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a Center for Reproductive Biology Research and Department of Obstetrics and Gynecology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2633
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Most investigators agree that complementary molecules on the surface of opposite gametes initiate sperm-egg recognition. Although the sequence of events leading to successful fertilization varies among species, the mechanisms underlying the events of sperm capacitation, sperm-egg binding, and the induction of the AR share many similarities. These events are best understood in the mouse [7, 8], although there is some information in other species, including the pig, guinea pig, hamster, and human [9]. In the mouse, the AR is triggered after irreversible binding of capacitated spermatozoa to the terminal sugar residues of mZP3, a complex binding process that appears to reflect the interaction of multiple receptors on the sperm plasma membrane with multiple ligands (glycans) on mZP3 [2, 10]. The ability of the mZP3 to serve as the primary binding site and the inducer of the AR is thought to depend on glycan chains as well as on the protein portion of the molecule [11]. Small glycopeptides, generated by pronase digestion of mZP3, bind to the plasma membrane of capacitated spermatozoa but do not trigger the AR unless the bound glycopeptides are cross-linked on the sperm surface by anti-mZP3 IgG [12]. These results emphasize the role of the protein backbone of mZP3 in cross-linking and aggregating sperm receptors before the induction of the AR.
In this study, we examined the effect of several free and protein-conjugated sugars (neoglycoproteins) on the induction of the AR in an attempt to gain insight into the chemical nature of bioactive sugar residues. Our results provide evidence strongly supporting the concept that sperm-egg interaction leading to the acrosomal exocytosis is a complex event that most likely reflects multiple sperm-surface receptors and multiple sugar residues on the ZP.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mature C57BL6 male mice (8–10 wk old) were obtained from Harlan Sprague-Dawley (Indianapolis, IN). The animals were housed under 16L:8D conditions with free access to food and water for at least 3 days before experiments were begun. All animals were killed by CO2 asphyxiation. All procedures using animals were approved by the Institutional Animal Care Review Board.
Chemicals
Unless otherwise stated, all chemicals, including p-aminophenyl-N-acetyl-ß-D-glucosaminide-BSA (N-acetylglucosamine-BSA), p-aminophenyl-N-acetyl-ß-D-galactosaminide-BSA (N-acetylgalactosamine-BSA), and p-aminophenyl-
-D-mannopyranoside-BSA (mannose-BSA) were purchased from Sigma Chemical Co. (St. Louis, MO) and were of highest purity available. Biggers, Whitten, and Whittingham (BWW) medium [13] was from Irvine Scientific (Santa Ana, CA). p-Acetylcyanomethyl-thioglucopyranoside-BSA (glucose-BSA) and p-acetylcyanomethyl-thiogalactopyranoside-BSA (galactose-BSA) were purchased from EY Laboratories Inc. (San Mateo, CA). Formaldehyde (20% stock solution) was from Electron Microscopy Sciences (Fort Washington, PA). Uridine diphosphate (UDP)-6-[3H]galactose (60 Ci/mM) was from American Radiolabeled Chemicals Inc. (St. Louis, MO).
Preparation and Capacitation of the Cauda Spermatozoa
Each cauda epididymidis was excised and freed of the fat-pad, blood vessels, and connective tissue under a dissecting microscope. The tissue was transferred to a 3-ml Petri dish containing 1 ml of BWW medium and was cut in several places with iridectomy scissors to release the spermatozoa. The concentration of the spermatozoa dispersed in the medium over a period of 1–3 min was assessed using a Neubauer hemocytometric chamber. The sperm concentration was adjusted to 1 x 106/ml with BWW medium containing 0.3% BSA, and the sperm were subjected to capacitation by incubation at 37°C for 30 min under 5% CO2 in air.
For enzyme assays, spermatozoa from caudae epididymidum of several mice were pooled and adjusted to a concentration of 1 x 108 sperm/ml in PBS.
Assessment of Sperm Motility
Progressive motility was used as a monitor of sperm viability. Spermatozoa were examined by phase-contrast microscopy, and the percentage of spermatozoa with progressive motility was determined subjectively by scoring 200 individual spermatozoa in each sample. Only samples of capacitated spermatozoa displaying > 80% motility were used in subsequent experiments.
Assessment of Acrosomal Status
The status of the sperm acrosome was assessed by using the Coomassie Brilliant blue G-250 dye method [14] with slight modifications. In brief, the dye solution was prepared by suspending 15 mg of Coomassie Brilliant blue G-250 dye in 10 ml of 3.5% perchloric acid and then filtering it through Whatman (Clifton, NJ) #1 filter paper. Multitest slides with fixed spermatozoa (see below) were incubated with the dye for 2 min and then washed in distilled water (three washes of 8 min each). The slides were covered with a coverslip over mounting solution (PBS containing 10% glycerol) and sealed. The cells were observed under a Zeiss (Oberkochen, Germany) brightfield microscope. The photographic images were recorded using an optronics noncooled 3-chip camera and were stored as TIF files using Adobe Photoshop software (Adobe, Mountain View, CA). All slides were randomized and scored blindly. The spermatozoa were scored for blue stain overlying the acrosome on the basis of criteria described by Moller et al. [14]. Bright-blue staining of the midpiece occurred in all spermatozoa and served as an internal control. Spermatozoa with an intact acrosome exhibited blue stain over the sperm head; such staining was not detectable on the sperm that had undergone the AR (Fig. 1). For every experiment, 200 spermatozoa were scored in duplicate, and the percentage of spermatozoa that had undergone the AR was calculated.
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Sperm Treatment
Aliquots (20 µl) containing 1 x 105 capacitated spermatozoa were transferred to microcentrifuge tubes with or without additions. The tubes were incubated at 37°C for 30 min. After this incubation, spermatozoa were fixed by adding a stock formaldehyde solution to a final concentration of 2% and incubated for 30 min at room temperature. After fixation, the spermatozoa were pelleted by centrifugation at 500 x g for 10 min. The supernatant was removed by aspiration and discarded. The pelleted spermatozoa were washed once by gentle suspension in 20 µl of ammonium acetate (0.15 M, pH 9.0) and centrifugation as above. The resulting pellet was suspended in 20 µl of PBS. Aliquots (10 µl) were transferred to multitest slides, air-dried, and stained; and the acrosomal status was assessed as above.
In competition studies, capacitated spermatozoa were transferred to a microfuge tube containing neoglycoproteins without (control) or with (experimental) competing sugars and were incubated at 37°C for 30 min. Spermatozoa were then fixed and evaluated.
Galactosyltransferase (GT) Assay
GT activity was quantified by measuring the amount of [3H]galactose transferred to an exogenous substrate as described previously [15]. Spermatozoa and neoglycoproteins were added to the assay mixture (100 µl) in various concentrations as described in Results. After the indicated time of incubation, the reaction was stopped by adding 1% phosphotungstic acid, and the samples were washed with 10% trichloroacetic acid and ethanol-ether as detailed previously [15, 16].
Statistical Analysis
Data were processed on a Macintosh (Apple Computers, Cupertino, CA) Quadra 660AV using the SPSS 6.1 program (SPSS Inc., Chicago, IL). Comparisons of the average values for the control and experimental groups were carried out by an independent two-tailed t-test to determine statistically significant differences (p < 0.001). For competition studies, mean values were compared by one-way ANOVA followed by Bonferroni-Tukey multiple comparison tests. The results are presented as mean ± SE; the number (n) of independent experiments is indicated.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although in vitro capacitation of mouse spermatozoa has been extensively studied, the conditions for optimal capacitation vary with the medium and time of incubation. Our first set of experiments was designed to establish capacitation conditions for determining the effect of free and protein-linked sugar on the AR. In these studies, mouse cauda spermatozoa were suspended in BWW medium supplemented without or with 3 mg BSA/ml, and then incubated at 37°C under 5% CO2 in air. At various times, aliquots were withdrawn, and the acrosomal integrity was assessed as described in Materials and Methods. Data presented in Figure 2 show that the number of spermatozoa undergoing a spontaneous AR is very low when the BWW medium is not supplemented with BSA. In this medium, the motility of sperm decreased with increasing time of incubation (data not included). In contrast, a higher number of spermatozoa showed a spontaneous AR in the medium supplemented with 0.3% BSA (Fig. 2). Incubation of spermatozoa in the presence of BSA for 30 min (capacitation) followed by treatment with calcium ionophore (experimental) for 30 min resulted in a significantly greater number of spermatozoa undergoing the AR as compared to the untreated control. Therefore, we used this time period in all studies described in this report.
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Effect of Free Sugars on the Induction of the AR
Since the glycan moieties of mZP3 are the primary binding sites for mouse sperm receptors [1, 5, 17], we examined the effect of free sugars on the induction of the AR. Capacitated spermatozoa were incubated without (control) or with (experimental) a monosaccharide (mannose), two amino sugars (N-acetylgalactosamine and N-acetylglucosamine), or a polymannosylated sugar (mannan). Some of these sugar residues present on mZP3 are believed to be recognized by sperm receptors during sperm-egg interaction [5]. After incubation at 37°C for 30 min, spermatozoa from the control and experimental groups were fixed and stained, and the status of the acrosome was examined as described in Materials and Methods. Data presented in Figure 3 demonstrate that whereas the calcium ionophore A23187 was effective in inducing the AR, the free sugars, even at high concentration, had no effect on the integrity of the acrosome.
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Induction of the AR with Neoglycoproteins
The protein portion of mZP3 plays an important role in the induction of the AR [18]. Therefore, we examined the effects of sugar residue(s) covalently linked to a protein (neoglycoprotein) on this process. In these studies, the capacitated mouse spermatozoa were incubated without or with varying concentrations (1, 5, 10 µg/ml) of a neoglycoprotein for 30 min at 37°C. After this incubation, the sperm were fixed, stained, and examined for acrosomal integrity. The results presented in Figure 4 show that a significantly greater number of spermatozoa underwent the AR in the presence of calcium ionophore, and in the presence of DMSO and PBS combined with 3 of the neoglycoproteins (mannose-BSA, N-acetylglucosamine-BSA, and N-acetylgalactosamine-BSA) at a concentration of 5 µg/ml or higher. Neither galactose-BSA nor glucose-BSA induced the AR, even at concentrations of 10 µg/ml (Fig. 4).
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The effect of the neoglycoproteins appears to be dose-dependent since an addition of low concentration (1 µg neoglycoprotein/ml) failed to induce the AR (Fig. 4). In addition, a combination of the three effective neoglycoproteins (mannose-BSA, N-acetylglucosamine-BSA, and N-acetylgalactosamine-BSA) at different concentrations (2, 5, or 10 µg each/ml) showed no significant additional increase in the percentage of acrosome-reacted spermatozoa compared to a single neoglycoprotein (data not shown).
Competition Studies
Next we examined whether unconjugated monosaccharides or polymannose oligosaccharide (mannan) could prevent induction of the AR by competing with neoglycoproteins. The effect of mannose-BSA, N-acetylglucosamine-BSA, and N-acetylgalactosamine-BSA was examined in the absence or presence of a high concentration (40 mM) of free sugars, namely, mannose, N-acetylglucosamine, N-acetylgalactosamine, or mannan (1 mg/ml). These sugars did not compete with neoglycoproteins as evidenced by their failure to prevent the neoglycoprotein-induced AR (Fig. 5).
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GT Assays
The participation of mouse sperm-surface GT in fertilization by binding to N-acetylglucosamine residues on ZP is well documented [19, 20]. It has also been reported that aggregation of the enzyme molecules on the mouse sperm surface induces the AR [21]. We therefore used neoglycoproteins as potential substrates (acceptor-glycoproteins) for the enzyme assays, using UDP [3H]galactose as the donor. The amount of radiolabeled galactose transferred to the acceptor neoglycoprotein was quantified as described in Materials and Methods. In preliminary studies, we used three neoglycoproteins (mannose-BSA, N-acetylglucosamine-BSA, and N-acetylgalactosamine-BSA) as acceptor substrates and quantified the amount of radioactivity transferred. These studies indicated that only N-acetylglucosamine-BSA was a substrate for the sperm-surface GT (Fig. 6). We then examined the kinetics of [3H]galactose transfer to the N-acetylglucosamine-BSA. The amount of [3H]galactose transferred was dependent on the concentration of the neoglycoprotein (data not shown). The reaction was linear for over 24 h (Fig. 7), and it was proportional to the concentration of up to 1 x 106 spermatozoa/assay tube (Fig. 8), suggesting that the transfer of [3H]galactose is specific and follows typical enzymatic kinetics. Moreover, a known inhibitor of GT (-lactalbumin) showed a dose-dependent inhibition of the transfer of [3H]galactose when N-acetylglucosamine-BSA was used as an acceptor (Fig. 9).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To discover optimal capacitation conditions, we incubated spermatozoa in BWW medium in the absence or presence of BSA, a protein known to facilitate capacitation by altering fatty acids and/or cholesterol from the sperm plasma membrane [26]. In the absence of BSA, capacitation appears to be very low over an extended period of time. Even after 5 h of incubation, the number of spermatozoa undergoing a spontaneous AR was low (Fig. 2). Furthermore, when the spermatozoa were treated with calcium ionophore, a reagent known to induce the AR in capacitated spermatozoa [11, 27], a negligible number of spermatozoa underwent the AR (data not shown). In contrast, when the BSA was included, a significant number of spermatozoa were acrosome-reacted after 2 h of incubation. Data presented in Figure 2 allowed us to establish that the total time of capacitation and treatment combined should not exceed 1 h. For instance, when the spermatozoa were capacitated for 30 min and treated with ionophore for 30 min, nearly 3 times the number of spermatozoa were acrosome-reacted than when the spermatozoa were treated with dimethyl sulfoxide (DMSO) or PBS (Fig. 4, see controls).
The carbohydrate portion of several glycoproteins is known to mediate cell-cell adhesion, including sperm-oviduct adhesion [28], sperm-egg interaction [4], and implantation of the embryo [29, 30]. It is generally accepted that interaction of the opposite gametes is a carbohydrate-mediated species-specific event. Several sperm-surface proteins from various species have been proposed to function as receptor molecules on spermatozoa (for review see [5]). Extensive studies in the mouse have resulted in the identification of several receptors on the male gamete and several glycan (ligand) residues on homologous ZP. The terminal sugar residues suggested to be recognized by the capacitated spermatozoa include mannosyl [31], sialyl [32], glucosaminyl [20], and -galactosyl [33]. Although a terminal fucosyl residue has not been implicated in sperm binding, its presence appears to be obligatory for an oligosaccharide to bind spermatozoa with high affinity [34]. Data presented in a recent study strongly suggest that the initial molecular interaction between sperm and the ZP is a complex binding event that involves multiple sperm proteins and multivalent ZP3 [10].
It is important to mention that although monosaccharides, glycans, and small glycopeptides bind to spermatozoa, they do not induce the AR unless they are linked by a protein backbone [12, 18]. We confirmed these studies using millimolar concentrations of monosaccharides, amino sugars, and polymannose in our assay conditions. Indeed, the unconjugated sugar residues did not induce the AR. Therefore, synthetic glycoproteins were used to mimic the protein backbone of mZP3, a glycoprotein of 83 kDa [35]. Although the subunit molecular mass of BSA (66 kDa) is somewhat larger than the 44-kDa mass of mZP3 protein backbone, we felt that these neoglycoproteins could be reasonable substitutes for the mZP3. Data presented here demonstrate that three neoglycoproteins (mannose-BSA, N-acetylglucosamine-BSA, and N-acetylgalactosamine-BSA) induced the AR in a dose-dependent manner. However, two other neoglycoproteins (galactose-BSA and glucose-BSA) had no effect on the induction of the AR. These results allow us to conclude that specific sugar residues covalently linked to a protein backbone can induce the AR.
Two recent studies demonstrated that mannose-BSA [36] and N-acetylglucosamine-BSA [37] induced the AR when incubated with capacitated human spermatozoa. However, N-acetylgalactosamine-BSA had no effect on the number of human spermatozoa undergoing the AR [38]. These differences between mouse and human suggest that different sugar residues (ligands) are important in the interaction of the opposite gametes and induction of the AR in different species.
In the next set of studies, we determined that the sugars mannose, glucosamine, galactosamine, and mannan, even at high concentrations, did not prevent the neoglycoprotein-induced AR. This result was surprising since inclusion of some of the monosaccharides and oligosaccharides in an in vitro sperm-egg binding assay inhibits binding of the gametes in the mouse [32, 31], rat [39, 40], and human [41,42]. A likely explanation could be that the sugars do compete with the neoglycoproteins for the complementary binding site(s) on sperm plasma membrane overlying the sperm head. However, a small amount of the added neoglycoproteins that bound to the sperm receptors(s) in spite of the competition is sufficient to induce the AR.
That the sperm-surface GT, at least in part, is a receptor for the mouse ZP is suggested by several publications [43–46]. In two recent publications, Shur and his associates used a gene disruption approach to address the in vitro and in vivo role of the sperm GT [47, 48]. The investigators generated GT null mice through targeted mutation. Spermatozoa from these mice failed to undergo the ZP-induced AR in vitro [48], a result suggesting the importance of the sperm GT in binding to its complementary glucosaminyl residue(s) on the ZP before undergoing the AR. In this report, we have presented evidence demonstrating that N-acetylglucosamine-BSA (but not mannose-BSA or N-acetylgalactosamine-BSA) is an exogenous substrate for the GT-mediated transfer of [3H]galactosyl residues. This result is consistent with the proposed role for the sperm-surface GT and suggests that the interaction of GT with the complementary sugar (protein-conjugated glucosaminyl) residue(s) may be important for induction of the AR. Interestingly, terminal glucosaminyl residues have been reported on mZP2 and mZP3 [49] as well as on porcine ZP3 [50–52]. The fact that two other neoglycoproteins that induce the AR failed to accept the [3H]galactosyl residues suggests that the induction of the AR by mannose-BSA and N-acetylgalactosamine-BSA is by a mechanism other than binding to the sperm-surface GT. It is interesting that despite the impaired response of the spermatozoa to the ZP-induced AR in vitro, the GT null mice were reported to be fully fertile [47]. This result, in conjunction with other evidence, strongly suggests the occurrence of multiple receptors on mouse spermatozoa.
In addition to GT, mouse spermatozoa possess -D-mannosidase [31], an exoglycohydrolase believed to have a receptor-like role by binding to its complementary substrate (high mannose/hybrid-type glycan) present on mZP2 and mZP3. The enzyme is a glycohydrolase that binds to its substrate, forming an enzyme:substrate complex [5, 53] before hydrolytic cleavage of the mannosyl residues. Thus, it is reasonable to suggest that binding of mannose-BSA to sperm-surface mannosidase is the mechanism by which it induces the AR. The evidence for the presence of high mannose/hybrid-type oligosaccharides on mZP2 and mZP3 [54] is consistent with this suggestion.
In conclusion, data presented in this report demonstrate that several neoglycoproteins when incubated with capacitated spermatozoa induce the AR. The fact that several sugar residues when conjugated to a protein backbone are effective suggests the occurrence of multiple sugar binding proteins (receptors) on the sperm plasma membrane. Understanding the mechanism(s) underlying sperm-neoglycoprotein interaction and induction of the AR awaits new approaches, including the use of the AR blockers. Combined, the studies will further our understanding of the molecular mechanisms underlying the AR.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 615 343 7797.
Accepted: August 17, 1998.
Received: June 26, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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