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Regular Article |
a Centre de Recherche en Biologie de la Reproduction and Département d'Obstétrique-Gynécologie, Faculté de Médecine, Université Laval, Ste-Foy, Quebec, Canada G1V 4G2
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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epididymis, gamete biology, male reproductive tract, sperm, sperm maturation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Using the hamster, we identified P26h, a sperm surface protein involved in zona pellucida (ZP) recognition [7–9]. P26h is glycosylphosphatidylinositol (GPI) anchored to the sperm surface during the epididymal transit. Membranous particles present in the luminal compartment of the epididymis are involved in the anchoring of P26h to the sperm surface and are involved in sperm maturation [10]. Ortholog proteins have been identified in other species such as the mouse [11], monkey [12], bull [13], and human [14–16]. The bovine and human proteins have been named P25b and P34H, respectively, and have been shown to be markers of male fertility [13, 17, 18]. As in hamster, P25b is GPI anchored to bovine spermatozoa, and epididymal membranous particles are involved in the transfer of this surface protein on male gametes [19]. These particles show similarities with prostasomes and have been named prostasome-like particles (PLPs).
Prostasomes are multivesicular particles first described as human semen constituents [20, 21]. Secreted by the prostate, they are also present in other male reproductive glands, including the epididymis [10, 19, 22, 23]. Prostasomes are multilamellar lipoprotein membrane particles with a diameter ranging between 50 and 500 nm. The cholesterol:phospholipid ratio can reach 2:1 in the human, with sphingomyelin being the major phospholipid [24]. Many proteins have been shown to be associated with prostasomes, including GPI-anchored proteins [10, 25, 26]. Prostasomes interact with spermatozoa, and different functions, such as modulation of the female's immune system, the regulation of complement, the enhancement of sperm motility, stabilization of the sperm plasma membrane, and the protection of sperm against the acidic female reproductive milieu, have been attributed to them [21]. The mechanisms underlying prostasome-spermatozoa interactions remain poorly documented. This is particularly true regarding PLP-spermatozoa interactions within the epididymal lumen and the importance of this phenomenon in sperm maturation.
Bovine epididymides were used in this study to document the mechanisms underlying the interaction of PLP with spermatozoa during epididymal maturation. Cauda epididymal PLP (PLPCd) were prepared and their surface proteins biotinylated (PLPCd-biot). The transfer of PLPCd biotinylated proteins to caput epididymal spermatozoa was then studied in vitro. These results are discussed with regard to the importance of PLP in epididymal sperm maturation.
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MATERIALS AND METHODS |
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Bulls were killed at a slaughterhouse; the testis and epididymis were rapidly chilled on ice and brought to the laboratory within 4 h. Only epididymides with swollen cauda distal tubules were used for this study. The epididymidis were dissected from the testis and kept on ice until used.
Intraluminal fluid from the cauda epididymidis was obtained by retrograde flushing by applying air pressure with a syringe in the proximal scrotal segment of the vas deferens. The cauda epididymal fluid was diluted with 0.15 M NaCl, centrifuged twice at 700 x g for 10 min to remove spermatozoa and one time at 3000 x g to remove remaining debris. The resulting supernatant was ultracentrifuged at 120 000 x g for 2 h. The pellet was suspended in 0.15 M NaCl and centrifuged a second time at 120 000 x g. The PLPCd were suspended in 250 µl of PBS (pH 7.4) containing 0.7 mg sulfo-NHS-LC-biotin (Pierce, Brockville, ON, Canada). The suspension was kept at room temperature for at least 30 min and then at 4°C for 8–10 h. The biotinylated PLPCd were diluted 15 times with 0.15 M NaCl and centrifuged at 120 000 x g for 2 h. The pellet was resuspended in a small volume of 0.15 M NaCl; the PLPCd suspension was aliquoted and kept at -80°C until use. To allow comparison with PLPCd, spermatozoa collected from cauda epididymal fluid were pelleted by centrifugation and surface proteins were biotinylated using the same protocol.
Isolation of the Caput Spermatozoa
The midparts of the caput epididymidis were carefully dissected. In one motion, 3–4 tubules were neatly cut with a razor blade and the fluid was recovered by applying a gentle pressure to the proximal portion of the dissected caput epididymidis. This operation was repeated 8–10 times for each epididymis, allowing recovery of 50–100 µl of intraluminal fluid per pair of caput epididymides. The fluid was diluted in 0.15 M NaCl; the spermatozoa were recovered by centrifugation at 700 x g for 5 min and were washed 2 more times in isotonic NaCl. Only preparations with no apparent contamination with blood were used. By this procedure, 100–200 million spermatozoa were recovered per bull.
Incubation of the Caput Spermatozoa with Biotinylated PLPCd
Caput spermatozoa (Cp spz) were resuspended at a concentration of 200 million/ml in MES-PIPES (10 mM each), at pH 5.5–7.5, containing 0.15 M NaCl and the PLPCd-biotin for 3–4 h at various temperatures and pH. Optimum pH for binding of PLPCd to caput spermatozoa was determined at 32°C and 37°C. Temperature-dependant experiments were performed in MES-PIPES, pH 6.5, at room temperature (22°C), 32°C, and 37°C during a coincubation period varying between 30 and 240 min.
The effect of divalent cations (0–2.5 mM) on PLPCd-biot binding to caput spermatozoa was evaluated at 37°C, pH 6.5, using the chloride salt of each metal tested. PLPCd-biot associations with spermatozoa were also performed in the presence of EDTA (3.3 mM) alone or in coincubation with 1 mM ZnCl2.
At the end of the incubation period, caput spermatozoa were diluted in 1 ml of 0.15 M NaCl and centrifuged at 700 x g for 5 min. The spermatozoa were washed 3 more times with isotonic NaCl.
Histochemistry
To visualize PLPCd association with spermatozoa, caput epididymal spermatozoa were incubated with PLPCd-biot at pH 6.5 as described above. After washing by centrifugation, caput spermatozoa were smeared on a microscopic slide and air dried. The biotin-labeled spermatozoa were incubated with the avidin/biotin-horseradish peroxidase reagent (Vectastain, Dimension Lab, Mississauga, ON, Canada) and stained using AEC/H2O2 in an acetate buffer, pH 5.2, according to the supplier's instructions.
Protein Extraction, Electrophoresis, Detection, and Analysis
Following coincubation with PLPCd in different conditions, caput spermatozoa were extracted with Triton X-100 (0.08% in water) for 15 min at room temperature and centrifuged 10 min at 5000 x g. Supernatants were precipitated with MeOH/CHCl3 according to Wessel and Flugge [27]. Extracted proteins were dissolved in sample buffer (2% SDS, 2% B-mercaptoethanol, 50 mM Tris, pH 6.8), electrophoresed by SDS-PAGE according to Laemmli [28], and transferred onto nitrocellulose membrane using a semidry milliblot-graphite electroblotter system at 4 mA/cm2 (Millipore). The membrane was blocked at least 1 h in PBS-0.1% Tween-20 containing 5% dried skim milk and then incubated with NA-HRP (neutravidin-conjugated horseradish peroxidase; Pierce) for 80 min in a 2% dried skim milk solution in PBS-Tween. The protein-biotin-NA-HRP complexes were revealed using a chemiluminescent peroxidase substrate according to the supplier's instruction (Roche Diagnostics, Laval, QC, Canada). No labeling was observed when caput spermatozoa were not incubated with PLPCd-biot. Quantities of biotinylated proteins associated with caput spermatozoa were determined by densitometry using a Gel Documentation System (Alpha Innotech Co., San Leandro, CA). Only the three major bands of molecular weight ranging from 28 to 34 kDa were used for the densitometric analysis of Western blots.
In order to identify PLPCd proteins interacting with caput spermatozoa, biotinylated PLPCd proteins as well as proteins extracted from caput spermatozoa preincubated with PLPCd were submitted to two-dimensional gel electrophoresis [29]. Isoelectric focusing was conducted under equilibrium and the second dimension under denaturating conditions. Biotinylated proteins were revealed on Western blots using neutravidin-conjugated horseradish peroxidase. Coomassie Blue-stained two-dimensional electrophoreses were run in parallel, and protein spots corresponding to PLPCd proteins transferred to caput spermatozoa were cut out and digested with trypsin for peptide mass spectrometric analysis using the Matrix-Assisted Laser Desorption Ionization Time-of-Flight technique (MALDI-TOF). Identification was performed by mass fingerprinting in a peptide mass computer database (www.expasy.ch/tools) at our core facility (Service Protéomique de l'Est du Québec, Ste-Foy, Canada).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To determine the localization of these transferred proteins, caput epididymal spermatozoa coincubated with PLPCd-biot were smeared and revealed with avidin/biotin-peroxidase. Light microscopic visualization revealed that the pattern of protein distribution varied from one sperm cell to the other, with a more intense labeling on the acrosomal cap (Fig. 3). Processing of spermatozoa unincubated with PLPCd-biot remained unlabeled (Fig. 3b).
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The quantity of 28–34 kDa PLPCd proteins transferred to a constant number of caput spermatozoa incubated with a constant amount of PLPCd-biot was determined by densitometry following coincubation performed in different conditions. The amount of proteins transferred to spermatozoa appeared to be pH sensitive. Between pH 5.5 and 7.5, a 2.5-fold difference in the amount of transferred proteins was observed, the optimum pH for protein transfer being 6.0–6.5 (Fig. 4). The time course of association of PLPCd-biot proteins to caput spermatozoa was performed at different temperatures. At 22°C, the amount of PLPCd-biot proteins remaining associated with caput spermatozoa was less efficient when compared with coincubations performed at 32 and 37°C (Fig. 5). The quantity of PLPCd-biot proteins transferred to a constant number of spermatozoa increased with time until it reached a plateau after 150–240 min of coincubation (Fig. 5).
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The effect of divalent cations on PLPCd-biot protein transfer to caput spermatozoa was investigated at pH 6.5. Whereas Mg2+ and Ca2+ at concentrations between 0 and 2.5 mM had no effect on the amount of protein remaining associated with spermatozoa following coincubation, Zn2+ had a beneficial effect (Fig. 6). At concentrations between 0.1 and 1.5 mM, Zn2+ present in the coincubation medium increased the amount of transferred proteins in a dose-dependent manner (Fig. 6). When 3.3 mM EDTA, a divalent cation chelator, was added to the coincubation medium together with 1.0 mM Zn2+, the amount of PLPCd-biot proteins transferred to caput spermatozoa was comparable with control medium without divalent cation (Fig. 7). EDTA per se has no detrimental effect when comparing the amount of protein remaining associated with spermatozoa following coincubation performed in 0.15 M NaCl with or without EDTA (Fig. 7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The applied biotinylation procedure used in this study allows labeling of proteins exposed at the surface of PLP. The electrophoretic pattern of cauda epididymal surface proteins appeared to be very complex (Fig. 1, lane C). When compared with biotinylated surface proteins of cauda epididymal spermatozoa, PLP surface proteins show a different electrophoretic pattern of comparable complexity. The pattern of proteins associated with PLP is probably much more complex considering that some of them are not exposed to the PLP surface and thus are not accessible to biotinylation. This is the case for P25b, a protein previously shown to be PLP associated and transferred to the sperm surface during epididymal transit [13, 19]. Therefore, the pattern of proteins associated with epididymal PLP is probably as complex as that for prostasomes, with which as many as 80 different proteins can be associated [22]. When biotinylated PLPCds are incubated with caput epididymal spermatozoa, only selected proteins are transferred to the male gamete (Fig. 1, lane A). The pattern of proteins transferred is the same from one PLP preparation to the other.
Two-dimensional electrophoretic analysis of biotinylated PLPCd proteins transferred to caput spermatozoa revealed proteins of molecular weight between 31 and 45 kDa with isoelectric point heterogeneity (Fig. 2). The MALDI-TOF analysis demonstrates that these PLPCd proteins transferred to caput spermatozoa are closely related, at least the four major protein spots analyzed. The protein transfer occurring during the epididymal transit appears to be quite selective and involves only a small proportion of proteins associated with prostasome-like particles. This might suggest that PLPs are not transferred or fused to spermatozoa as an intact entity. Another possibility is that the PLP population is heterogeneous and that proteins associated with these membranous vesicles vary from one subpopulation to the other. Only this subpopulation of PLP would then be involved in adding proteins to epididymal spermatozoa. For the moment, we cannot discriminate between these two possibilities.
In our experimental design, cauda PLP are coincubated with caput spermatozoa. It is thus possible that we are looking at only a very small fraction of the transfer going on since cauda vesicles already have exchanges with spermatozoa within the epididymis. We prefer to think that PLPs are in excess in the epididymal lumen. This is also true for prostasomes or prostasome-like particles prepared from semen or from all epididymal segments. In this regard, classical experiments on interactions between spermatozoa and prostasomes used ejaculated spermatozoa and prostasomes prepared from semen. Even though spermatozoa have already been in contact with these vesicles in semen, transfer of macromolecules still occurs in vitro [20, 21].
The subcellular localization of PLPCd proteins transferred to the sperm surface can be visualized by revealing smears of spermatozoa with avidin/biotin peroxidase preformed complexes. Distributions of PLP transferred proteins show heterogeneity from one sperm cell to the other (Fig. 3). Some spermatozoa remain unstained, but the majority of them are labeled on the middlepiece and/or the acrosomal cap. This labeling variability probably reflects the heterogeneity of an epididymal sperm population. In fact, it has been reported that the rat epididymal fluid contains two populations of vesicles differing in their ultrastructure and enzymatic composition [33]. The fact that PLPCd proteins are transferred to defined membrane domains suggests that interactions between PLP and spermatozoa are highly orchestrated and selective. This is well illustrated by the restricted distribution of P26h and P25b on the acrosomal cap of hamster and bull spermatozoa, respectively. Those sperm surface proteins have been shown to be transferred from epididymal PLP to spermatozoa [10, 13, 19].
It has been reported that fusion of spermatozoa with prostasomes isolated from human semen is favored at a slightly acidic pH [24]. Even though our results did not allow us to discriminate between fusion and another mechanism of protein transfer from cauda epididymal PLP to caput spermatozoa, this phenomenon is pH sensitive. Whereas the pH of normal human semen is of 7.2 or higher [34], the bovine epididymal fluid has a pH around 6.5 [35]. Coincubations of PLP with caput spermatozoa were performed at pH between 5.5 and 7.5, more extreme pH being considered as physiologically irrelevant. Interestingly, maximum transfer was reached at pH 6.0–6.5, with a 2.5-fold increase compared with levels reached at pH 7.5 (Fig. 4). At pH 6.5, the physiological pH of epididymal fluid, the transfer appears to be temperature and time dependent (Fig. 5). Temperature sensitivity of protein transfer suggests that the interaction between PLP and the sperm surface is not a simple binding phenomenon and that membrane fluidity of sperm plasma membrane and/or PLP may influence the protein transfer.
The concentration of zinc in the epididymis is surpassed only by the prostate [36] and has also been reported to be present in high concentrations in prostasomes prepared from human seminal fluid [37]. The epididymal luminal zinc concentration is not regulated by androgens, and other lumicrine factors appear to be involved [38, 39]. Even though the physiological significance of this divalent cation concentration in the excurrent ducts remains to be determined, it has been hypothesized to be important in epididymal sperm maturation [40, 41]. In fact, zinc has a great effect on the amount of protein transfer from PLPCd to caput spermatozoa (Fig. 6). This appears to be specific since the beneficial effect of zinc is completely abolished by a divalent cationic chelating reagent (Fig. 7). At concentrations varying in the range of intraluminal epididymal concentrations [35, 42], calcium and magnesium have no effect on sperm protein acquisition involving PLPCd (Fig. 6). Considering that some transfer of PLPCd protein to the epididymal spermatozoa is necessary for male gamete fertilizing ability [10, 19, 26] and that this phenomenon is favored by zinc, this cation probably plays an important role in sperm maturation.
Taken together, these results show that the interaction between PLPCd and spermatozoa is an orchestrated phenomenon with optimal efficiency in physicochemical conditions (pH, zinc concentration, and temperature) present in the epididymal intraluminal compartment. In these conditions, PLPCd transfer selected proteins becoming associated with selected sperm surface domains. These membranous vesicles thus appear to be involved in the acquisition of fertilizing ability of the male gamete during its transit along the reproductive tract.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants from the National Sciences and Engineering Research Council of Canada and Formation Chercheurs et Aide à la Recherche of the province of Quebec to R.S. 
2 Correspondence: Robert Sullivan, Unité d'Ontogénie-Reproduction, Centre de Recherche, Centre Hospitalier de l'Université Laval, 2705 Blvd. Laurier, Ste-Foy, PQ, Canada G1V 4G2. FAX: 418 654 2765; robert.sullivan{at}crchul.ulaval.ca
Accepted: February 6, 2002.
Received: November 8, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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