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BOR - Papers in Press, published online ahead of print September 29, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.029330
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BIOLOGY OF REPRODUCTION 72, 346–357 (2005)
DOI: 10.1095/biolreprod.104.029330
© 2005 by the Society for the Study of Reproduction, Inc.

Dynamics of Carbohydrate Affinities at the Cell Surface of Capacitating Bovine Sperm Cells

Edita Sostaric2,3, Chris H.A. van de Lest3, Ben Colenbrander2, and Bart M. Gadella1,2,3

Department of Farm Animal Health2 Departments of Biochemistry and Cell Biology,3 Faculty of Veterinary Medicine, Utrecht University, 3584 CL Utrecht, The Netherlands


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In vivo capacitation of eutherian sperm cells coincides with changes in carbohydrate-dependent interaction with the oviduct epithelia (fucose-dependent for bovine). Heparin-like glycosaminoglycans (GAG) secreted by the oviduct compete for sperm-oviduct binding and are believed to release capacitated sperm cells from oviduct epithelia. A biochemical assay to quantify the specificity and dynamics of carbohydrate-mediated bovine sperm-oviduct binding is developed. Sperm apical plasma membranes (SPM) were purified by a factor eight and biotinylated carbohydrate probes were used for quantitative evaluation of carbohydrate binding. SPM of fresh sperm showed >12 times higher binding capacity for biotinylated fucose than for LewisA. SPM from fresh sperm also efficiently bound biotinylated fucoidan and mannan. Binding of biotinylated fucose could be inhibited by various mono- and oligosaccharides such as fucoidan, mannan, heparin, maltose, and, to a lesser extent, glucose (50% binding at 0.2 mM, 2 mM, 0.3 µg/ml, 15 mM, 50 mM, respectively). SPM from sperm cells that were in vitro capacitated for 4 h in bicarbonate-enriched media (either with or without 10µg/ml heparin) showed a 70–85% reduction in fucose binding. This was also achieved by follicular fluid or by GAG, both obtained from dominant follicles. Total follicular fluid was much more potent in competing with fucose for sperm binding than the isolated GAG moieties (50% competition at 0.02 µg/ml, 20 µg/ml based on number of GAG moieties, respectively). These results support the hypothesis that in vivo capacitation of sperm cells is regulated by carbohydrate moieties similar to those regulating sperm-oviduct adhesion.

calcium, carbohydrate-affinity, female reproductive tract, follicular fluid, gamete biology, sperm capacitation, sperm motility and transport, sperm-oviduct adhesion


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Sperm cells are stored in the oviduct of mammalian species during estrus. Bovine sperm cells actively attach to ciliated epithelial cells with the apical part of the sperm head surface [1]. Sperm cells remain attached until the time of ovulation, when, under not-yet-characterized conditions, they will be released and are capable of fertilizing the oocyte (capacitated) [2]. Indeed, only uncapacitated and acrosome-intact sperm cells appear to bind to the oviductal epithelium in vitro [3, 4] and in vivo [5]. Bovine, equine, and porcine sperm cells released from the oviductal epithelium have a capacitation-specific chlortetracycline (CTC) staining pattern [3, 6, 7]. Apical head plasma membranes of rabbit spermatozoa can bind to isolated apical plasma membranes of oviductal epithelial cells. Incubation of rabbit, bovine, or equine sperm with apical plasma membranes of oviductal epithelia prolonged the sperm longevity [8, 9], probably by keeping the cytoplasmic free Ca2+ levels in these sperm cells low [10].

Initial steps have been made to characterize the sperm cell binding to the epithelia of the oviduct. This interaction seems to be mediated by carbohydrate ligands in a species-specific manner: The sperm cells contain lectin-like molecules on their cellular surface with affinities for certain carbohydrate moieties on the apical surface of oviduct epithelial cells. Indirectly, these interactions can be studied by the use of competitive carbohydrate-binding inhibition assays. For instance, fetuin and sialic acid [11] specifically block binding of the hamster sperm to the oviduct epithelium, whereas, in equine fetuin, asialofetuin, and galactose have been reported to inhibit binding of the sperm to the oviductal epithelium [12]. In bovine sperm, binding to the oviductal epithelium can be inhibited by fucose and is even more pronounced by fucoidan (a multimeric fucose moiety). Pretreatment of the bovine oviduct epithelium with fucosidase also reduces sperm binding [13]. From different fucose-containing oligosaccharides, only trisaccharide LewisA ({alpha}-l-Fuc[1, 4]-ß-D-Gal[1, 3]-d-GlcNAc) significantly reduced binding. This indicates that not only the presence of fucose in a carbohydrate moiety per se but also its position within the carbohydrate moiety determines functional binding [14].

Although initial attempts have been made to characterize molecules involved in sperm-oviduct binding, still very little is known about the mechanisms involved in sperm release after ovulation. Hormonal changes at the time of ovulation are likely to affect sperm release. Preliminary data suggested that these changes induce secretory activity in the oviduct, most predominantly in the ipsilateral oviduct to the ovulatory follicle [15]. Furthermore, follicular fluid will enter the oviduct at ovulation [16, 17]. Both the oviduct secretory products and components in the follicular fluid induce sperm capacitation [18, 19], support sperm motility [20], induce acrosome reaction [21], and enhance affinity for the zona pellucida [22]. In addition, proteins from oviductal fluid can bind to sperm [23, 24], thus affecting sperm motility, viability [25], and fertilizing ability [26]. Follicular and oviductal fluids are rich sources of glycosaminoglycans (GAG) that may affect sperm capacitation in bovine [19]. In fact, follicular fluid is included in in vitro fertilization (IVF) media to increase the capacitation of sperm cells and the developmental capacity of the oocytes to be fertilized [27, 28]. Despite the importance of follicular fluid for IVF and for in vitro sperm capacitation, the effects on carbohydrate-binding affinities of the sperm cell (and thus its affinity for the oviduct epithelial cells) have still to be elucidated.

It should be noted that sperm capacitation normally occurs in the female genital tract [29], but for IVF, sperm can be capacitated in chemically defined media [30]. The addition of heparin is a species-specific adaptation for bovine sperm capacitation/IVF media [31, 32]. Sulfated glycoconjugates have been suggested to inhibit sperm binding and quickly induce sperm release from the oviductal monolayers and so may act as signals that induce sperm release and migration from the oviduct sperm reservoir [33].

As described above, initial results indicate that uncapacitated sperm cells bind to oviduct epithelial cells in a carbohydrate-dependent manner and are released as capacitated sperm cells after a certain period of attachment. However, the specificity, competition, and dynamics in carbohydrate-mediated interactions between sperm cells and epithelial cells of the oviduct remain, to a large extent, terra incognita. Due to technical limitations, it is difficult to follow this process in situ. In this study, we used biotinylated carbohydrate probes to detect carbohydrate affinity of sperm cells before and after capacitation in vitro. To this end, the specific oviduct-binding area of the sperm surface (the apical sperm head plasma membrane) with the carbohydrate binding ligands was isolated. With this approach, we were able to quantify affinity and binding capacity of bovine sperm cells for various carbohydrate probes. We were also able to study carbohydrate-binding competition of unlabeled sugar moieties (including heparin and GAG from follicular fluids) using these materials. The monitored effects of in vitro capacitation of bovine sperm on carbohydrate-binding characteristics may well relate to those occurring during in vivo capacitation of sperm cells. It is likely that the altered carbohydrate-binding properties of capacitating bovine sperm cells at least partly represent alterations of sperm surface molecules involved in sperm-oviduct interaction.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Media

Polyacrylamide-biotin (PAA-biotin) as well as {alpha}-L-fucose-polyacrylamide-biotin (fucose-PAA-biotin) and LewisA-polyacrylamide-biotin (LeA-PAA-biotin) were obtained from Glycotech Corporation (Moscow, Russia). Both biotinylated probes consist of 20 mol% carbohydrate, 5 mol% biotin, and 75 mol% acrylamide, which equals the conjugation of four carbohydrate moieties and one biotin moiety to one polyacrylamide molecule. Neutravidin horseradish peroxidase conjugated (neutravidin-HRP), biotin-hydrazide (EZ-Link Biotin-lethal concentration-hydrazide) and N-hydroxysulfosuccinimide (NHS)-biotin were from Pierce (Rockford, IL). Percoll and Sephadex (G-50 medium grade) were from Pharmacia (Uppsala, Sweden). Protease inhibitors (Comlate Mini, EDTA-free) were from Roche (Mannheim, Germany). 1,9-Dimethyllene blue (DMMB) (80% pure) was from Aldrich Chem. Co. (Bornem, Belgium). Polystyrene 96-well microtiter plates (maxisorb) were from NUNC (Roskilde, Denmark). Hematoxylin was from J.T. Baker (Chemicals B.V., Deventer, The Netherlands), DePeX mounting medium from BDH Laboratory Supplies (Poole, England). The tetramethylbenzidine was from Merck (Darmstadt, Germany). Chlortetracycline (CTC), Toluidine Blue, N-{alpha}-benzoyl-L-arginine-p-nitroanilide (4.0 mM), cartilage chondroitin 4-sulphate, heparin-albumin-biotin (heparin-BSA-biotin), inositol, D(+)-mannose, maltose, mannan (from Saccharomyces ceravisiae), D(+)-glucose, fucose, fucoidan, 1-O-methyl-{alpha}-D-glycopyranoside and bovine serum albumin (BSA) as well as all other chemicals were from Sigma (St. Louis, MO) if not indicated otherwise. The investigations reported in this manuscript were conducted after approval of the Ethical Committee for Animal Experiments of Utrecht University and were in accordance to the International Guiding Principles for Biomedical Research Involving Animals as promulgated by the Society for the Study of Reproduction.

Semen Collection and Preparation

Semen was obtained from three fertile bulls housed at the Faculty of Veterinary Medicine, Utrecht University, The Netherlands. Freshly collected semen was diluted 10 times in saline medium (containing 137 mM NaCl, 2.5 mM KCl, 10 mM glucose, 20 mM Hepes) of 38°C [34] and transported to the laboratory, avoiding cold-shock damage of the sperm. All buffers were iso-osmotic (285–300 mOsm) with pH 7.4 unless indicated otherwise.

Semen was washed over a discontinuous Percoll density gradient. For this purpose, sperm cells were first layered on top of 35% and 70% isotonic Percoll layers diluted with saline medium as described by Harrison et al. [34] and subsequently centrifuged at 350 x g for 10 min and 20 min at 700 x g at room temperature to remove saline medium and seminal plasma. Traces of Percoll were removed by washing the pellet four times with Hepes buffered saline (HBS; containing 5 mM Hepes, 2.7 mM KCl, 146 mM NaCl, 5 mM CaCl2) (700 x g, 10 min). The final pellet was diluted in HBS and sperm motility was estimated (approximately 85% of the cells were progressively motile). To assess sperm cell concentration, sperm was diluted in fixative (0.5% formaldehyde in saline) and counted in a Bürker-Turk cell chamber. Sperm motility and concentration were estimated under a phase contrast microscope, BH2 Olympus (Tokyo, Japan).

Sperm Capacitation

Fresh sperm cells were collected and prepared as described above. Sperm was incubated [32] with one of four media: i) Tyrode medium displaying minimal supportive effects on sperm capacitation (T–BIC; 115 mM NaCl, 3.1 mM KCl, 0.3 mM NaH2PO4, 20 mM Hepes, 2 mM CaCl2, 0.4 mM MgSO4, 21.6 mM sodium lactate, 1 mM sodium pyruvate, 6 mg/ ml BSA, 5 mM glucose, and 1 µg/ml gentamycin; pH 7.4, 290 mOsm/ kg). ii) Tyrode medium displaying maximal effects on sperm capacitation (T+BIC; 99 mM NaCl, 25 mM NaHCO3, 3.1 mM KCl, 0.3 mM NaH2PO4, 20 mM Hepes, 2 mM CaCl2, 0.4 mM MgSO4, 21.6 mM sodium lactate, 1 mM sodium pyruvate, 6 mg/ml BSA, 5 mM glucose, and 1 µg/ ml gentamycin; pH 7.4, 290 mOsm/kg). iii) T+BIC supplemented with 10 µg/ml heparin (T+BIC+Hep). iv) T+BIC+Hep with 10 mM glucose as final concentration; the higher amount of glucose is thought to inhibit capacitation (T+BIC+Hep+Glu). Sperm was incubated in capacitating media (all T+BIC media) for 4 h in a humidified atmosphere at 38°C and 5% CO2, and sperm diluted in T–BIC medium was incubated in a humidified atmosphere at 38°C in air (containing 0.05% CO2) to prevent bicarbonate buffering of CO2. Sperm concentration in the media was 20 x 106 sperm/ml. Following incubation, 10-µl aliquots from the incubated sperm samples were used to assess the capacitation status of the sperm cells by CTC staining [35]. The remaining part of the incubated samples was concentrated by centrifugation at 800 x g for 10 min at room temperature. The obtained pellets were resuspended in 10 ml HBS (room temperature) and recentrifuged at 800 x g for 10 min, and this washing procedure was repeated three times. The resulting pellets were resuspended in HBS and plasma membranes were isolated (see below).

Sperm Plasma Membrane Isolation

Bull sperm plasma membranes (SPM) were isolated according to a method described for boar sperm [36], with slight modifications. Briefly, sperm cells (approximately 5 x 108 cells/ml) were subjected to a nitrogen pressure of 45 Bar in a cell-disruption bomb (Parr Instrument Company, Moline, IL) on ice. After 10 min, sperm cells were slowly extruded into a 50-ml polyethylene tube into which protease inhibitors were added. All further isolation steps were performed on ice or at 4°C. This procedure resulted in the exclusive removal of the apical sperm head membrane from sperm cells (cf., porcine sperm cells [36]; data not shown). The cavitate was centrifuged for 10 min at 1000 x g and the pellet was washed with 5 ml HBS and centrifuged for 10 min at 1000 x g. Supernatants were combined and centrifuged for 10 min at 6000 x g. The 6000 x g supernatant was further centrifuged for 70 min at 285 000 x g. The 285 000 x g pellet was washed in cold HBS and centrifuged for 40 min at 285 000 x g. The final pellet, containing SPM, was diluted in cold HBS with protease inhibitors. SPM was frozen in liquid nitrogen and stored at –20°C till use.

Biochemical Analyses

The degree of purification of the isolated SPM relative to the total sperm sample was estimated by quantifying their specific SPM and intracellular marker enzyme activities. i) Protein contents were determined according to Lowry [37], with minor modifications using bovine serum albumin as the standard. Samples were boiled for 10 min in 30 mM SDS, 160 mM Na2CO3, and 12 mM KNa-tartrate in 80 mM NaOH and then incubated for 10 min in 1.5 mM CuSO4. Folin-Ciocalteu phenol reagent (final concentration 0.1 M) was added for 30 min and color formation was measured in a Beckman DU-62 spectrophotometer (Beckman Instruments Nederland B.V., Mijdrecht, The Netherlands) at 750 nm. ii) Alkaline (EC 3.1.3.1) and acid (EC 3.1.3.2) phosphatases were assayed according to Soucek and Vary [38]. iii) 5'-Nucleotidase (EC 3.1.3.5) activity was determined by incubation of samples in a buffer containing 0.1 M glycine, 10 mM MgCl2, and 5 mM 5'-AMP (pH 8.5) for 30 min at 37°C. The reaction was stopped by addition of phosphate reagent [39], and phosphate was measured. iv) Acrosin (EC 3.4.21.10) activity was determined by its esterolytic activity on N-{alpha}-benzoyl-L-arginine-p-nitroanilide. The formation of the product p-nitroaniline was measured continuously at 410 nm ({epsilon}410 = 9.9/mM) [40] during the incubation in a buffer containing 0.1 M Tris and 67 mM NaCl (pH 8.0) at 25°C.

Biotinylation of Mannan and Fucoidan

Biotinylated mannan was prepared by biotinylation of a mannan-BSA conjugate, which was previously prepared [41]. One hundred millimoles NHS-biotin (in DMSO) was diluted to a final concentration of 5 mM in the mannan-BSA solution (10 mg/ml in 0.1 M H3BO3, pH 9), and this mixture was incubated at room temperature for 2 h.

Fucoidan was biotinylated with biotin-hydrazide after partial oxidation of the carbohydrate moieties according to the manufacturer's instructions. Briefly, 10 mg fucoidan was dissolved in 200 µl demineralized water and partially oxidized by the addition of 50 µl of 100 mM NaIO4 (containing 100 mM CH3COONa, pH 5.5). After 15 min, the excess periodate was removed with a Sephadex G50 desalting column that was equilibrated with 100 mM sodium acetate buffer (pH 5.5). To the eluted fucoidan, biotin-hydrazide was coupled (final concentration 5 mM) by agitation for 2 h at room temperature. Nonconjugated biotin was removed from both fucoidan-biotin and mannan-BSA-biotin by means of a Sephadex G 50 desalting column.

Follicular Fluid Collection

Bovine ovaries were obtained in a local slaughterhouse (Gosschalk and Zn BV, Epe, The Netherlands) and transported to the laboratory in PBS at 22°C within 2 h postmortem. Fluids from dominant follicles (>15 mm) were aspirated into a sterile 15-ml plastic tube. Follicular fluid of 10 dominant follicles was pooled and centrifuged at 1300 x g for 15 min, and the supernatant was subsequently stored at –20°C until use.

Isolation of Glycosaminoglycans from Follicular Fluid

GAGs were extracted from follicular fluid by alkaline borohydride [42]. To 1 ml of follicular fluid, 3 ml borohydride buffer (containing 15 mM NaBH4 dissolved in 1 M NaOH) was added. After 1 h at 73°C, the mixture was neutralized by addition of 6 M HCl. Proteins were precipitated by addition of trichloroacetic acid to a final concentration of 6% (w/ v). The mixture was incubated for 2 h at –20°C, and proteins were pelleted by centrifugation at 2000 x g for 15 min at 4°C. The supernatant was diluted in 100% ethanol in a ratio 1:5 to precipitate GAG and incubated for 18 h at –20°C. GAGs were pelleted by centrifugation at 25 000 x g for 30 min at 4°C. The resulting pellet was dried and GAG were redissolved in demineralized water and stored at 4°C over night.

Quantification of Total Sulfated Glycosaminoglycans

Concentrations of total sulfated GAG in follicular fluid and GAG isolated from follicular fluid were determined using a DMMB assay [43]. To 10 µl of sample, 200 µl of DMMB reagent (46 µM DMMB, 40 mM glycine, and 42 mM NaCl, pH 3.0) was added and absorbance was measured at 525 nm. Chondroitin 4-sulfate was used as a standard.

Enzyme-Linked Carbohydrate-Binding Assay

Binding properties of bovine SPM to carbohydrates were analyzed by an enzyme-linked carbohydrate-binding assay (ELCBA). Polystyrene 96-well microtiter plates were coated with SPM samples (protein range from 1 ng to 8 µg per well) diluted in coating buffer (containing 0.1 M Na2CO3, pH 9.6) and incubated overnight at 4°C. All further procedures were carried out at room temperature. The coating buffer was discarded, and the wells were blocked for 10 min with 0.3% (v/v) Tween-20 in HBS, followed by 0.05% Tween-20, 1% BSA in HBS for 1 h. After removing the blocking solution, 50 µl of biotin-conjugated carbohydrates (fucose-PAA-biotin, LeA-PAA-biotin, mannan-BSA-biotin, or fucoidan-biotin) were allowed to bind to the coated plates for 2 h in incubation buffer (containing 0.05% [v/v] of Tween-20 in HBS) as added in serial dilution starting at 20 ng/well. After binding, the fluids were removed and the wells were washed five times in washing buffer (containing 10 mM Tris, 150 mM NaCl, 5 mM CaCl2, and 0.05% [v/v] Tween-20, pH 7.5). The amount of immobilized biotin conjugates was quantified using neutravidin-HRP. A final concentration, 0.2 µg/ml, of neutravidin-HRP was prepared in washing buffer and 50 µl/well was added. After 1 h of incubation, free neutravidin-HRP was removed and the plates were washed five times in washing buffer. Immobilized enzyme activity was quantified by adding 150 µl/ well tetramethylbenzidine reagent (containing 100 µg/ml tetramethylbenzidine, 1 mM H2O2 in 0.1 M citric acid buffer, pH 4.0). The reaction was stopped by adding 50 µl of 2 M H2SO4 per well. Absorbance was measured at 450 nm, using a microtiter plate reader (Benchmark, Bio-Rad Laboratories B.V., Veenendaal, The Netherlands). For each set of the experiments, ELCBA was assayed using three separate SPM isolates.

Divalent Cation Requirement for Fucose-PAA-Biotin Interactions with SPM

Microtiter plate wells were coated with SPM (0.5 µg protein/well) and blocked as described above. Each well was incubated for 2 h with 50 µl/ well of fucose-PAA-biotin (final concentration 0.25 µg/ml) in incubation buffer supplemented with different concentrations of divalent cations (serial concentrations in the range of 0–20 mM of either CaCl2, MnCl2, MgCl2, ZnCl2, or 10 mM EDTA). Binding of fucose-PAA-biotin was detected as described above.

Characterization of the SPM Binding to the Carbohydrate Probes by Saccharide Competition Assay

Competition of SPM binding to the various prepared biotinylated carbohydrate conjugates was tested in polystyrene 96-well microtiter plates. The plates were coated with 0.5 µg SPM protein/well diluted in coating buffer and plates were blocked as described for ELCBA. Various concentrations from 0 to 100 mM of inhibitory carbohydrate solutions in 50 µl of incubation buffer were added per well for 1 h. Subsequently, the inhibition on immobilization of biotinylated carbohydrates was quantified as described above.

Staining of the Uncapacitated and Capacitated Sperm with Biotinylated Carbohydrate Probes

Sperm samples were washed twice in HBS containing BSA (HBS+B; 1% [w/v] BSA in HBS; centrifugation for 5 min at 300 x g) and 90-µl aliquots (containing 20 x 106 sperm cells/ml) were incubated for 15 min at room temperature with the biotinylated carbohydrate-conjugate of choice by adding 10 µl containing 1 mg/ml conjugate. Subsequently, 100 µl of 5 ng/ml of streptavidin-fluorescein isothiocyanate (FITC) was added for 15 min. Washed sperm cells were also incubated in the absence of biotinylated carbohydrate conjugates with 100 µl of streptavidin-FITC (negative control). The specimens were counterstained for cell viability using propidium iodide and only propidium iodide-negative cells (living cells) were imaged (single scans were made to freeze motile cells). Staining of the sperm was visualized using an inverted spectral emission confocal laser-scanning microscope (CLSM) (Leica TSC-SP GmbH, Heidelberg, Germany) or alternatively analyzed by flow cytometry. Flow cytometric analysis was performed on a FACScan flow cytometer equipped with a 100-mW argon laser excitation at 488 nm (Becton Dickinson, San José, CA). Five minutes before analysis, sperm cells were counterstained with propidium iodide for simultaneous detection of carbohydrate binding and viability status of individual sperm cells [31]. Cell deterioration was minimal and deteriorated cells show similar FITC fluorescence as positive stained cells (data not shown). After gating out nonsperm and aggregated events (on FSC and SSC scatter properties) and deteriorated cells (on FL-3 fluorescence properties; 630 nm long-pass filter), fluorescein-conjugated carbohydrates bound to the live sperm cells were quantitated on the FL-1 detector (530/30-nm band-pass filter). Replicate experiments (n = 3) were performed on separate days using fresh sperm from two bulls.

Toluidine Blue Staining of the Oviducts

Oviducts were obtained from a local slaughterhouse (Gosschalk and Zn BV) and the oviduct connected to the ovary with the dominant follicle was used. The isthmus and ampulla were dissected into 50-mm pieces and placed into Bouin fixative for 24 h. After fixation, tissue was dehydrated, embedded in paraffin, and cut into 10-µm sections. Sections were mounted on poly-L-lysine-coated slides, deparaffinized in xylene, and rehydrated in graded ethanol dilutions (100%, 90%, 80%, 70%, and 50%). Rehydrated sections were stained with toluidine blue (0.1% [w/v] in phosphate buffer, pH 5.5) for 5 min [44]. After rinsing in water, slides were dehydrated in a graded series of ethanol (70%, 80%, 90%, and 100%) and in xylene. Slides were mounted with DePeX mounting medium and analyzed using bright-field illumination under a BH-2 microscope (Olympus). The resulting toluidine blue-stained histological slides of isthmus and ampulla of cow oviducts were used to detect the distribution of sulfated GAG (pink staining) on a blue background staining for other cellular material [44].

Statistics

For statistical analysis, the data were fitted into a one-site binding curve (i.e., E450 = Bmax x [fucose-PAA-biotin]/(Kd + [fucose-PAA-biotin]) where Bmax is the maximal binding and Kd is the concentration of fucose-PAA-biotin required to reach half-maximal binding). The individual curves were compared with a curve of the combined data using F-statistics, in which the sum of squares of the individual curves were compared with the sum of squares of the curve of the combined data set. Statistical significance was considered when P < 0.05.

Unconjugated carbohydrates were tested for binding competition to biotinylated carbohydrates and 50% binding inhibition (EC50) was determined using a one-site competition curve (E450 = minE450 + (maxE450 – minE450)/(1 + 10([carbohydrate]-log(EC50))). The software used was GraphPad Prism (GraphPad Software, Inc., San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Sperm Plasma Membrane Isolation

SPMs obtained from fresh sperm immediately after Percoll washing were purified at least with a factor eight (see Table 1) as the specific activity of alkaline phosphatase, acid phosphatase, and 5'-nucleotidase were respectively 8.5, 8, and 9, times higher in isolated plasma membranes when compared with complete sperm cavitate. The purified SPM isolate contained less than 7% of contamination with acrosome material as detected with the specific activity of acrosin recovered in the SPM (Table 1). SPMs, obtained after the sperm was incubated for 4 h in one of four different Tyrode media (T–BIC, T+BIC, T+BIC+Hep, T+BIC+Hep+Glu), showed similar purification rates from plasma membrane material and identical low amounts of acrosomal contamination (P > 0.05; n = 3 for all each treatment (Table 1).


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  		TABLE 1. Specific activity of the enzymes in sperm cells and isolated sperm plasma membranes (SPM) and purification of SPM obtained in different Tyrode media

Enzyme-Linked Carbohydrate Binding Assay

Serial dilutions of isolated SPM (starting with 2 mg/ml) were immobilized on polystyrene microtiter plates and varying concentrations of biotinylated carbohydrate probes (initially from 20 mM) were allowed to bind to SPM. Both fucose-PAA-biotin and LeA-PAA-biotin were captured by immobilized SPM in a concentration-dependent manner. Fucose-PAA-biotin presented approximately 12 times higher binding capacity for the isolated SPM. Maximal binding (Bmax) was 4.71 ± 0.48 and 0.40 ± 0.03 (mean arbitrary units ± SD, n = 3; P < 0.001) for fucose-PAA-biotin and LeA-PAA-biotin, respectively. SPM proteins had approximately two times lower affinity for fucose-PAA-biotin when compared with LeA-PAA-biotin. Half maximal binding (Kd values) was obtained at 2.13 ± 0.35 µg/ml and 1.16 ± 0.15 µg/ml (mean values ± SD, n = 3; P < 0.01) for fucose-PAA-biotin and LeA-PAA-biotin, respectively (Fig. 1). From the result of this experiment, we decided to use 0.5 µg SPM per well and only to use fucose-PAA-biotin for quantification of carbohydrate binding because SPM had a higher binding capacity for this conjugate than LeA-PAA-biotin.



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  		FIG. 1. Binding of fucose-PAA-biotin and LewisA-PAA-biotin (both 0.0– 2.0 µg/ml) to the SPM (0.125 µg/well) from uncapacitated sperm cells. Both fucose-PAA-biotin (Fuc) and LewisA-PAA-biotin (LeA) bind to the SPM in a concentration-dependent manner. The data indicated in the figure is corrected for nonspecific binding of the biotinylated carbohydrate to noncoated wells. Results shown are representative of three independent experiments, each done in triplicate. Mean values ± SD (n = 3) are indicated

The dependency of binding on divalent cations was tested using a range of 0–20 mM of either Ca2+ Mn2+, Mg2+, Zn2+, or EDTA. Binding of fucose-PAA-biotin to the SPM was dependent on divalent cations and present in all cation buffers tested. Binding was blocked completely in the absence of divalent cations (i.e., in the presence of 10 mM EDTA; P < 0.0001, n = 3; Fig. 2) and decreased 20% in the presence of 5 mM Zn2+ (P < 0.01, n = 3; Fig. 2), while the other divalent cations did not affect fucose-PAA-biotin binding when compared with Ca2+ (P > 0.05, n = 3; Fig. 2).



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  		FIG. 2. Binding of fucose-PAA-biotin to SPM in the presence of different divalent cations and EDTA. Microtiter plate wells were coated with SPM (0.5 µg/well) and incubated with fucose-PAA-biotin (0.25 µg/ml) in the presence of 5 mM CaCl2 (Ca), 5 mM MgCl2 (Mg), 5 mM MnCl2 (Mn), 5 mM ZnCl2 (Zn), or 10 mM EDTA. Results shown are representative of three independent experiments, each done in triplicate. Mean values ± SD (n = 3) are indicated. **, Significant reduction of binding in the presence of Zn2+, P < 0.01; and EDTA, P < 0.001)

Carbohydrate Binding Competition

Fucose-PAA-biotin binding to immobilized SPM was challenged by preincubating the SPM-coated wells (0.5 µg SPM per well) with various concentrations (0–100 mM) of carbohydrates before addition of 0.25 µg/ml fucose-PAA-biotin per well. The competition of fucose-PAA-biotin binding by fucose, glucose, maltose, mannose, galactose, inositol, 1-O-methyl-{alpha}-D-glycopyranoside, mannan, and fucoidan (where mannan and fucoidan concentration was calculated from the values of mannose or fucose units, respectively) were tested. All tested carbohydrates inhibited fucose-PAA-biotin binding to immobilized SPM (P < 0.001, n = 3 for all inhibiting carbohydrates tested, Fig. 3). The exception was inositol, which did not exert any binding competition even at the highest tested dose (P > 0.05, n = 3; Fig. 3). Galactose and 1-O-methyl-{alpha}-D-glycopyranoside only slightly inhibited this binding at concentrations higher than 10 mM, and 50% binding inhibition (EC50) was reached over 400 mM for both sugars. Fucoidan showed the highest competition for fucose-PAA-biotin binding to SPM (EC50 values of 41.7 ± 1.6 µM), followed by mannan, which also effectively competed for this binding (EC50 values of 1.67 ± 0.02 mM) (Fig. 3). Lower inhibition of fucose-PAA-biotin binding was achieved at >10 mM concentrations of maltose, mannose, glucose, and fucose with EC50 values of 31.12 ± 3.6, 218.6 ± 41.4, 85.81 ± 4.2, 162.6 ± 28.1 mM, respectively (Fig. 3).



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  		FIG. 3. Carbohydrate competition of fucose-PAA-biotin binding to SPM from uncapacitated sperm cells. SPM (0.5 µg/well) were incubated with 0.25 µg/ml of fucose-PAA-biotin in the presence of increasing concentrations (0.0–100 mM) of fucose (Fuc), glucose (Glu), maltose (Mal), mannose (Man), galactose (Gal), mannan (Mann), inositol (Ino), 1-O-methyl-{alpha}-D-glycopyranoside (1-O-m), and fucoidan (Fdan). The data indicated in the figure are corrected for nonspecific binding of biotinylated carbohydrate to noncoated wells. Results shown are representative of three independent experiments, each done in triplicate. Mean values ± SD (n = 3) are indicated

Mannan and fucoidan were the most efficient carbohydrates blocking fucose-PAA-biotin binding to immobilized SPM. Therefore, the binding affinities of immobilized SPM for biotinylated mannan and fucoidan were tested. Polymeric biotinylated conjugates of mannose (mannan-BSA-biotin) and fucose (fucoidan-biotin) showed a high binding affinity to SPM from uncapacitated sperm (Fig. 4). Both biotinylated oligosaccharides reached their maximum binding (Bmax) at a concentration of approximately 0.25 µg/ml, with Bmax values for mannan-BSA-biotin and fucoidan-biotin of 2.25 ± 0.07 and 2.96 ± 0.09 mean arbitrary units ± SD, n = 3, respectively. However, fucoidan-biotin presented the strongest binding affinity to immobilized SPM (mean Kd ± SD from three experiments were 1.40 ± 0.37 and 27.88 ± 3.83 µM, respectively). When the competition in this binding with fucose and mannan was evaluated, fucose failed to compete in fucoidan-biotin binding to immobilized SPM, whereas mannan clearly inhibited this interaction (Fig. 5), presenting an inhibitory effect at concentrations higher than 5 µg/ml (EC50 values for mannan was over 200 mg/ml).



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  		FIG. 4. Binding of fucoidan-biotin and mannan-BSA-biotin (both 0.0– 0.5 µg/ml) to the SPM (0.125 µg/well) from uncapacitated sperm. Both fucoidan-biotin (Fdan) and mannan-BSA-biotin (Mann) did bind to the SPM in a concentration-dependent manner. The data indicated in the figure are corrected for nonspecific binding of biotinylated carbohydrate to noncoated wells. Results shown are representative of three independent experiments, each done in triplicate. Mean values ± SD (n = 3) are indicated



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  		FIG. 5. Inhibition of fucoidan-biotin binding to SPM from uncapacitated sperm cells with increasing concentrations of fucose and mannan. SPM (0.5 µg/well) were incubated with 0.25 µg/ml of fucoidan-biotin in the presence of increasing concentrations (0.1–100 mg/ml) of fucose (Fuc) and mannan (Mann). Fucoidan-biotin binding can be inhibited with mannan in concentrations higher than 5 µg/ml, fucose had no inhibitory effect. The data indicated in the figure are corrected for nonspecific binding of biotinylated carbohydrate to noncoated wells. Results shown are representative of three independent experiments, each done in triplicate. Mean values ± SD (n = 3) are indicated

Heparin, a well-described capacitation factor for bovine sperm [30], strongly inhibits sperm binding to the oviductal epithelial monolayers [33]. Here, the competition of heparin for fucose-PAA-biotin or for mannan-BSA-biotin binding to immobilized SPM was tested. Immobilized SPM was incubated with increasing concentrations of heparin (0.001– 100 µg/ml) and a constant concentration of fucose-PAA-biotin or mannan-BSA-biotin (0.25 µg/ml). Heparin strongly inhibited binding of both biotinylated carbohydrates to the immobilized SPM in a concentration-dependent manner (Fig. 6). This inhibitory effect was already present at a concentration as low as 0.09 µg heparin/ml (EC50) values for inhibition of fucose-PAA-biotin binding at approximately 2 mM and at 9 mM for mannan-biotin). Note that we can only claim that heparin showed binding competition to both biotinylated carbohydrate probes; however, the differences in EC50 values are likely due to differences in the stoichiometry of carbohydrates to the conjugated molecule: One fucose-PAA-biotin molecule contains four fucose and one biotin groups, whereas, for the self-prepared mannan-BSA-biotin, the exact number of mannan and biotin groups per molecule are unknown (we estimate 20 mannan groups and 1 biotin group per albumin molecule).



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  		FIG. 6. Inhibition of fucose-PAA-biotin and mannan-biotin binding to the SPM from uncapacitated sperm cells by heparin. SPM (0.5 µg/well) were incubated with either 0.25 µg/ml of fucose-PAA-biotin (Fuc) or mannan-BSA-biotin (Mann) in the presence of increasing concentrations (0.001–100 µg/ml) of heparin. The data indicated in the figure are corrected for nonspecific binding of biotinylated carbohydrate to noncoated wells. Results shown are representative of three independent experiments, each done in triplicate. Mean values ± SD (n = 3) are indicated

Influence of Capacitation on SPM-Carbohydrate Binding

The effect of in vitro capacitation on sperm-carbohydrate interaction was studied using bicarbonate-enriched Tyrode media (T+BIC) either supplemented with heparin (T+BIC+Hep) or with heparin and glucose (T+BIC+Hep+Glu) and compared with Tyrode medium without bicarbonate (T–BIC). The subpopulation of sperm cells that showed signs of capacitation following incubation was quantified by the CTC-staining assay (for reference and staining patterns observed for bovine sperm, see [35]). Incubation for 4 h in capacitation media resulted in capacitation-specific CTC staining of approximately 75% of the cells (75% ± 2.6% for T+BIC+Hep and 75% ± 4.2% for T+BIC), whereas only 35% ± 3.4% in control medium (T–BIC) showed this CTC-labeling pattern (P < 0.001; n = 3). Higher glucose concentration (10 mM instead of 5 mM) in the capacitation media (T+BIC+Hep+Glu) partially inhibited the heparin effect, as only 56% ± 3.5% of sperm cells showed CTC capacitation staining in T+BIC+Hep+Glu (significantly different from the other capacitation media, n = 3; P < 0.05). Under all conditions, <5% of total cells showed signs of acrosome disruption with the exception of the T+BIC treatment, where approximately 8% of the total cells showed signs of acrosome disruption detected by the CTC-staining assay [35]. The SPM fractions of these four incubated sperm samples were isolated and immobilized on 96-well plates. Fucose-PAA-biotin showed the highest binding affinity for control samples: immobilized SPM isolated from freshly collected sperm cells (Fig. 7). This binding significantly decreased (P < 0.03) after incubation of 4 h in T–BIC. A much more pronounced reduction in fucose-PAA-biotin binding was found to take place on immobilized SPM from sperm cells incubated in T+BIC (P < 0.01) as well as T+BIC+Hep (P < 0.01) when compared with T–BIC. Inclusion of glucose did not prevent fucose-PAA-biotin binding to the immobilized SPM.



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  		FIG. 7. Effect of sperm capacitation on fucose-PAA-biotin binding to the SPM. SPM were isolated from the fresh sperm (SPM) and sperm incubated for 4 h in four different media: tyrode medium without bicarbonate (T–BIC), tyrode medium with bicarbonate (T+BIC), tyrode medium with bicarbonate and with 10 µg/ml heparin (T+BIC+Hep), and tyrode medium with bicarbonate, 10µg/ml heparin and 5mM glucose (T+BIC+Hep+Glu). Constant concentrations of each SPM (0.5 µg/well) were incubated with increasing concentrations of fucose-PAA-biotin. Results shown are representative of three independent experiments, each done in triplicate. Mean values ± SD (n = 3) are indicated

Binding of Biotinylated Carbohydrate Probes During In Vitro Capacitation on Living Sperm Cells

Uncapacitated sperm cells (T–BIC) and capacitated sperm cells (T+BIC, T+BIC+Hep) were incubated with biotinylated carbohydrate conjugates and bound conjugates were further visualized with streptavidin-FITC and detected under a CLSM. Uncapacitated sperm cells were stained with fucose-PAA-biotin, heparin-BSA-biotin, fucoidan-biotin, or mannan-BSA-biotin. In all cases, a strong staining in a rim-like area of the anterior region of the sperm head was observed in approximately 60% of the sperm cells (Fig. 8). The remaining part of the cell was unstained or showed only very weak labeling. This staining pattern was similar but weaker when sperm cells were stained with LeA-PAA-biotin (Fig. 8). The percentage of cells showing streptavidin-FITC labeling remained unaltered during capacitation in vitro. Control incubations with PAA-biotin (with no conjugated carbohydrate) and streptavidin-FITC showed no staining of the sperm cells (Fig. 8D). The amount of streptavidin-FITC labeling per positive sperm cells was determined by flow cytometry and was normalized for each biotinylated glycoconjugate to 100%. The amount of FITC labeling (indicating the amount of carbohydrate binding) decreased for biotinylated LeA and heparin carbohydrates (P < 0.01, n = 3; Table 2) when sperm cells were treated with T+BIC while the labeling with fucose-PAA-biotin and mannan-BSA-biotin binding remained unaltered (P > 0.05, n = 3; Table 2). Heparin had a more ubiquitous effect and severely decreased binding of all biotinylated carbohydrates tested (P < 0.001, n = 3; see Table 2 and Fig. 8C). Higher levels of glucose in the T+BIC+Hep buffer did not significantly reduce binding of biotinylated carbohydrates (Table 2; P > 0.05). However, the oligosaccharide binding of mannan-BSA-biotin remained unaltered after T+BIC treatment (P > 0.05, n = 3) and only partly decreased after T+BIC+Hep and T+BIC+Hep+Glu capacitation incubations (P < 0.005, n = 3) when compared with T–BIC treatment (Table 2).



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  		FIG. 8. Live uncapacitated sperm cells stained with biotinylated carbohydrate probes as detected by confocal laser-scanning microscope. Sperm cells were stained with one of four different carbohydrate probes (fucose-PAA-biotin, LeA-PAA-biotin, fucoidan-biotin, mannan-BSA-biotin) and visualized by streptavidin-FITC. Immediately before microscopic evaluation sperm cells were counterstained with propidium iodide and only propidium iodide-negative (live) sperm cells were imaged (see Material and Methods section). As presented for LeA-PAA-biotin (A) and mannan-BSA-biotin (B), sperm showed strong staining in a rim-like area of their anterior region after incubation in T–BIC. C) Staining of the LeA-PAA-biotin was strongly diminished after sperm were incubated in T+BIC ±Hep for 4 h. A similar pattern was observed for all carbohydrate probes (fucose-PAA-biotin, LeA-PAA-biotin, fucoidan-biotin, mannan-BSA-biotin). D) Sperm cells stained with PAA-biotin (without conjugated carbohydrates conjugated) showed no streptavidin-FITC labeling


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  		TABLE 2. Effects of different capacitation media on glycoconjugate binding to the bovine sperm after 4 h of incubation.*

Competition of SPM-Carbohydrate Binding by Follicular Fluid and Corresponding Isolated Follicular Glycosaminoglycans

The presence and localization of sulfated GAG in the oviduct was detected with toluidine staining, which, in the presence of GAG, changes its color from blue to pink. GAGs were highly abundant in both the isthmus (Fig. 9, A and B) and the ampulla (Fig. 9, C and E), and localized on the luminal surface of the oviductal mucosa. GAGs were equally distributed over the apical plasma membranes of the secretory and ciliated oviductal epithelial cells, in the form of a thin layer. In the ampulla, they were also found within apical protrusions of the secretory cells, indicating their secretory origin (Fig. 9). The secretory activity of the ampulla is maximal around ovulation [15] and probably increases the amount of GAG in the isthmus, where it may facilitate sperm release. A concentration of 403.5 µg/ml of sulfated GAG was found in the fluid of dominant follicles. Increasing concentrations of isolated GAG from follicular fluids as well as complete corresponding follicular fluid were highly potent in inhibiting fucose-PAA biotin binding to SPM (Fig. 10). This inhibitory effect was most predominant with whole follicular fluid, where inhibition was already visible at concentrations lower than 0.01 µg GAG/ ml and reached its maximum at about 0.3 µg GAG/ml. The inhibitory effect of the isolated GAG fraction from native glycosylated components in follicular fluid was visible at concentrations from 1 µg GAG/ml with EC50 values of 47.2 ± 6.7 µg/ml, which was about a 50 times higher level than the EC50 values for the native follicular fluid GAG (0.25 ± 0.04 µg/ml; Fig. 10).



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  		FIG. 9. Light microscopic micrograph of toluidine blue staining of cow oviducts. Pink-stained glycosaminoglycans (GAG) can be found on the apical surface of the epithelium equally distributed over the whole luminal side (arrows) of the isthmus (A, B, C) and the ampulla (D, E). Within the ampulla (E) GAG were detected within the apical protrusion of the secretory cells (circle) and so indicating their origin



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  		FIG. 10. Inhibition of fucose-PAA-biotin binding to the SPM from uncapacitated sperm cells by complete follicular fluid or by GAG isolated from follicular fluid. SPM (0.5 µg/well) were incubated with 0.25 µg/ml of fucose-PAA-biotin in the presence of increasing concentrations of either follicular fluid (FF) or GAG (GAG). The data indicated in the figure are corrected for nonspecific binding of biotinylated carbohydrate to noncoated wells. Results shown are representative of three independent experiments, each done in triplicate. Mean values ± SD (n = 3) are indicated


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have developed a new assay to biochemically quantify carbohydrate affinity of the apical head plasma membrane of bovine sperm. The SPM was purified and immobilized and biotinylated carbohydrate probes were used for quantitative detection of binding as well as binding competition. The effects of in vitro capacitation factors such as bicarbonate and heparin, as well as endogenous proteoglycans present in the follicular fluid and oviduct, on regulation of carbohydrate affinity were tested. The assay confirmed involvement of fucose in the sperm-oviduct binding in the bovine species and suggested a role for mannan in this binding. The relevant carbohydrate binding sites appear to be exclusively localized in the apical tip of the sperm head surface area of uncapacitated bovine sperm cells, and not only the sugar but also the sugar conformation seems to be an important binding factor. We should consider here that we visualized the binding of multivalent biotinylated carbohydrate probes on living sperm cells, which may well cause patching or capping of the sperm lectins of interest. This process may even lead to cell deterioration that was not observed, in fact only live sperm cells were imaged. Moreover, paraformaldehyde-fixed cells showed similar distribution of biotinylated carbohydrate probes (results not shown), which would at least indicate against a lateral movement of the sperm lectins from a distinct surface area of the sperm head toward the apical tip. Nevertheless, at the spatial resolution limit of CLSM, it is not possible to discriminate whether or not our labeling protocol may have caused sperm lectin aggregation within the stained apical tip area of the sperm head. Bicarbonate is a primary trigger for reduction of carbohydrate staining. Heparin and endogenous GAG (especially in the native proteoglycan state) additionally decreased carbohydrate staining. Although it is unclear how much follicular fluid really enters the oviduct at the time of ovulation, follicular fluid can directly affect the sperm release from the oviductal epithelium even at low concentration. Moreover, the oviduct has been shown to actively secrete GAG that may function in sperm release from the oviduct epithelia.

Sperm-oviduct interactions cannot be investigated in situ at the biochemical level. However, several in vitro studies have shown that mammalian sperm binding to the oviduct is mediated by carbohydrate interactions. For bovine species, the animal model of the current investigation, this interaction appears to be fucose specific [13, 14]. We have set up a biochemical assay to quantify the binding affinity of the apical surface of the sperm head, and for this purpose, we first isolated this specific plasma membrane area (SPM) and constructed biotinylated carbohydrate conjugates to quantify SPM carbohydrate-binding capacity and the potency of unconjugated carbohydrates to compete in this binding. Specifically, binding of fucose-PAA-biotin and LeA-PAA-biotin to immobilized SPM was investigated. The fact that immobilized SPM had high binding capacity for both biotinylated probes and that this binding was confined to the apical tip area of the sperm head confirms that sperm bind fucosylated molecules at their surface area involved in sperm-oviduct binding. However, the observation that fucose-PAA-biotin showed higher binding specificity for isolated SPM than LeA-PAA-biotin is in contrast with previously published data [14]. The difference may arise from experimental differences as, in our approach, isolated and immobilized SPM were used as binding templates for fucose- or LeA-PAA-biotin (each polyacrylamide-biotin conjugate contains approximately 20 mol% of fucose or LeA). In contrast, Suarez et al. [14] used a bioassay in which unconjugated (free-form) fucose and LeA was used to block live sperm binding to oviductal epithelium. Alternatively, the difference can be due to the different spatial presentations of these two carbohydrates (fucose and LeA) attached to the PAA molecule. The presentation of the sugar residue within a larger molecule determines the binding affinity of the lectin [45].

Various mono- and oligosaccharides affect the binding affinity of fucose-PAA-biotin to SPM. Fucose, mannose, glucose, and maltose only weakly inhibited this binding. This lack in fucose-binding competition contrasts with the significant inhibitory effect of extracellular fucose on sperm-oviduct binding [13]. Possibly, the absence of inhibition of free fucose on fucose-PAA-biotin binding to SPM is due to conformation of this conjugate that contains four fucose residues. The most potent inhibitor for sperm-oviduct binding was fucoidan [13], and indeed, polysaccharides like mannan and fucoidan were also highly effective in preventing fucose-PAA-biotin binding to immobilized SPM. In fact, mannan-BSA-biotin and fucoidan-biotin had higher affinity for immobilized SPM than fucose-PAA-biotin. We should consider here that the conjugates used differ in stoichiometry of carbohydrate and biotin moieties. The high inhibitory effect of mannan and its high binding affinity for the isolated SPM indicates that bovine sperm cells have high affinity for mannan containing glycoconjugates. Mannan binding may either take place during sperm-oviduct binding in bovine species or later, for example, during ZP binding, as has been suggested by Revah et al. [31]. It would be of interest to evaluate the role of mannan in bovine sperm binding to oviduct explants as well as to the zona pellucida.

Heparin, another polysulfated glycoconjugate, is widely used to capacitate bull sperm and induces capacitation-specific CTC staining in 60–70% of the sperm cells [32]. Although heparin had no effect on the binding of the uncapacitated sperm to the oviductal epithelium [13], it was recently shown that heparin acts as a strong inhibitor of sperm binding and also a quick releaser of sperm bound to the oviduct epithelium (this study, [33]). Heparin (and fucoidan) induce an increase in flagellar-beat frequency and also high linear motility during sperm release from the bovine oviduct [33]. We demonstrated that heparin is a very strong inhibitor of fucose-PAA-biotin binding to SPM even at concentrations as low as 0.01 µg/ml (1000 times lower than the standard heparin concentration at capacitation conditions). This inhibitory effect is most likely due to competitive binding of heparin to the fucose-binding ligand present in the SPM. In this respect, indeed, the binding competition between fucose and heparin was independent from sperm capacitation. Therefore, our experiments support the observation that heparin interferes in binding of the uncapacitated SPM to the specific carbohydrate moieties of the oviductal epithelial cells [14]. Interestingly, a bull sperm protein, PDC-109, has been described as being involved in sperm binding to the oviduct [46] and to have affinity for both fucose as well as heparin [32]. This may explain why heparin interferes with bovine sperm-oviduct binding. An issue for further research is to decipher whether the sperm release is caused i) directly by extracellular GAG or ii) indirectly via sperm capacitation that induces sperm surface changes and subsequently causes the release, or iii) a combination of both processes.

Bicarbonate-enriched Tyrode media induced capacitation of approximately 40% of the bovine sperm cells, as also did heparin during the 4-h incubation, as observed by CTC staining (this study, [32]). This indicates that bicarbonate primarily is responsible for the capacitation changes demonstrated by the CTC staining (see also [47]). High levels of glucose only partly inhibited the capacitation response (this study) but did not block capacitation as reported earlier [48]. It is likely that this discrepancy is caused by the method of detecting capacitation: Medeiros and Parrish [48] used a panel of lectins to detect heparin-induced surface changes in lectin-binding topology and intensity. Probably, such changes in lectin interactions are in part a sequential response occurring later than the CTC response (see [47]). More importantly, heparin itself competes with lectins for binding to the sperm surface. Moreover, bicarbonate and heparin-induced capacitation reduced carbohydrate-binding properties of the bovine sperm surface (this study). Indeed, capacitated sperm had a lower binding rate for fucose-BSA-biotin ([31], using a flow cytometric assay) than did uncapacitated sperm. We were able to repeat this result not only for fucose-PAA-biotin but also for LeA and heparin. The effect, however, was solely a result of bicarbonate and was not dependent on the presence of heparin or glucose. Therefore, bicarbonate seems responsible for the reduced fucose-binding capacity. We demonstrated a high affinity of the sperm cells for mannan-biotin in the absence of heparin (both in the flow cytometric assay as well as on immobilized SPM). Only a partial decrease in mannan-biotin affinity was observed after capacitation in the presence of heparin. This supports the hypothesis [31] that mannose (mannan is a polymere of this sugar) plays a role in sperm-zona binding and the absence of mannan binding would distort this important prefertilization event. Sperm interactions with heparin-like GAG may facilitate capacitation in vivo by inducing loss or modification of sperm surface molecules involved in sperm-oviduct binding [33]. We in fact showed that GAG from fluids derived from dominant follicles were very efficient in competing for carbohydrate binding to the sperm surface (valid for all biotinylated carbohydrates tested). A chemically prepared GAG fraction from follicular fluid was less efficient in competing for carbohydrate binding to the sperm surface than unprocessed follicular fluid. The tertiary structure of glycoproteins (containing GAG), which is lost after purification, is probably important for optimal bioactivity. This finding may indicate that the complete tertiary structure of glycosylated proteins is required for full bioactivity of glycosidic structures involved in sperm adhesion to the oviduct epithelial cells. It is also worth noting that various proteoglycans from follicular fluid promote capacitation and acrosome reaction in bovine spermatozoa [49, 50]. In fact, in fertility clinics, follicular fluid from dominant follicles is used for optimizing IVF [27]. In vivo, however, these processes take place in the oviduct. Two obvious changes in the oviduct just before ovulation are i) GAG secretion especially at the ipsilateral side to the ovulating follicle in the isthmus [15] and ii) infiltration of follicular fluid in the ampulla of the ipsilateral oviduct. Therefore, GAG are likely involved in modulating sperm-oviduct interactions (sperm release) and probably facilitates sperm activation and surface remodeling, which is important for sperm-zona interaction.

In conclusion, the newly developed biochemical assay for the quantitative detection of carbohydrate binding properties of the SPM did elucidate how carbohydrate moieties are involved in the sperm-oviduct adhesion as well as in the in vivo capacitation-induced release of activated sperm cells. The assay can additionally be used to determine exposed carbohydrate moieties that are involved in sperm-zona binding.


   FOOTNOTES
 
1 Correspondence: Bart M. Gadella, Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Yalelaan 2, P.O. Box 80.176, 3508 TD Utrecht, The Netherlands. FAX: 31 30 2535492; B.Gadella{at}vet.uu.nl Back

Received: 8 March 2004.

First decision: 29 March 2004.

Accepted: 3 September 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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