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a Department of Anatomy, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1-6]GlcNAc-O-Me), only Lewis-a significantly reduced binding, and it did so in a dose-dependent fashion (p = 0.009 at 12.5 mM). Ca2+ dependency of binding was examined. Sperm were incubated with explants in Tyrode's albumin lactate pyruvate (TALP) containing 2 mM CaCl2 or lacking CaCl2 and containing 2 mM EGTA. Sperm-binding density was reduced significantly in EGTA (p < 0.03) but could be restored by readdition of CaCl2. Also, live sperm were labeled with the oligosaccharide ligand Lewis-a conjugated to fluorescein isothiocyanate-tagged polyacrylamide. Sperm exhibited labeling on the head only in the presence of Ca2+. Labeling could be blocked by fucose or Lewis-a-polyacrylamide. It was concluded that bull sperm bind to an oligosaccharide ligand on the oviductal epithelium that resembles Lewis-a and that binding is Ca2+-dependent.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There is evidence in several mammalian species that the oviductal reservoir is created by binding of sperm to oviductal epithelium. Motile sperm have been observed to bind to the apical surface of the oviductal epithelium in cattle [3], mice [12], hamsters [13], pigs [14], and horses [15]. The trapping action of the sperm-binding moieties may be enhanced by the narrow lumen of the uterotubal junction and isthmus. The bovine lumen is particularly tortuous and narrow in the uterotubal junction and caudal isthmus, the sites of sperm storage, due to mucosal folding and flexure of the tube [16, 17]. There is an extensive vascular plexus in the wall that resembles erectile tissue and a thick muscular layer that could further compress the lumen during estrus [16, 18]. The narrowness of the lumen is especially apparent in frozen sections, because tissue does not shrink as it does during preparation of paraffin-embedded sections [17]. Thus, sperm entrapment in the reservoir may be due to a number of factors that enhance the trapping effectiveness of adhesive molecules on the epithelium, particularly by increasing contact of sperm with the mucosal surface.
Sperm binding to oviductal epithelium involves carbohydrate recognition. The first evidence for this came from the hamster, in which fetuin and its terminal sugar, sialic acid, inhibited binding of sperm to the epithelium when infused with sperm into oviducts [19]. Binding of stallion sperm to explants of epithelium was blocked by asialofetuin and its terminal sugar, galactose [20]. Bull sperm binding to explants of oviductal epithelium was blocked by fucoidan and its component fucose [21]. Pretreatment of bovine epithelium with fucosidase reduced binding [21]. Thus, in cattle, binding is mediated by a carbohydrate moiety that contains or resembles fucose.
The present study was undertaken in order to further characterize the binding interaction between bull sperm and the mucosal epithelium of the bovine oviduct. In vivo, the interaction would involve a carbohydrate moiety on membrane glycoproteins or glycolipids. Fucose is rarely linked directly to a protein core [22, 23]. For this reason, the relative binding inhibition capacities of carbohydrates containing fucose in various linkages commonly found in vivo were determined. Also, because many animal lectins require Ca2+ for binding, the Ca2+ dependency of sperm binding was examined.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All chemicals and media were obtained from Sigma Chemical Company (St. Louis, MO) with the exception of those noted here. BSA (fraction V, catalog no. 12659) and HEPES buffer were purchased from Calbiochem-Novabiochem Corporation (La Jolla, CA). Lewis-a, Lewis-X, lacto-N-fucopentaose I, and 3-fucosyllactose were from V Labs, Inc. (Covington, LA). Lewis-a was also obtained from Toronto Research Chemicals, Inc. (Toronto, ON, Canada), as was GlcNAcß1-4[Fuc1-6]GlcNAc-O-Me. Lewis-a-PAA-FITC (a synthetic carbohydrate probe consisting of a polyacrylamide [PAA] matrix in which every fifth amide group of the polymer chain is N-substituted by Lewis-a on a spacer arm and fluorescein isothiocynate [FITC] is substituted on approximately every 20th group) was purchased from Glycotech Inc. (Rockville, MD).
A modified Tyrode's balanced salt solution, termed sperm-TALP [24, 25], sterilized through a 0.2-µm Nalgene cellulose nitrate sterilization filter (Nalge Co., Rochester, NY), was used as the medium for sperm and oviductal explants. TALP consisted of 99 mM NaCl, 3.1 mM KCl, 25 mM NaHCO3, 0.35 mM NaH2PO4, 10 mM HEPES, 2 mM CaCl2, 1.1 mM MgCl2, 21.6 mM sodium lactate, 1.1 mg/ml sodium pyruvate, 6 mg/ml BSA, and 1 µg/ml gentamycin (pH 7.4, 290 mOsm/kg). TALP was equilibrated in a humidified atmosphere at 39°C containing 5% CO2 before use. In order to test the Ca2+ dependence of sperm labeling and binding to oviductal explants, TALP with low Ca2+ was prepared by omitting the calcium salt (TALP-0Ca). Calcium-free medium was obtained by adding 2 mM EGTA (ethylene glycol-bis-N,N'-tetraacetic acid) to TALP without calcium salt (TALP-EGTA). The pH was adjusted to 7.4 by adding NaOH. The concentration of NaCl was adjusted to maintain osmolality at 290 mOsm/kg.
PBS, used for transport of oviducts, consisted of 136 mM NaCl, 2.68 mM KCl, 0.49 mM MgCl2, 9.58 mM NaH2PO4, 1.47 mM KH2PO4, and 0.88 mM CaCl2, pH 7.4, 290 mOsm/kg. PBS was sterilized as described above and stored at 4°C for several weeks.
Sperm Preparation
Semen was prepared and generously donated by Genex Cooperative, Inc. (Ithaca, NY). Samples from three fertile Holstein bulls were diluted in egg yolk extender, pooled, loaded into 0.5-ml artificial insemination straws at a concentration of 50 x 106 sperm per straw, frozen in liquid nitrogen (LN2) vapor, and held in LN2 tanks until use. For some experiments, fresh semen from individual bulls was used. In this case, the semen was diluted 1:1 with TALP shortly after collection and transported to the laboratory in an insulated container at room temperature.
For each treatment, one or two straws of frozen semen were thawed by immersion in a water bath at 37°C for 30 sec, diluted into 5 ml of TALP solution, and centrifuged at 350 x g for 5 min. All but 1 ml of the medium overlying the sperm pellet was removed, and the sperm were resuspended into the medium. The 1-ml sperm suspension was divided into 4 aliquots for selection of motile sperm by swim-up. Each 250-µl aliquot was layered under 1 ml of TALP in a 15-ml centrifuge tube. After incubation for 1 h at 39°C, 750 µl was removed from the top of each tube. Sperm were concentrated by centrifugation at 350 x g for 5 min and adjusted to a concentration of 5 x 106/ml for testing binding to epithelium or 12 x 106/ml for labeling with fluorescent ligand. Motility ranged from 80-90%.
When fresh semen was used, the sperm were washed three times by 1:10 dilution in TALP, followed by centrifugation at 170 x g for 5 min. The sperm concentration was then adjusted to 12 x 106/ml for labeling.
Preparation of Oviductal Explants
Oviducts associated with large follicles (> 15 mm diameter) were collected at an abattoir and transported on ice to the laboratory in sterile PBS (pH 7.4) with penicillin (100 µg/ml) and streptomycin (50 µg/ml). Upon arrival, they were thoroughly washed in PBS/penicillin/streptomycin, then dissected free of the surrounding tissues. The isthmus was identified by its narrow width and thick, muscular walls. It was separated from the remaining oviduct and straightened by cutting it free from mesentery. The oviductal epithelium was obtained as previously described [26, 27] by gently milking the isthmus or ampulla with tweezers from the uterotubal junction towards the ovary. Epithelium, which emerged in sheets, was collected in TALP and disaggregated into small pieces by drawing them into a 1-ml pipette tip. Within 30 min of disaggregation, the clumps of epithelial cells formed everted vesicles with apical surfaces facing outward, henceforth referred to as explants. Explants were washed twice in TALP by centrifugation (80 x g, 20 sec), then allowed to settle in a dense layer at the bottom of a 35 x 10-mm Petri dish (Becton Dickinson Labware, Rutherford, NJ).
Binding Inhibition Studies
For binding inhibition experiments, 10-µl aliquots were taken from the dense layer of explants and transferred to 50-µl droplets of sperm-TALP under silicon oil. The droplets contained the monosaccharides or oligosaccharides to be tested. The droplets of medium had been equilibrated under oil at 39°C in a humidified atmosphere containing 5% CO2. After 10 min, 20 µl sperm was added to the droplets, such that the final droplet volume was 80 µl, the final sperm concentration was 1.25 x 106 sperm/ml, and the final concentration of test substances was 30 mM for monosaccharides and 12.5 mM for oligosaccharides. After 15 min of coincubation, the explants were washed free of loosely attached sperm by drawing them up into a pipet and transferring them into fresh 80 µl sperm-TALP droplets three times. This washing step was found to reduce variation within groups. The explants were then transferred to slides and covered with coverslips supported by silicon grease for videotaping and analysis of density of sperm binding.
Videomicroscopy and Image Analysis
Slides containing the oviductal explants with sperm were transferred to a 39°C stage on a Zeiss Axiovert microscope (Carl Zeiss Inc., Thornbrook, NY). All explants on each slide (representing 0.5-1.0 mm2 of surface area) were videotaped using a 30x Hoffman modulation contrast objective (Modulation Optics, Greenvale, NY). Superimposed on the recorded image was time-date information provided by a videotimer (Model VTG 33; For-A Co., Ltd., Newton, MA). A black-and-white video camera (model CCD72; Dage-MTI, Inc., Michigan City, IN) was used with a Panasonic AG-7300 SuperVHS video cassette recorder (Panasonic Industrial Co., Secaucus, NJ). Videotaping of each slide was completed in approximately 2 min. For the analyses of attached sperm, the videotapes were reviewed by video monitor (model TR-196M; Panasonic), and the sperm attached to the side of the explant facing the camera were counted. The surface areas of those regions of the explants were determined from the videotapes using a digital-imaging system (Power Macintosh 7100AV; Apple Corp., Cupertino, CA) and image analysis software (IPLab Spectrum H-LG3; Signal Analytics Corporation, Vienna, VA).
Experimental Design and Statistics
In the first set of experiments, a variety of monosaccharides were tested for binding inhibition activity, in order to verify that fucose was the most effective monosaccharide inhibitor. In the second set of experiments, commercially available oligosaccharides representing the following fucose linkages were tested: lacto-N-fucopentaose I for Fuc1-2Gal; 3-fucosyllactose for Fuc1-3Glc; Lewis-X for Fuc1-3GlcNAc; and Lewis-a for Fuc1-4 GlcNAc. In the third set of experiments, the inhibitory activity of Fuc1-6 GlcNAc, in the form of GlcNAcß1-4[Fuc1-6]GlcNAc-O-Me, was compared with that of fucose and Lewis-a. Treatment effects on sperm binding per unit area of epithelium were analyzed by ANOVA using Systat [28] (SPSS Inc., Chicago, IL). Post-hoc pair-wise comparisons of means were made with Tukey's HSD test [29]. Three to five replicates were performed for each set of experiments. For each replicate, a different oviduct was incubated with frozen/thawed sperm from the same pool of 3 bulls.
In a fourth set of experiments, a dose-response curve was constructed for Lewis-a trisaccharide. A linear regression test was used to detect an effect of Lewis-a concentration on sperm-binding density (Systat, SPSS).
Labeling of Sperm
Fresh bull sperm of superior motility were prepared as described above. After adjusting the sperm concentration to 12 x 106 sperm/ml, 85 µl were incubated with 10 µl of 600 mM D(+)mannose (final concentration, 60 mM), 600 mM L(-)fucose, 1.0 mg/ml Lewis-a-PAA-biotin in PBS, or PBS (pH 7.4) for 15 min at room temperature. Then 5 µl Lewis-a-PAA-FITC (1.0 mg/ml in PBS, stock) was added, and samples were incubated at room temperature in the dark for 30 min. After washing in 5 ml TALP (170 xg, 5 min), the supernatant was drained down to 50 µl. Ten-microliter aliquots were placed on a slide and viewed by laser scanning confocal microscopy (Bio-Rad, Richmond, CA). Labeling was also carried out using frozen sperm that had been prepared by swim-up as described above. In order to investigate Ca2+dependence of labeling, experiments were repeated using TALP, TALP without CaCl2 (TALP-0Ca), and TALP without CaCl2 and with 2 mM EGTA (TALP-EGTA) in all dilution and washing steps. Motility of the sperm was checked every 30 min.
Testing Ca2+ Dependence of Binding
To investigate the Ca2+ dependence of sperm binding, oviductal explants were washed rapidly three times in TALP, TALP-0Ca, or TALP-EGTA. Frozen/thawed sperm were prepared as described above, with the exception that TALP-0CA or TALP-EGTA was used in all washing and dilution steps. Then, 10 µl of explants were transferred to 50-µl droplets of TALP, TALP-0Ca, or TALP-EGTA that had been equilibrated at 39°C in a humidified atmosphere containing 5% CO2. After 10 min, 10 µl of sperm (concentration 5 x 106 sperm/ml) were added. After 15 min of coincubation, the explants were transferred to slides and covered with coverslips supported by silicon grease. They were not rinsed again, because rinsing was discovered to be detrimental to ciliary motility in the absence of Ca2+. Videomicroscopy and analysis of sperm binding was performed as described above. Statistical analysis was as described above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the first set of experiments, five monosaccharides (fucose, mannose, sialic acid, glucose, N-acetyl glucosamine, and galactose) were tested for binding inhibition at 30 mM. Only fucose significantly reduced sperm-binding density compared to control (p = 0.03; Fig. 1). In the second set of experiments, four oligosaccharides were tested for binding inhibition at 12.5 mM (lacto-N-fucopentaose I for Fuc1-2Gal, 3-fucosyllactose for Fuc1-3Glc, Lewis-X for Fuc1-3GlcNAc, and Lewis-a for Fuc1-4 GlcNAc (Fig. 2). Only Lewis-a (-L-Fuc[1-4]-[ß-D-Gal-(1-3]-D-GlcNAc), produced significantly lower sperm-binding density than the control and the other oligosaccharides (p = 0.009; Fig. 3A). In the third set of experiments, Lewis-a and GlcNAcß1-4[Fuc1-6]GlcNAc-O-Me were tested. Only Lewis-a and fucose produced significantly lower sperm-binding density than the control (Lewis-a: p=0.001; fucose: p = 0.003; Fig. 3B).
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A significant dose effect was observed for binding inhibition by Lewis-a (multiple regression R2 = 0.64; p < 0.001). The binding inhibition curve was shifted to the left of the curve produced by fucose and reported in an earlier publication [21] (Fig. 4).
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Both fresh and frozen/thawed live bull sperm treated with Lewis-a-PAA-FITC showed bright, punctate labeling over the acrosomal ridge and some even, fainter labeling over the postacrosomal region (Fig. 5A). Acrosomal labeling was blocked by Lewis-a-PAA-biotin (0.1 µg/µl) and L(-)fucose (Fig. 5, B and C), but not by D(+)mannose (mannose data not shown). These results indicate that there was a lectin on the surface of sperm that bound Lewis-a. If sperm were smeared on slides, frozen and thawed, acrosomes were missing and there was no labeling. Labeling was not reduced in TALP-0Ca; however, there was no labeling at all in TALP-EGTA.
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The experiments testing the Ca2+ dependence of sperm binding revealed by direct observation that merely omitting the calcium salt from the medium did not reduce binding. If the calcium chelator EGTA was added to the medium lacking calcium salts, binding was significantly reduced (p = 0.03; Fig. 6). As preliminary experiments had shown that complete preparation of explants in TALP-0Ca or TALP-EGTA caused the cilia of the explants to cease beating, the explants were prepared in complete TALP and then washed rapidly through 3 droplets of TALP-0Ca or TALP-EGTA just before sperm were added. This enabled cilia to remain beating during the experiment. To test the reversibility of the effect of EGTA, explants incubated with sperm in TALP-EGTA were transferred to TALP medium containing Ca2+ and a fresh supply of sperm. The sperm rapidly attached to the explants.
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Sperm motility was not affected by TALP-0Ca or TALP-EGTA for at least 2 h. After 2 h, motility in both media soon declined from 80-90% to about 40%. Because the binding inhibition and labeling experiments required less than two hours to complete, it is unlikely that a decrease in sperm motility affected the results.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1-4 linkage to N-acetylglucosamine, as contained in Lewis-a, is a more effective binding inhibitor than fucose alone. We therefore propose that there is a lectin on sperm that recognizes fucose in an 1-4 linkage to N-acetylglucosamine. The evidence indicates that the lectin is on the surface of sperm. The Lewis-a bound to live sperm with intact acrosomes. When we tried labeling fixed sperm that had acrosomes removed via freezing and thawing, there was no labeling. Also, in previous experiments, enzymatic removal of fucose from the epithelium reduced binding dramatically [21]. In the case of hamster sperm, there appears to be a lectin on the sperm that recognizes a carbohydrate moiety that contains sialic acid. Colloidal gold-labeled fetuin bound to the heads of fresh epididymal hamster sperm but did not bind to the apical regions of the heads of sperm incubated under capacitating conditions until they were hyperactivated [19]. Hyperactivated/capacitated hamster sperm did not bind to epithelium when perfused into the oviduct [13]. Taken together, these data indicate that there is a lectin on the surface of sperm that is lost or modified during capacitation to allow sperm to release from the epithelium.
Sperm would not label in the complete absence of Ca2+. EGTA was required to obtain these results; simply leaving Ca2+ out of the medium did not lower available Ca2+ sufficiently to reduce labeling. The apparent Ca2+-dependence of sperm binding to Lewis-a and to oviductal epithelium indicates that the lectin on sperm may be a C-type animal lectin. Sequence information is needed to confirm this classification. It may be noted that the binding densities obtained in these experiments were much higher than those obtained for the competitive inhibition experiments. This is because the explants were washed extensively for the binding inhibition experiments but could not be washed without harm in low Ca2+. We had previously adopted the triple washing procedure after determining that it reduced within-group variability. This was the technique of choice for comparing inhibitory activities of fucose in various linkages but not for testing Ca2+-dependence of binding.
Other forms of heterotypic binding between cells involve carbohydrate recognition. Examples are the selectins, which mediate leukocyte binding to endothelium [30], and glycolipid ligands on ciliated respiratory cells, which are recognized by mycoplasmas [31]. Selectins mediate temporary or intermittent binding between the two cell types, just as binding between sperm and epithelium is temporary. In each of the three species we have studied so far, a different sugar inhibited binding most effectively: sialic acid in hamsters [19], galactose in horses [20, 32], and fucose in cattle [21]. These species differences may not seem unusual when one considers that a single amino acid residue can determine the ligand specificity of a selectin [33, 34] and that closely related animal lectins have different carbohydrate specificities (reviewed in [35]). Carbohydrate recognition is also implicated in sperm/zona binding (reviewed in [36]) and sperm/Sertoli cell binding [37]. Lectins with different specificities could easily have arisen to regulate sperm attachment to these different surfaces.
In summary, we have shown that Lewis-a oligosaccharide is an effective inhibitor of sperm binding to oviductal epithelium and that Lewis-a binds to sperm. This binding is Ca2+-dependent. We propose that there is a C-type (Ca2+-dependent) lectin on the surface of bull sperm that binds Lewis-a with greater avidity than fucose, that is responsible for binding bull sperm to the oviductal epithelium, and that plays a role in the formation of the bovine oviductal reservoir of sperm.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: (607) 253-3541;sss7{at}cornell.edu
Accepted: February 18, 1998.
Received: December 29, 1997.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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