Molecular Human Reproduction, Vol. 5, No. 9, 809-815,
September 1999
© 1999 European Society of Human Reproduction and Embryology
Regulation of sperm function |
Characterization of human semen 
-L-fucosidases
1 Department of Chemistry, 111 Research Drive, Lehigh University, and 2 Department of Biological Sciences, Lehigh University, Bethlehem, PA 18015, USA
Abstract
Human semen contains a large amount of 
-L-fucosidase activity, the great majority of which is found in the seminal fluid. Immunocytochemical studies indicate that a small amount of semen fucosidase activity is present on the sperm plasma membrane, primarily in the posterior head region. Subcellular fractionation studies also indicate that sperm -L-fucosidase is present in the plasma membrane-enriched fraction. Comparative characterization of human seminal fluid and sperm -L-fucosidases indicates that seminal fluid -L-fucosidase has a broad pH optimum curve with a number of near-equal maxima between pH 4.8 and 7.0 while sperm fucosidase has a major optimum between pH 3.4 and 4.0. Isoelectric focusing indicates that seminal fluid -L-fucosidase contains three to six isoforms with isoelectric points (pI) of 5–7 while sperm fucosidase contains two distinct isoforms with pI values of 5.2 ± 0.2 and 7.0 ± 0.2. Western blotting indicates that seminal fluid fucosidase contains a major protein band with a molecular mass ratio (Mr) of ~56 kDa while sperm fucosidase contains a major protein band of ~51 kDa. The overall results indicate the presence of a low-abundance, plasma membrane-associated human sperm -L-fucosidase, which is different in its properties from human seminal fluid -L-fucosidase(s), and whose function is not yet known.
-L-fucosidase/semen/seminal fluid/spermatozoa
Introduction
Significant evidence indicates that sperm–egg interactions in numerous species including several mammals are mediated by protein receptors on the sperm plasma membrane attaching to carbohydrate-containing molecules on the surface (e.g. vitelline coat, zona pellucida) of oocytes (Benoff, 1997
; Shalgi and Raz, 1997; Tulsiani et al., 1997). Among the proposed sperm receptors are a number of saccharide-binding proteins including fucose-binding protein (Huang et al., 1982), galactose-binding protein (Cheng et al., 1994) and mannose-binding protein (Benoff et al., 1993), as well as a number of glycosyltransferases and glycosidases including ß-1, 4-galactosyltransferase (Miller et al., 1992), fucosyltransferase (Ram et al., 1989), -D-mannosidase (Tulsiani et al., 1989) and ß-glucuronidase (Brandelli et al., 1996).
Evidence has been accumulating for a role of L-fucose and/or -L-fucosidase in sperm–egg interactions. A number of studies have suggested that L-fucose and/or L-fucose-containing molecules inhibit sperm–egg interactions in a number of mammals (Huang et al., 1982) including hamsters (Ahuja, 1982), mice (Boldt et al., 1989), rats (Shalgi et al., 1986) and humans (Lucas et al; 1994; De Cerezo et al., 1996). A recent paper has provided good evidence for the requirement of a fucosyl residue on a zona pellucida ligand for high-affinity sperm binding in the mouse (Johnston et al., 1998). A previous study gave evidence for fucosyl sites present on the vitelline coat of ascidian Ciona intestinalis eggs (DeSantis et al., 1983) and suggested that a sperm surface -L-fucosidase might bind to these sites (Hoshi et al., 1983). In addition, treatment of eggs with -L-fucosidase has been shown to significantly reduce sperm binding (Blithe, 1993). Several studies have documented the presence of plasma membrane-associated -L-fucosidases on spermatozoa from bulls (Jauhiainen and Vanha-Pertulla, 1986), from the mollusc bivalve Unio elongatulus (Focarelli et al., 1997) and from rats (Avilés et al., 1996; Abascal et al., 1998). In addition, a deficiency of -L-fucosidase in English Springer spaniels with fucosidosis results in acrosomal dysgenesis and impaired sperm maturation (Veeramachaneni et al., 1998), suggesting an important role for -L-fucosidase in sperm development.
Previous immunocytochemical studies on the rat demonstrated the presence of a novel -L-fucosidase on the plasma membrane of the convex region of the principle segment of testicular and cauda epididymal spermatozoa (Avilés et al., 1996). Subcellular fractionation studies in sucrose density gradients provided further evidence for a plasma membrane localization of the -L-fucosidase. Isoelectric focusing studies of rat sperm -L-fucosidase during epididymal maturation indicated that the isoform composition of this enzyme changed in the distal half of the cauda at which location a significant enrichment of -L-fucosidase activity was found (Abascal et al., 1998). The change involved the appearance of a second, acidic isoform, with an isoelectric point (pI) of almost 5, in addition to the major neutral isoform (with a pI near 7) which was seen in all spermatozoa. The more acidic isoform appears to be derived by sialylation of the neutral isoform. The first appearance of the acidic isoform near the end of epididymal maturation when spermatozoa first become motile and fertilization-competent suggests a potential role for this fucosidase isoform in sperm–egg interactions (Abascal et al., 1998).
In the present study, the -L-fucosidases of human semen (spermatozoa and seminal fluid) have been investigated. Semen is very rich in -L-fucosidase activity, the great majority of which is found in seminal fluid. A very small amount of activity was found, by immunocytochemical and subcellular fractionation studies, to be associated with the sperm plasma membrane. Comparative characterization of seminal fluid and sperm -L-fucosidases with regard to their kinetic properties, and isoform and protein band compositions, indicated that sperm -L-fucosidase is different from the seminal fluid enzyme. The finding of two isoforms of human sperm -L-fucosidase with pI values near 5 and 7 is very similar to previous findings of two fucosidase isoforms at comparable pI values in rat spermatozoa (Abascal et al., 1998).
Materials and methods
Human semen and spermatozoa
Semen samples were obtained from male donors who were apparently healthy and fertile. The sperm parameters of the semen were within the normal ranges for morphology, motility and numbers based on World Health Organization (WHO) criteria. Specimens were screened for the presence of antisperm antibodies (Tang and Bean, 1998), and all studies were performed on antisperm-negative specimens. After liquefication (~30 min at room temperature), semen was diluted with 3.5 ml of Dulbecco's phosphate-buffered saline (PBS), pH 7.4, per ml of semen and centrifuged at 450 g for 8 min at 20°C. The supernatant (i.e. seminal fluid) was carefully removed and the pelleted spermatozoa were washed three times by resuspension in 3.5 ml PBS and by recentrifugation at 450g for 8 min at 20°C. In some experiments, the spermatozoa were further enriched and separated from contaminating debris by putting them through a 47/90% (2 ml and 1.5 ml respectively) Percoll gradient. In some experiments, the seminal fluid and/or washed spermatozoa were stored for up to 48 h at 2–4°C until used. For some experiments, semen from two to five donors was combined. All procedures which involved semen donations were approved by an Institutional Human Subjects Committee, informed consent was given by each donor and anonymous number designations were used.
Assays for -L-fucosidase, phosphodiesterase and protein
-L-fucosidase activity was assayed as described (Abascal et al., 1998) using 1 mmol/l 4-methylumbelliferyl--L-fucopyranoside (Sigma Chemical Co, St. Louis, MO, USA) as substrate in 0.1 mol/l NaH2PO4/Na2HPO4 buffer, pH 7.0. Fluorescences were read on a Turner Model 450 Fluorometer (using wavelengths of 360 nm and 430 nm for excitation and emission respectively) and corrected by subtracting tissue and substrate blanks. One unit of -L-fucosidase activity is defined as the amount of enzyme necessary to hydrolyze 1 nmole of substrate/min at 37°C under the above-defined conditions. Phosphodiesterase activity was used as a plasma membrane marker (Touster et al., 1970) and assayed as described previously (Tulsiani et al., 1990) using 1 mmol/l p-nitrophenyl-(PNP)-5'-thymidylate (Sigma ) as substrate in 50 mmol/l Tris–HCl buffer, pH 9.0, containing 0.2% (v/v) Triton X-100. Incubations were for 60 min at 37°C and reactions were terminated by addition of 0.9 ml 0.133 mol/l glycine/0.083 mol/l Na2CO3 buffer containing 0.067 mol/l NaCl and adjusted to pH 10.7 with NaOH. Absorbances were read at 400 nm and corrected for tissue and substrate blanks. One unit of phosphodiesterase activity is defined as the amount of enzyme necessary to release l µmol PNP/h at 37°C under the above-defined conditions.
Protein concentration was determined using the method of Bradford which employs treatment with the Coomassie Brilliant Blue reagent for 5 min at 21°C followed by reading absorbances at 595 nm (Bradford, 1976). Human serum albumin (Sigma) was used as the standard for the protein assays.
Stability, pH optimum and isoelectric focusing
The stability of -L-fucosidase activity from seminal fluid and spermatozoa was determined after storage of aliquots at either 2–4°C or –20°C for various lengths of time up to 8 days. The pH activity curves for -L-fucosidase from seminal fluid and spermatozoa were determined as described (Abascal et al., 1998) using three buffers: 0.1 mol/l oxalic acid/sodium oxalate; 0.1 mol/l citric acid/sodium citrate; and 0.1 mol/l NaH2PO4/Na2HPO4. Assays were performed in duplicate for 20 min at 37°C, and actual pH values of a third set of mock tubes were recorded. Fluorescences were corrected for tissue and substrate blanks, and the data were plotted as percentage maximal activity versus pH.
Isoelectric focusing was performed at 2–4°C essentially as described (Abascal et al., 1998) on aliquots of -L-fucosidase from seminal fluid and sperm suspensions containing 3–40 units of -L-fucosidase activity. Focusing was carried out at 600 V (2–2.5 mA) for 17.5–24 h in a 40 ml column with 2% (v/v) Ampholine ampholytes (pH range 5–8; Pharmacia LKB Biotechnology, Bromma, Sweden) and a 0–67% (w/v) sucrose gradient. After focusing, fractions (0.3–0.4 ml) were collected, their pH values determined and 25 µl aliquots of each fraction were assayed for -L-fucosidase activity for 45 min at 37°C. The data were plotted as -L-fucosidase activity versus isoelectric point (pI).
Release of -L-fucosidase from intact spermatozoa
Intact, washed spermatozoa were shaken gently (75 revolutions/min) for 30 min at 20°C as described (Avilés et al., 1996) in PBS alone or containing 0.5 mol/l NaCl and/or 0.5% (v/v) Triton X-100. After shaking, the suspensions were centrifuged (10 000 g for 10 min), the resultant supernatant fluids and pellets were assayed for -L-fucosidase activity, and the relative amounts of recovered fucosidase activity in the two fractions were calculated.
Western analysis
Slab sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) was carried out on -L-fucosidase from seminal fluid, spermatozoa and a sperm plasma-membrane enriched fraction using the method of Laemmli (Laemmli, 1970). Briefly, 4% stacking and 10% separating gels were run at room temperature using 25 mmol/l Tris/0.2 mol/l glycine buffer, pH 8.3, containing 0.1% SDS and 2.5% (v/v) 2-mercaptoethanol for 1.5 h at 75 V and for 5 h at 140 V respectively. Molecular mass standards (BioRad Labs, Hercules, CA, USA) were run and included myosin (200 kDa), ß-galactosidase (116 kDa), phosphorylase b (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), lysozyme (14.4 kDa) and aprotinin (6.5 kDa). The standard markers were stained with 0.1% Coomassie Blue (BioRad Labs) in methanol/acetic acid/water (50:10:40, by vol) and destaining was accomplished in the same solvent system without Coomassie Blue.
Proteins were electrotransferred from the slab gel to polyvinylidene difluoride (PVDF) membrane (Hybond-P, Amersham, Buckinghamshire, UK) at 156 mA for 1 h, and blocked overnight in 5% non-fat dry milk (Carnation, Glendale, CA, USA) in Tris-buffered saline-Tween-20 (TBST) containing 20 mmol/l Tris, 0.9% (w/v) NaCl and 2% (v/v) Tween-20. The membrane was incubated for 1 h in a 200-fold dilution of the immunoglobulin G (IgG) fraction of anti-(human liver -L-fucosidase) polyclonal antibody from a goat (Andrews-Smith and Alhadeff, 1982) and washed three times with TBST. The secondary antibody, biotin-conjugated donkey anti-goat IgG antibodies (Jackson ImmunoResearch, West Grove, PA, USA), was diluted 104-fold, incubated with the membrane for 40 min and washed three times with TBST. The membrane was incubated with a preformed avidin and biotinylated horseradish peroxidase macromolecular complex which was prepared from Vectastain Elite ABC reagent (Vector Laboratories, Burlingame, CA, USA), and development was accomplished using enhanced chemiluminescence (ECL) (following instructions provided by the manufacturer, Amersham) for 1 min, and the membrane was exposed to Kodak X-OMAT LS film.
Immunocytochemical localization studies
The immunocytochemical studies were carried out using a modified Seidman procedure on spermatozoa prepared from liquefied semen as follows: liquefied semen was diluted in 5 vol PBS, pH 7.4, mixed gently and centrifuged (350 g, 8 min, 20°C) (Seidman, 1997). The pellet was resuspended in 5 ml PBS, recentrifuged as above, and the spermatozoa in the resulting pellet were adjusted with PBS to a concentration of 20–40x106/ml. Spermatozoa were smeared on clean glass slides, dried at room temperature for 3 h, and fixed with 4% paraformaldehyde, pH 7.2, for 15 min. The slides were incubated for 15 min in 0.25% hydrogen peroxide in PBS at room temperature and then were washed three times in PBS, pH 7.4. The slides were incubated for 60 min with a 100-fold dilution of the IgG fraction of anti-(human liver -L-fucosidase) polyclonal antibodies (Andrews-Smith and Alhadeff, 1982), and were washed three times in PBS, pH 7.4. Negative controls were carried out in which the slides were incubated in PBS buffer without the anti-fucosidase antibodies or incubated with goat serum instead of the anti-fucosidase antibodies. The slides were incubated for 40 min with a 103-fold dilution of biotin-conjugated donkey anti-goat IgG antibodies (Jackson ImmunoResearch) and washed three times in PBS. The slides were incubated for 30 min with a preformed avidin and biotinylated horseradish peroxidase macromolecular complex (Vector Laboratories), developed for 5–10 min in 3,3'-diaminobenzidine (DAB) reagent (Sigma) and H2O2 following instructions from the supplier, and washed twice in double-distilled H2O. The slides were dehydrated in 70, 95 and 100% ethanol (3 min/solution) and incubated twice (2 min/time) in Hemode detergent solution (Fischer Scientific, Fair Lawn, NJ, USA). One drop of Permount toluene solution (Fisher Chemical) was placed on each slide, a coverslip was added, and the covered slides were photographed using an Olympus Model IMT2-RFC Fluorescence Microscope.
In some experiments, unfixed spermatozoa were gently shaken for 60 min with 200-fold diluted anti-fucosidase antibodies or with buffer alone (negative control). These spermatozoa were washed in PBS as described above, resuspended in PBS containing 500-fold diluted biotin-conjugated donkey anti-goat IgG antibodies, and incubated for 40 min. The spermatozoa were washed two times in PBS, and endogenous peroxidase was blocked with 0.6% H2O2 in methanol for 20 min. The spermatozoa were rewashed in PBS, incubated with biotinylated horseradish peroxidase macromolecular complex and developed in DAB and H2O2 (as described above). The reaction was stopped with distilled H2O, and sperm suspensions were put on slides and studied microscopically (as described above).
Preparation of plasma membrane-enriched fraction
A plasma membrane-enriched fraction was prepared from spermatozoa (Tulsiani et al., 1990), using the procedure for human spermatozoa. All procedures were at 2–4°C unless otherwise specified. Liquefied semen was put in 5 volumes PBS, pH 7.4, centrifuged at 600g for 20 min and the supernatant fluid was recentrifuged at 105 000 g for 30 min. The sperm-containing pellets from the two centrifugations were combined, resuspended in 2 ml PBS, pH 7.4, added carefully on top of 10 ml of 11% dextran solution, and centrifuged at 600 g for 20 min to remove soluble seminal plasma components and unidentified vesicles. The sperm pellet at the bottom of the tube was added to 4 ml PBS, which was subjected to nitrogen cavitation in a Model 4639 Cell Disruption Bomb (Parr Instruments Co, Moline, IL, USA) for 10 min at 600 psi. The disrupted sperm suspension was vortexed for 1 min and centrifuged at 6000 g for 10 min. The pellet was resuspended in 4 ml PBS and subjected to a second round of nitrogen cavitation. The two supernatants (from the 6000 g spins) were combined, centrifuged at 105 000 g for 30 min, and the pellet (crude membrane fraction) was added to 1 ml 0.25 mol/l sucrose in 10 mmol/l PIPES buffer (piperazine-N-N'-bis 2-ethane sulphonic acid, pH 7.0). The crude membrane/0.25 mol/l sucrose suspension was placed carefully on top of a prepared sucrose gradient tube (14x89 mm polyallomer, Beckman, Palo Alto, CA, USA) (containing 3 ml each of 1.6, 1.3 and 1.0 mol/l sucrose in 10 mmol/l PIPES buffer) which was centrifuged at 100 000 g for 90 min. The white band on top of the 1.0 mol/l sucrose fraction (putative plasma membrane fraction) was collected, added to 4 ml PBS and centrifuged at 105 000 g for 30 min. The resulting pellet (plasma membrane-enriched fraction) and the initial sperm pellet were assayed for -L-fucosidase and phosphodiesterase activities and protein concentrations (so that specific activities and enzyme-enrichment factors could be calculated). A second, alternative procedure was employed for purifying human sperm plasma membranes as described in detail (Xu et al.; 1994). In brief, this procedure involved preparation of spermatozoa by centrifugation at 6000 g for 30 min at 4°C using a 47/90% Percoll gradient. The spermatozoa (at the bottom of the 90% Percoll layer) were washed and centrifuged (600 g, 30 min, 4°C) twice, the final sperm pellet was suspended in 5 ml 0.24 mol/l sucrose in 10 mmol/l PIPES, pH 6.8, containing 1 mmol/l phenylmethylsulphonyl fluoride (PMSF; Sigma) and 2 µg/ml aprotinin (Sigma), and subjected to nitrogen cavitation (as described above) at 600 psi for 10 min. The cell debris was centrifuged at 6000 g for 10 min at 4°C and the supernatant fluid (containing sperm membrane vesicles) was removed and centrifuged at 20 000 g for 30 min at 4°C. The supernatant fluid from this spin was further centrifuged at 100 000 g for 60 min at 4°C to yield the final membrane-enriched pellet which was assayed (as described above) for fucosidase and phosphodiesterase activities and protein concentration.
Results
Initial stability studies were carried out on preparations of -L-fucosidase from human spermatozoa and seminal fluid after storage of aliquots at either 2–4°C or –20°C for various lengths of time. Both preparations maintained complete fucosidase activity for at least 8 days at 2–4°C and –20°C. Therefore, proteinase inhibitors were not needed to preserve fucosidase activity. Most studies on the fucosidase preparations were performed immediately, and no study was carried out after storage of -L-fucosidase for >2 days.
A large amount of -L-fucosidase activity was present in human semen. Table I summarizes the fucosidase units/ml semen, the total units in the semen, and the percentage of the semen fucosidase activity found in the supernatant fluid (i.e. seminal fluid) after centrifugation of semen at 450 g for 8 min for seven semen donors. It can be seen for the individuals (1, 2) who donated more than once that the fucosidase units/ml semen varied up to about two-fold, and the total semen fucosidase activity varied up to about seven-fold for a given donor on different days. The reasons for this variability are unknown but may include the time period of abstinence prior to ejaculation and hormonal fluctuations which could effect the secretion of -L-fucosidase from various glands (e.g. prostate, seminal vesicle, Cowper's) into the semen. For the different donors there was an ~10-fold range in the fucosidase units/ml semen, and an ~20-fold range in total semen fucosidase units. The great majority (97–99.9%) of semen -L-fucosidase activity for all donors was found associated with the seminal fluid while only a small, but reproducible, amount of fucosidase activity (0.1–3.0%) was found associated with the thoroughly washed spermatozoa. Similar results were found for spermatozoa prepared by Percoll gradients. This sperm-associated fucosidase activity was not released after gentle shaking for 30 min in PBS containing 0.5 mol/l NaCl or 0.5% (v/v) Triton X-100 or in PBS containing both of these additives.
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pH activity curves for
-L-fucosidase from human seminal fluid and spermatozoa prepared from the same semen sample were run for five semen specimens, and representative curves for one specimen are depicted in Figure 1. The seminal fluid fucosidase curve (panel A) has significant activity (>50% maximal) over a broad pH range between 4 to 7.8, with three apparent optima at pH values near 4.8, 6.0 and 7.0. The sperm fucosidase curve (panel B) is quite different from that of seminal fluid fucosidase, with a very acidic optimum near pH 3.4 and an apparent second minor optimum centred around pH 7.0. All five sperm -L-fucosidase samples had acidic pH optima of 3.4–4.0, and minor neutral optima of 6.3–7.3.
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Isoelectric focusing profiles for
-L-fucosidase from human seminal fluid and spermatozoa prepared from the same semen sample were run for eight semen specimens, and representative isoform profiles are depicted in Figure 2. Seminal fluid -L-fucosidase (panel A) contained several possible isoform peaks with pI values from 5.0–7.0. All eight of the seminal fluid samples which were studied contained multiple (3 to 6) fucosidase isoforms with pI values between 5.0 and 7.3. The sperm -L-fucosidase profile (panel B) is different from the seminal fluid profiles and contains two distinct isoforms with pI values near 5.4 and 6.7, with the more acidic isoform present in larger amounts. Six of the eight sperm fucosidase isoform profiles were very similar to the one depicted in Figure 2B (with a major isoform of pI 5.0–5.4 and a minor isoform of pI 6.6–7.3) while in the other two profiles the more neutral isoform was the predominant species.
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The results of the immunocytochemical localization studies are shown in Figure 3
. Strong staining of the spermatozoa for -L-fucosidase can be seen, primarily in the posterior head region (panel A). Weaker staining (which is difficult to see in the Figure) was also observed in the anterior head region of the sperm plasma membrane. This staining is absent in the negative control (Figure 3B) in which the slides were treated identically except that the primary antibodies against -L-fucosidase were omitted from the PBS buffer. In addition, another negative control in which goat serum was substituted for the anti-fucosidase antibodies showed no staining of the spermatozoa (data not shown). Results comparable to those shown in Figure 3 for paraformaldehyde-fixed spermatozoa were obtained when unfixed spermatozoa were used for the localization studies, indicating that fixation did not alter sperm antigencity.
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Several subcellular fractionation studies of human spermatozoa after N2 cavitation were carried out in sucrose gradients. An established technique for human spermatozoa (Tulsiani et al., 1990
) was used and yielded a plasma membrane-enriched fraction which contained -L-fucosidase activity and phosphodiesterase activity (which was used as a marker for plasma membranes) (Touster et al., 1970; Tulsiani et al., 1990). The specific activity (units activity/mg protein) of phosphodiesterase was increased 8–11-fold (compared with the sperm pellet) while the largest increase of fucosidase specific activity was three-fold. The average recoveries of -L-fucosidase and phosphodiesterase activities in the plasma membrane-enriched fraction (compared with sperm pellet values) were very low (0.3 and 1% respectively). An average 61% of the -L-fucosidase activity was recovered in the initial sperm membrane fraction (after cavitation and centrifugation) but only an average 9% of the fucosidase activity was recovered in the crude membrane fraction (after centrifugation at 105 000 g). Analysis of plasma membrane-enriched fractions by Western blotting for six different experiments gave consistent results and indicated the presence of small amounts of fucosidase antigen. The relevant region of one such ECL image, after scanning and enhancement of the signal-to-noise ratio, is shown in Figure 4. Two immunoreactive bands near 56 and 51 kDa, as determined by molecular mass ratio (Mr) markers, can be seen for the plasma membrane-enriched fraction (lane 5) which run very close to the corresponding bands for authentic human liver -L-fucosidase (lanes 1, 6, 7). Similar results were found for all experiments, but the 56 kDa protein band was found in increased relative amounts in four of six experiments. Seminal fluid -L-fucosidase (from the same semen specimen) gave a single immunoreactive band near 56 kDa (lanes 2, 4) (which was consistent for all six experiments) while the sperm pellet (from which the plasma membranes were prepared) contained two bands (lane 3) near 56 and 51 kDa (and comparable to the two bands in the plasma membrane-enriched fraction) (lane 5). In three of six experiments, only the 51 kDa protein band was detected in the sperm pellet. The buffer blank negative control had no immunoreactive bands (lane 8). A second subcellular fractionation procedure for the preparation of human sperm plasma membranes (Xu et al., 1994) was also employed since the first method gave such low recoveries of enzymatic activities. The results for fucosidase and phosphodiesterase were similar to those found when the procedure of Tulsiani et al. (1990) was employed. The specific activities of fucosidase and phosphodiesterase were increased 2.1-fold and 10.7-fold respectively, and the recovery of activity for both enzymes was 0.6%.
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Discussion
As described in the Introduction, previous studies have provided evidence for a plasma membrane-associated sperm -L-fucosidase in several organisms including the mollusc bivalve Unio elongatulus, the ascidian Ciona intestinalis, bull and rat. In addition, indirect evidence has been presented which is consistent with the presence of a plasma membrane-associated sperm fucosidase in mice. In the present study, we have extended our study on rats (Avilés et al., 1996; Abascal et al., 1998) to humans and characterized the large amount of -L-fucosidase activity found in human semen. After centrifugation of liquefied semen the great majority of -L-fucosidase activity is found in the seminal fluid, with only a small percentage of the recovered activity found associated with thoroughly washed spermatozoa. These results are similar to previous studies (Tulsiani et al., 1990) which found only 9% of human semen -L-fucosidase associated with spermatozoa. Spermatozoa prepared using Percoll gradients gave similar results in our studies and those of Tulsiani et al. (1990), suggesting that the small amount of fucosidase activity associated with spermatozoa is real and not artefactual. Immunocytochemical and subcellular fractionation studies provided evidence that the small amount of sperm fucosidase activity was associated with the plasma membrane. Using the IgG fraction of antibodies against human liver -L-fucosidase, immunoreactive material was found on the plasma membranes of human spermatozoa, primarily in the posterior head region. Our results are consistent with other studies (DeCerezo et al., 1996) which found that fucosylated glycoconjugates bind to human sperm plasma membranes with high density in the post-acrosomal sheath. However, the precise localization of fucosidase to the posterior head region on human sperm plasma membranes is different from that found previously for rat sperm plasma membranes in which fucosidase was enriched on the convex region of the principle segment (Avilés et al., 1996). An enrichment of fucosidae in the posterior head region on human sperm plasma membranes suggests that any proposed role for fucosidase in binding to the egg zona pellucida is more likely to be through secondary binding rather than through attachment/adhesion or primary tight binding. Subcellular fractionation studies of cavitated spermatozoa in sucrose gradients indicated that small amounts of fucosidase activity were present in the plasma membrane-enriched fraction, as previously shown by Tulsiani et al. (1990) in their studies on a human sperm plasma membrane-associated -D-mannosidase. The plasma membrane-enriched fraction contained a significant enrichment of the plasma membrane marker enzyme phosphodiesterase suggesting that the fraction contained an enrichment of sperm plasma membranes. The reason for the low recovery (in this study) of fucosidase activity in this fraction is not clear, but was comparable to the low recovery found by Tulsiani et al. (1990). It may be that the fucosidase is loosely associated with the sperm plasma membrane and much of it dissociates during the procedures and conditions of subcellular fractionation. The complementary technique of Western blotting indicated that small amounts of fucosidase antigenic material were present in the plasma membrane-enriched fraction.
Comparative characterization of the properties of seminal fluid and sperm -L-fucosidases provided evidence for a sperm fucosidase which was different from that of seminal fluid -L-fucosidase. Kinetic analysis indicated that seminal fluid -L-fucosidase had an unusual pH activity curve with a number of near-equal maxima between 4.8 and 7.0. These results suggest that more than one fucosidase may be present in seminal fluid, possibly originating from the major glands (e.g. prostate, seminal vesicle, Cowper's) which secrete material into semen (Eliasson and Johnsen, 1986). The sperm fucosidase pH activity curves were different from those of seminal fluid fucosidase and always had an acidic pH optimum between 3.4 to 4.0 with a second minor neutral pH optimum between 6.3 to 7.3. These findings are similar to those previously found for rat sperm -L-fucosidase except that in rat spermatozoa the major pH optimum was neutral (between 6.9 and 7.1) with a minor second acidic optimum (between pH 3 and 4) (Abascal et al., 1998). Isoelectric focusing of seminal fluid -L-fucosidase always revealed several (three to six) isoform peaks with pI values of 5–7, suggesting (like the pH optima data) the presence of more than one fucosidase in this fluid. On the other hand, the sperm fucosidase contained two distinct isoforms with pI values of 5.2 ± 0.2 and 7.0 ± 0.2, with the more acidic isoform usually being the predominant species. For rat testicular and epididymal spermatozoa, we previously found (Abascal et al., 1998) a single fucosidase isoform with a pI near 7 until late in epididymal maturation (distal half of cauda) at which time a second, more-acidic isoform with a pI of 5–6 was first observed. It seems more than coincidental and may be significant that rat and human sperm fucosidases have two isoforms with similar pI values. The larger relative amount of the more acidic isoform in human spermatozoa may be an evolutionary adaptation of an isoform with an important function, for example an involvement in sperm–egg interactions. However, it must be pointed out that ejaculated human sperm fucosidase is being compared to non-ejaculated (testicular and epididymal) rat sperm fucosidase, and the differences found between human and rat sperm fucosidases may be due to this fact. Further studies on ejaculated rat spermatozoa and/or testicular and epididymal human spermatozoa are needed to clarify the significance of the differences found for rat and human spermatozoa. Western blotting of seminal fluid -L-fucosidase indicated the presence of a single protein band with an Mr near 56 kDa while sperm -L-fucosidase contained a major protein band near 51 kDa and a minor protein band near 56 kDa. Rat sperm -L-fucosidase contained two closely-spaced protein bands near 53 ± 3 kDa (Abascal et al., 1998). Our results for human sperm -L-fucosidase are similar to those of other workers (Focarelli et al., 1997), who found different polypeptide compositions for seminal fluid (56 kDa) and sperm plasma membrane (68 kDa) -L-fucosidases from Unio elongatulus.
The overall results indicate the presence of a low-abundance human sperm -L-fucosidase which is loosely associated with the plasma membrane and which is different in its properties from human seminal fluid -L-fucosidase(s). In addition, the similarities which exist between human and rat sperm plasma membrane-associated fucosidases lead us to postulate that this enzyme, particularly the more acidic isoform, may be involved in sperm–egg interactions. The presence of -L-fucosidase, an enzyme which is normally found as a soluble component of the lysosome (reviewed in Alhadeff, 1998), on the plasma membrane of spermatozoa from at least five species argues for an important biological function for this novel fucosidase.
Acknowledgments
This research was supported in part by a grant for Undergraduate Education in Biological Sciences from the Howard Hughes Medical Institute.
Notes
3 To whom correspondence should be addressed 
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Submitted on February 9, 1999; accepted on June 9, 1999.
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