. The blots were blocked with 20 mM Tris-HCl, 150 mM NaCl, pH 7.3 (TBS) supplemented with 0.5% Tween 20 and 5% goat serum. The second antibody was a goat polyclonal anti-rabbit antibody conjugated with peroxidase (Sigma; dilution 1/2000 to 1/5000) and was revealed with 4-chloro-
-naphthol.
Production of Antibodies
Serum from different female rabbits was tested on fluids and sperm extract from various epididymal regions by Western blotting before the first injection. Only rabbits presenting no reactions were kept for immunization, which was performed either with N-octyl-ß-D-glucopyranoside-solubilized sperm proteins or with proteins from the cauda epididymal fluid. Four injections of about 100 µg of protein each were performed. The initial injection included Freund's complete adjuvant; 3 wk later the first boost included incomplete adjuvant, and this was followed by two more boosts at intervals of 15 days. The last serum was used diluted 1/5000. The serum of the two rabbits immunized with the sperm extract showed only slight differences in the antigens recognized when probed on a Western blot of cauda epididymal fluid. They were therefore mixed in order to increase the chances of obtaining cross-reacting antigens with the fluids.
The same protocol was followed with the gel electrophoresis-purified 17-kDa protein for the preparation of the anti-17-kDa polyclonal antibody (see below). This polyclonal antibody was further blot-affinity purified [27] using bands of the 17-kDa region of a blot made from a 15% gel separation of the sperm plasma membrane extract. The final solution of antibody contained 50–80 µg/ml of protein. This affinity-purified antibody was diluted 5-fold for blots and used directly for immunolocalization.
Triton X-114 Partitioning
A stock solution of Triton X-114 was prepared as previously described [28]. Cauda epididymal proteins (10 mg of CEP) were dissolved in 1 ml of Tris-HCl (50 mM, pH 6.9) and mixed with 1 ml of a 20% Triton X-114 solution (made from the stock solution in Tris-HCl, 50 mM, pH 6.9) and left to stand at 4°C for 30 min. The solution was then heated at 37°C for 5 min and centrifuged at 2000 x g for 10 min. The supernatant was again extracted by 1 ml of 10% Triton X-114. Both final Triton X-114 phases were mixed and centrifuged at 10 000 x g through a 6% sucrose (w:v) layer; the oily phase recovered at the bottom of the tube is referred to as the TX-114 extract.
Reverse-Phase HPLC Purification of the 17-kDa Protein and Its Peptide Fragments
Based on their hydrophobic properties (see Results), the 17- and 23-kDa proteins were extracted from the cauda epididymal proteins by methanol-chloroform extraction [29]. A solution at 10 mg/ml of CEP was made with deionized water and mixed with 4 volumes of a chloroform-methanol solution (1:2 v:v). After 2 h at -20°C to allow precipitation of proteins, the solution was centrifuged at 15 000 x g; the supernatant was removed and centrifuged again at 15 000 x g. This second supernatant was then vacuum dried. About 10% of the weight of the initial CEP was recovered as dried material.
This residue was resuspended either directly in gel sample buffer for electrophoretic purification (see below) or dissolved in 10% methanol-deionized water (1/5 of the extracted volume) for separation by reverse-phase HPLC. In the latter case, the solution was centrifuged at 15 000 x g, filtered at 0.2 µm, and injected on a reverse-phase column (Pharmacia pro-RPC, C1-C8) on an HPLC system (Prosys; Biosepra, Cergy Pontoise, France). Elution was performed with a water-methanol gradient (10–100%) in the presence of 0.1% trifluoroacetic acid. The 17- and 23-kDa proteins were recovered in the fractions between 90% and 100% methanol (see Results).
Purification of the 17-kDa Protein by Electrophoresis
The methanol-chloroform extract was subjected to electro-endosmotic preparative electrophoresis (ELFE system; Genofit, Geneva, Switzerland). The dried material was dissolved in sample buffer and deposed on top of a 20% polyacrylamide tube gel (10 cm long, 1 cm in diameter) and electrophoresed for 10 h at 10 mAmp. Fractions were collected every 10 min (about 1 ml/fraction) after exit of the front blue dye. Each fraction from the separation was submitted to gel electrophoresis (20% SDS-PAGE), and an equivalent gel was transferred to nitrocellulose to be probed with the polyclonal antibody against the sperm proteins. The 17-kDa immunoreactive fractions from different separations were pooled, dialyzed against deionized water, and injected into a rabbit.
N-Terminal Amino Acid Sequence Analysis
The N-terminal amino acid sequences of the protein or fragments separated by reverse-phase HPLC were obtained on a Porton sequencer (Model LF3000, Beckman Instruments, Palo Alto, CA) using the reagents and methods recommended by the manufacturer. Homology was sought using BLAST and FASTA software [30].
Immunolocalization
Localization of the 17-kDa protein in the epididymis and on the spermatozoa was performed as previously described [31]. Briefly, tissues from the various epididymal regions were fixed at 4°C in 0.3% glutaraldehyde, 4% paraformaldehyde, and 0.1 M phosphate buffer, pH 7.4. After several washes in cold buffer containing 15% sucrose, the tissues were frozen and sectioned at 5–6 µm in a cryostat. The sections were preincubated in 10% goat serum-PBS and then incubated overnight in anti-17-kDa antibody. After three washes in PBS, the slices were incubated with the peroxidase-conjugated goat anti-rabbit IgG (dilution 1/100) and revealed with 4-chloro--naphthol. Sections were mounted in glycerol before observation. So that the positive reaction could be clearly seen, no counterstaining was used.
Spermatozoa carefully washed in PBS were layered on polylysine-coated slides and fixed in 2% paraformaldehyde in PBS. After three washes with PBS, they were incubated with the anti-17-kDa antibody and then further treated as described for tissue sections.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cauda epididymides were retroperfused with PBS, and sperm were separated from the cauda fluid. The spermatozoa plasma membrane proteins were further treated with a N-octyl-ß-D-glucopyranoside solution to solubilize the plasma membrane proteins. The fluid and this sperm extract served to immunize rabbits, and the polyclonal antisera obtained were used to search for immunorelated compounds between epididymal fluids and sperm membrane.
Epididymal fluids probed with polyclonal antibody against cauda epididymal sperm proteins (anti-CESP) Several immunoreactive bands with molecular masses ranging from 17 to 111 kDa were observed in fluids obtained by perfusion at different sites along the epididymis (Fig. 1A and Table 1). We observed variations in the intensity of the different antigens between rams and to a lesser extent in their regionalization. However, the extensive reaction between 17 kDa and more than 40 kDa in the cauda region (zones 7–9) was always present. When a smaller quantity of cauda fluid was loaded on the gel, the intensity of the reaction was decreased, and two main reactive bands were visualized at 17 kDa and 23 kDa (see below). This pattern was unchanged whether the cauda fluid was reduced by ß-mercaptoethanol or not.
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Other antigens were situated at about 105–94 and 85 kDa from zones 1/2 to 9. These bands presented a molecular mass shift of about 5–10 kDa between zones 2 and 3. These proteins were recently identified as the soluble form of the germinal angiotensin I-converting enzyme released from the sperm membrane in the first zones of the epididymis [10].
More faintly reactive antigens were also commonly observed in the various epididymal series tested: i.e., the 115-kDa band from zones 4 to 9 or the doublet at 38 kDa and 43 kDa from zones 3 to 8 (see Table 1). The most variable antigen was the doublet seen in regions 3–5 at 15–20 kDa, which was highly reactive in this series.
Sperm plasma membrane extract probed with polyclonal antibody against cauda epididymal fluid protein (anti-CEP) Only a few reactive bands were observed within the proteins extracted from spermatozoa from the various epididymal regions (Fig. 1B). The two main antigens were situated at 17 kDa and 23 kDa and were restricted to the cauda region (zones 7–9). Another slightly reactive band at about 27 kDa was observed in regions 1 and 2. The same pattern was obtained with all the rams tested. This low number of reactive bands was not due to a lack of proteins, as demonstrated by Ponceau red staining and also by the large number of reactive bands observed when the blot was probed with anti-membrane antibodies. In the latter case, the 17- and 23-kDa antigens specifically restricted to the cauda region were also observed (not shown).
Analysis of the Antigens by Two-Dimensional Gel Electrophoresis
The cauda epididymal fluid and sperm membrane extract were separated by two-dimensional gel electrophoresis and transferred to nitrocellulose. When the cauda fluid was probed with the anti-CESP antibody (Fig. 2A), reactive bands situated at 17 kDa and 23 kDa trailed from the left (acidic) to the right (basic) side of the gel. This striking pattern was obtained with all ram cauda epididymal fluids tested and was not due to a problem of gel electrofocalization, since the 95-kDa and 85-kDa antigens were well focalized. Other very faintly reactive spots were also visible, corresponding to the other antigens observed on one-dimensional gel.
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When the first dimension was not equilibrated (the isoelectrofocalization was stopped after 2 h of run), an intense immunoreactive area from 17 to almost 40 kDa was observed on the acidic side of the blot (not shown).
When the sperm membrane extract from the cauda epididymidis was probed with the anti-CEP antibody (Fig. 2B), two reactive smears at 17 and 23 kDa ranging from pH 4.0 to more than 8.0 were observed. This pattern was very similar to that obtained with the fluid probed by the anti-CESP, showing that the membrane 17- and 23-kDa antigens had the same behavior as those of the fluid. No other reactive antigens could be seen.
The 17- and 23-kDa Antigens Came From the Epididymal Tissue and Were Androgen Dependent
The supernatants from the incubation of epididymal tissues from orchidectomized rams were tested with the anti-CESP polyclonal antibody. No trace of reactive proteins was found in the epididymal supernatant before the beginning of supplementation (not shown) or after 3 days of testosterone injection (Fig. 3, 3d). After 8 days of supplementation, a slight reaction at about 17–23 kDa occurred in zone 8 of the cauda region, while the reactive bands at 17 kDa and 23 kDa were clearly regained after 20, 40, and 60 days of treatment, with the same distribution as in a normal ram (Fig. 3, 40d). No other reactive bands were visible.
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Biochemical Characterization of the 17- and 23-kDa Fluid Antigens
In order to isolate the 17- and 23-kDa compounds from the cauda epididymal fluid, several properties of these antigens were first tested. The SDS-PAGE behavior suggested that the 17- and 23-kDa polypeptides were glycoproteins; then the cauda epididymal proteins were chemically deglycosylated by trifluoromethanesulfonic acid treatment [32] or by sequential action of N- and O-glycosidase. A shift in the molecular mass of the reactive antigens of about 3–5 kDa was visible on gel electrophoresis after each treatment (not shown), indicating that at least O-glycosylation existed on these proteins. On this basis and because of their acidic isoelectric point suggesting potential sialic acid residues, the cauda proteins were loaded on lectin columns (wheat germ agglutinin and concanavalin A). Although the 17- and 23-kDa proteins bound to these columns to some extent, not all of these antigens were retained. Moreover, a large number of other cauda epididymal proteins remained as contaminants since they were also glycosylated (results not shown). On classical cation exchange column (DEAE), the 17- and 23-kDa proteins bound when loaded at pH 7.4, but they were not released as a single peak; instead they were found in a very large number of fractions.
Ethanol fractionation of the CEP was then performed, and we observed that the antigens remained in the supernatant even when the concentration reached more than 80% ethanol. The same result was obtained with methanol, suggesting that these proteins either were hydrophobic or were surrounded by a hydrophobic environment. This was further confirmed when a CEP solution was subjected to Triton X-114 phase partitioning. This detergent has been used to discriminate between hydrophobic (membrane) and hydrophilic (soluble) proteins [28]. Only a small number of proteins were recovered in the Triton X-114 phase, and the 17-kDa antigen was retrieved among them (Fig. 4).
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On the basis of these findings, we treated the CEP by a method described to extract proteolipids [29]. After centrifugation, the solvent phase was vacuum dried; the residue was separated by gel electrophoresis and probed by the anti-CESP polyclonal antibody (Fig. 5). We observed on the silver-stained gel that only a fraction of proteins with molecular masses in a range of 15–30 kDa were not precipitated by the chloroform-methanol treatment (Fig. 5, lanes A and B). This extracted material contained the antigens, as shown by the reaction with the anti-CESP antibody (Fig. 5, lanes C and D); and in general, almost all the reactive 17- and 23-kDa compounds present in the CEP solution were extracted.
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Purification and N-Terminal Sequences of the Fluid 17- and 23-kDa Antigens
Purification In an initial attempt to purify the proteins, the chloroform-methanol extract was loaded on a 20% tube gel using a preparative electrophoresis apparatus (see Materials and Methods). The fractions were blotted and probed with the anti-membrane antibody. The less reactive 23-kDa compound could not be detected, while the most reactive fractions containing the 17-kDa protein were pooled, dialyzed, and injected into a rabbit. The polyclonal antibody obtained was further blot-affinity purified against the membrane 17-kDa protein [27]: this antibody became highly specific for the 17-kDa protein both from the fluid and from the membrane in the cauda epididymal region (Fig. 6, A and B). This affinity-purified antibody was used to further purify the 17-kDa protein from the fluid.
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The methanol-chloroform extract was loaded on a C1-C8 reverse column and eluted with various solvents: propanol, acetonitrile, and methanol. The best separation was obtained with a methanol gradient in the presence of trifluoroacetic acid (Fig. 7A). SDS-PAGE and Western blotting with the affinity-purified antibody showed that the 17-kDa protein was separated from the rest of the protein extract when 90–95% methanol was reached (Fig. 7B).
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We also observed that a protein of 23 kDa was enriched in the fractions before the 17-kDa protein (Fig. 7). This protein was concentrated and tested with the anti-CESP polyclonal antibody. We then observed that this 23-kDa protein was immunoreactive (Fig. 8). Although the blot revealed that the 17-kDa protein contaminated this fraction, it was kept for N-terminal sequencing.
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Protein sequencing The N-terminal of the 17-kDa protein was not blocked, and 23 amino acids were obtained. Following are the N-terminal sequences of the proteins: 17-kDa protein, NH2-V-N-S-P-S-G-P-A-V-C-A-A-D-A-P-P-G-R-P-S-C-M-D-. The N-terminal sequencing of the 17-kDa protein was performed twice until the 24th amino acid, and at least five times for the 10 first amino acids. The serine in positions 3, 5, and 20 showed a striking pattern on the chromatogram, suggesting that it could be posttranscriptionally modified. Sequencing cycles 10 and 21 did not produce any amino acid peaks, and they were putatively assigned as cysteine.
The 17 kDa was subjected to various protease treatments in order to obtain internal sequences. With trypsin and endo-Lys C, no degradation was observed by SDS-PAGE. With papain, which is a less specific protease, the 17 kDa was proteolyzed, and a main fragment of about 10 kDa was visualized on a 20% SDS-PAGE. After reverse-phase HPLC, three peptide fragments could be separated and sequenced. One gave 6 amino acids, another 9 amino acids, and the last one 5 amino acids. All started from the N-terminal sequence, except that an alanine was observed in position 3 instead of the serine observed after direct sequencing. This N-terminal sequence showed no evident homology with any known protein in the current databases.
When the 23-kDa protein was submitted to N-terminal sequencing, two sequences were obtained, as expected: one corresponding to the 17-kDa contaminating protein and the other certainly to the 23-kDa protein: NH2-R-C-R-T-F-Q-S-Y-K-K-P-Q-Q- (this sequence was performed twice). The N-terminal sequence of this 23-kDa protein stopped abruptly after the 13th amino acid. This sequence showed no homology with proteins in the various databases consulted. The low amount of protein and its contamination with the 17 kDa precluded attempts to obtain an internal sequence.
Tissue and Spermatozoa Immunolocalization of the 17-kDa Protein
The affinity-purified anti-17-kDa antibody was used to localize the protein on the spermatozoa and in the epididymal epithelium. All sperm were completely negative from the caput to the corpus epididymidis (zones 0–6) (Fig. 9a shows sperm from zone 6). As soon as the sperm entered the cauda epididymal region (zone 7), all of them became positive. Labeling was very high throughout the principal piece of the flagellum, while the head remained completely negative (Fig. 9b). The membrane localization of the antigen was assessed by treating the sperm with 0.01% Triton X-100: removal of the plasma membrane resulted in the complete loss of labeling. We recently raised a second anti-17-kDa antibody from the protein purified by HPLC. This antibody also intensely labeled the flagellar piece of the cauda and ejaculated sperm (not shown).
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As for spermatozoa, no positive immunoreaction was observed on tissue slices from the caput to the corpus regions of the epididymis (Fig. 9c shows zone 6). The transition from nonreactivity to reactivity occurred at the beginning of the cauda region in zone 7. Thereafter the epithelium was labeled uniformly from zones 7 to 9 (Fig. 9d shows zone 7). The reaction was clearly visible around the microvilli and the luminal ridge of all cells lining the duct. No clear labeling of the cytoplasm or of cytoplasmic structures was visible, nor was labeling at the apical side of the cells. No differences in labeling between cells could be observed.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Among these epitopes, two compounds of 17 and 23 kDa were clearly present in both the fluid and the membrane. These compounds were specifically restricted to the cauda epididymidis. These antigens disappeared from the cauda fluid of the castrated ram, but they were recovered after testosterone supplementation, with the same epididymal distribution. These results strongly suggest that these components were androgen-dependent proteins, specifically secreted by the ram cauda epididymal epithelium and not the result of sperm protein release or the degradation of epididymal protein from a previous region.
N-Terminal sequencing indicated that the 17- and 23-kDa proteins are two different proteins and that they have no homology with proteins from databases. These proteins are therefore new epididymal proteins, which remain to be fully characterized.
The fluid and membrane 17- and 23-kDa proteins showed the same striking electrophoretic behavior, certainly due to glycosylation and perhaps to the presence of sialic acid residues, which could explain the acidic isoelectric point observed. The very low isoelectric point of these two antigens was also confirmed when the cauda fluid was submitted to liquid isoelectrofocalization. In this case, these two reactive compounds were found to be highly accumulated in the most acidic compartment (pH about 2), and to a lesser extent in fractions from pH 2 to 8 (unpublished results). The different techniques used to purify the 17- and the 23-kDa compounds established that they have hydrophobic properties and behave like membrane proteins or lipoproteins, although they appear to be under a soluble form in the fluid.
The polyclonal antiserum obtained with the purified 17-kDa fluid protein recognized the protein on membrane extracted from extensively washed cauda epididymal sperm, confirming that the protein is strongly linked to or inserted into the sperm plasma membrane. This was also confirmed by the immunolocalization of the protein on the sperm flagellar membrane.
Proteins secreted by the epididymal epithelium in different species have been shown to integrate the sperm membrane, and two mechanisms have been described for such membrane integration. One is linkage of the protein via a phosphatidylinositol anchor [33], and the other is proteolytic processing that allows inclusion of part of the protein in the plasma membrane (for reviews, see [4, 11]). The 17-kDa and the 23-kDa proteins retain the same apparent molecular masses in both the fluid and the plasma membrane, indicating that no proteolytic processing is involved in their integration. We also observed that a simple monophosphatidylinositol tail did not attach the 17-kDa protein to the membrane, since this protein was not released when ejaculated sperm were treated with phosphatidylinositol phospholipase C. Meanwhile the existence of a complex anchor structure could render this protein tail insensitive toward the phospholipase C, as recently reported for the human CD52 protein [33].
Another hypothesis comes from recent findings suggesting that vesicular prostasome-like vesicles ([34, 35]) might be also present in the epididymal fluid, where they may transfer proteins from the epididymal epithelium to the sperm membrane [33, 36]. This could explain some of our results concerning the high hydrophobicity observed for the fluid 17-kDa and 23-kDa proteins. However, it remains to be clearly demonstrated that such a transfer mechanism exists in the cauda.
Previous studies have shown that a 24-kDa protein, which was one of the major proteins iodinated in the cauda epididymal fluid, was able to bind to the sperm surface [13, 17]. This compound was not further characterized, and its relationship with the 23-kDa protein reported here has yet to be established.
A 18-kDa ram epididymal cauda sperm antigen, named ESA152, has been studied in detail [24, 37]. ESA152 is an 18-kDa glycoprotein with sialic acid residues. This antigen is mainly localized at the head of the sperm and redistributed to the anterior part after induction of the acrosomal reaction [23, 37]. This sperm localization therefore appears different from that found for the 17-kDa protein in the present study. ESA152 antigen was found to be accumulated only in certain types of epithelial cells of the ram cauda epididymidis, whereas the 17-kDa protein was present on all the cells lining the tubule. The 18-kDa-ESA152 protein was not purified or sequenced, but the compound showed striking biochemical similarity with the 17-kDa antigen reported in the present study.
The role of both the 17- and 23-kDa proteins in sperm maturation remains to be understood. The immunolocalization of the 17-kDa protein on the sperm tail suggests a role in motility. The cauda epididymidis is the place where the ram sperm acquires the capacity for straight-line displacement. The hydrophobic properties of these proteins could also suggest a role in transport of lipids and transfer or transformation, as shown for several other proteins present in the epididymal fluid and seminal plasma [38–40]. At the least, it is interesting to note that the 17- and 23-kDa proteins reported here present biochemical characteristics very similar to those of the human CD52 glycoprotein, which is also secreted in the cauda epididymidis where it binds to the sperm plasma membrane [33].
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by AIP-INRA BIOLOG 02, grant ACC-SV no. 9504155, by the CNRS and by Région Centre. X.D. was recipient of a thesis grant from the Région Centre. 
2 Correspondence. FAX: 33 247 427 743; gatti{at}tours.inra.fr
Accepted: November 22, 1999.
Received: September 3, 1999.
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