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a Center for Reproductive Biology Research and Departments of Obstetrics and Gynecology and Cell Biology, 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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-D-mannosidase, two carbohydrate-recognizing enzymes thought to be sperm surface receptors, suggests that the reagents block the AR by a mechanism other than binding to the active site of the enzymes.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previously, we demonstrated that specific sugar residues, when covalently linked to a protein backbone, mimicked mouse ZP3 glycoprotein (mZP3) and induced the AR in capacitated mouse spermatozoa [8]. A significantly greater number of spermatozoa were found to undergo the AR in the presence of mannose-BSA, N-acetylglucosamine-BSA, and N-acetylgalactosamine-BSA than in their absence. Glucose-BSA or galactose-BSA had no effect on the induction of the AR. These data were consistent with a recent report strongly suggesting that the initial binding of sperm (receptor) and ZP (ligand) is a complex binding event that reflects interaction of multiple sperm proteins with multiple sugar residues on the ZP3 [9].
The interaction of sperm surface receptor(s) with the terminal sugar residue(s) of the bioactive glycan on ZP3 is thought to start a cascade of signal events that trigger the AR [1, 6, 7]. Evidence thus far available strongly suggests that rises in sperm cytoplasmic Ca2+ and pH, regulated in part by membrane channel proteins, are important changes preceding the AR [1, 7]. These channel proteins are believed to be involved in the transport of ions across sperm plasma membrane and outer acrosomal membrane that are important in regulating cytoplasmic levels of Ca2+ ions and pH (for review see [6]). In this study, we have examined the neoglycoprotein-induced AR in the absence and presence of reagents known to prevent the AR by blocking specific Ca2+ channels. Our data demonstrate that the neoglycoprotein-induced AR can be prevented by selectively blocking the L-type Ca2+ channel(s).
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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
Neoglycoproteins p-aminophenyl-N-acetyl-ß-D-glucosaminide-BSA (N-acetylglucosamine-BSA), p-aminophenyl-N-acetyl-ß-D-galactosaminide-BSA (N-acetylgalactosamine-BSA), p-aminophenyl--D-mannopyranoside-BSA (mannose-BSA), and blocking reagents, including amiloride hydrochloride hydrate, diltiazem hydrochloride, genistein, and okadaic acid, were purchased from Sigma Chemical Co. (St. Louis, MO). Biggers, Whitten, and Whittingham (BWW) medium was from Irvine Scientific (Santa Ana, CA). Nifedipine, nitrendipine, and pertussis toxin were purchased from Calbiochem-Novabiochem Corp. (San Diego, CA). N-(2-[Methylamino]ethyl)-5-isoquinolinesulfonamide (H 8), 3-quinuclidinyl benzilate, and tyrphostin A 48 were purchased from ICN Biomedicals (Aurora, OH). 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). Mannose-labeled oligosaccharide ([3H]Man9GlcNAc) was prepared as described previously [10]. All other chemicals were obtained commercially and were of the highest purity available.
Preparation and Use of the AR-Blocker Solutions
A 100-fold-concentrated stock solution was prepared by dissolving the reagents either in distilled water (diltiazem, pertussis toxin, verapamil) or in dimethyl sulfoxide. Aliquots of the stock solution were mixed with the capacitated sperm solution to achieve the desired concentration.
Sperm Treatment
Mouse cauda epididymal spermatozoa, 1 x 106 sperm/ml in BWW medium containing 0.3% BSA, were incubated at 37°C for 25 min under 5% CO2 in air. After this incubation, aliquots (198 µl) were withdrawn and mixed with 2 µl of the medium without (control) or with the blocking reagent (experimental), and the mixture was incubated for an additional 5 min at 37°C as above. Aliquots (20 µl) from the control and experimental spermatozoa were transferred to a microfuge tube that either contained or did not contain a neoglycoprotein, and the mixture was incubated at 37°C for 30 min. Spermatozoa were then fixed by adding a stock formaldehyde solution to a final concentration of 2% and incubation for 30 min at room temperature. The fixed spermatozoa were processed as described previously [8].
Assessment of Acrosomal Status
The status of the sperm acrosome was assessed as described previously [8]. In brief, multitest slides with fixed spermatozoa were incubated with 0.15% Coomassie brilliant blue G-250 solution for 2 min followed by washing in distilled water. The slides were covered with a coverslip over mounting solution (PBS containing 10% glycerol) and sealed. The cells were observed under a Zeiss (Carl Zeiss, Thornwood, NY) brightfield microscope. All slides were randomized and scored blindly. For every experimental parameter evaluated, 200 spermatozoa were scored in duplicate, and the percentage of spermatozoa that had undergone the AR was calculated. The Coomassie blue stain procedure has been shown to correlate well with the chlortetracycline assay [11].
Assessment of Sperm Motility
Motility was used as a monitor of sperm viability. Spermatozoa were examined by phase-contrast microscopy, and the percentage of motile sperm was determined by scoring 200 spermatozoa in each sample. Only samples of capacitated spermatozoa displaying > 80% motility were used in subsequent experiments.
Enzyme Assays
Galactosyltransferase (GT) activity was quantified by measuring the amount of [3H]galactose transferred to an exogenous substrate as described previously [8, 12]. Briefly, cauda spermatozoa in the assay mixture without or with the Ca2+ channel blocker were incubated for 15 min at 4°C. After this incubation, the reaction was started by adding donor sugar (UDP[3H]Gal) and acceptor protein (N-acetylglucosamine-BSA). After the indicated time of incubation at 37°C, the reaction was stopped by adding 1% phosphotungstic acid, and the samples were washed with 10% trichloroacetic acid and ethanol-ether before quantifying the amount of [3H]galactose transferred to the exogenous substrate as described previously [8, 12].
[3H]Man9-mannosidase activity was assayed by our published procedure [10, 13]. Briefly, the incubation mixture (100 µl) contained ~4000 cpm of [3H]Man9GlcNAc, 50 mM sodium cacodylate buffer, pH 6.4, 0.2% Triton X-100, and spermatozoa (1 x 106) in the absence or presence of the Ca2+ channel blocker. After incubation for 3 h at 37°C, the reaction was stopped by heat treatment. The released [3H]mannose was separated from the oligosaccharide by gel filtration on a column of Bio-Gel P-2 and quantitated by scintillation counting [10]. One unit of the activity is the amount of enzyme that catalyzes the release of 1000 cpm of free [3H]mannose per hour at 37°C.
Statistical Analysis
The data were processed on a Macintosh Quadra 660AV (Cupertino, CA) 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 a paired two-tailed t-test to determine statistically significant differences (p < 0.05). 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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Previously, we reported that a significantly greater number of spermatozoa underwent the AR in the presence of a calcium ionophore (A23187) or several neoglycoproteins (mannose-BSA, N-acetylglucosamine-BSA, or N-acetylgalactosamine-BSA) than in their absence, a result analogous to the ZP (ZP3)-induced AR [6–8]. Evidence accumulated over the years suggests that the ZP3-induced AR is regulated by several ion channel proteins present on the sperm plasma membrane and on the outer acrosomal membrane (for review see [6]). The ion channels are believed to regulate the ion specificity and the direction of their movement across the sperm membranes. These are important contributing factors that elevate intracellular Ca2+ and pH preceding the ZP3-induced AR. Our first set of experiments was directed toward obtaining insight into the involvement of ion channels or sperm surface receptors during neoglycoprotein-induced AR. In these studies, the number of capacitated mouse spermatozoa undergoing the neoglycoprotein-induced AR was compared in the absence and presence of reagents known to block the ZP3-induced AR. The results from these studies, presented in Table 1, show that with the exception of verapamil [14, 15] and diltiazem [15], these reagents failed to prevent induction of the AR by mannose-BSA, N-acetylglucosamine-BSA, and N-acetylgalactosamine-BSA. These preliminary studies provided evidence suggesting that two out of three reagents that prevent ZP3-induced AR by blocking the L-type Ca2+ channels were effective in preventing the induction of the AR by the three neoglycoproteins.
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Effect of Verapamil and Diltiazem on Neoglycoprotein-Induced AR
In these studies, we examined the effect of various concentrations of verapamil and diltiazem in blocking neoglycoprotein-induced AR. Mouse spermatozoa were capacitated and then incubated with or without various concentrations of verapamil (Fig. 1) or diltiazem (Fig. 2) as described in the figure legends, followed by incubation without (control) or with (experimental) a neoglycoprotein. After incubation at 37°C for 30 min, spermatozoa were fixed and stained, and the status of the acrosome was examined as described in Materials and Methods. Data presented in Figures 1 and 2 demonstrate that whereas low concentrations (10 nM) of verapamil or (10 nM and 50 nM) of diltiazem had no significant effect in preventing the AR, the high concentrations (50 nM and 100 nM) of verapamil or (100 nM) of diltiazem were quite effective in preventing the induction of the AR by the three neoglycoproteins. These data demonstrate that both reagents prevent the neoglycoprotein-induced AR in a dose-dependent manner.
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Effect of L-type Ca2+ Channel Blockers on Mouse Sperm Surface GT or -D-Mannosidase Activity
Next, we examined whether verapamil and diltiazem could interfere with the active sites(s) of mouse sperm surface GT and -D-mannosidase, two sugar-binding enzymes believed to be recognized by N-acetylglucosamine-BSA and mannose-BSA, respectively [8]. First, we quantified the amount of radioactive [3H]galactose transferred to N-acetylglucosamine-BSA in the absence and presence of the L-type Ca2+ channel blockers by the procedure described in Materials and Methods. These reagents did not inhibit GT activity as was evident by their failure to prevent radiolabeled galactose transfer to the acceptor neoglycoprotein (Fig. 3).
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We then examined whether the channel blockers inhibit mouse sperm surface -D-mannosidase activity perhaps by binding to the active site of the enzyme. In these studies, we assayed the release of [3H]mannose from [3H]man9GlcNAc (Man9-mannosidase activity) in the absence and presence of two concentrations of verapamil and diltiazem as described in Materials and Methods. Results from these studies presented in Figure 4 show that the two reagents failed to block the active site of the sperm surface mannosidase as evident by their noninhibitory effect on the enzyme activity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In a previous report, we demonstrated that a significantly greater number of capacitated mouse spermatozoa underwent the AR in the presence of several neoglycoproteins, synthetic glycoproteins containing a known monosaccharide covalently linked to BSA, than in their absence [8]. Although the mechanism underlying the neoglycoprotein-induced acrosomal exocytosis is not yet known, it seems likely that binding of the sugar moiety of the neoglycoprotein to the complementary sugar-recognizing molecules (receptors) on the plasma membrane overlying the acrosome causes aggregation of the receptors and triggers the acrosomal exocytosis as has been suggested for sperm surface GT [17]. The studies reported here were undertaken to examine the involvement and functional significance of voltage-sensitive ion channels that are responsible for regulating sperm cytoplasmic levels of Ca2+. The entry of Ca2+ via these channels is known to mediate a variety of cellular responses including acrosomal exocytosis [6, 8, 9].
In preliminary studies, we incubated capacitated mouse spermatozoa and the neoglycoprotein in the absence and presence of various concentrations of reagents (channel blockers and inhibitors of enzymes) known to prevent the ZP3-induced AR. Data from these studies, presented in Table 1, provided evidence suggesting that with the exception of two L-type channel blockers (see below), the reagents did not prevent the neoglycoprotein-induced AR.
Next, we examined the induction of the AR by three neoglycoproteins (mannose-BSA, N-acetylglucosamine-BSA, and N-galactosamine-BSA) in the absence or presence of various concentrations of verapamil and diltiazem, reagents known to prevent ZP3-induced AR by blocking L-type Ca2+ channels. Indeed, the reagents blocked the induction of the AR by three neoglycoproteins in a concentration-dependent manner (Figs. 1 and 2). These data provided the first evidence suggesting that the neoglycoprotein-induced AR may be regulated by L-type Ca2+ channel protein(s).
Although verapamil and diltiazem blocked the induction of the AR, nitrendipine, another L-type Ca2+ channel blocker [14, 15], showed no effect on the neoglycoprotein-induced AR (Table 1). A plausible explanation could be the use of different medium and capacitation conditions during these studies. Alternatively, the differences may reflect contribution by multiple factors, including multiple sugar residues on ZP3 as well as differences in the polypeptide portion of the ZP3 and neoglycoproteins. Since many details of sperm capacitation, sperm-zona/neoglycoprotein interaction, and sperm activation are still lacking, additional studies are needed to address the differences between ZP and neoglycoprotein agonists. Regardless of these differences, data reported in this manuscript provide additional information on the functional significance of the protein-conjugated sugar residues during mammalian fertilization. The observed inhibition of the neoglycoprotein-induced AR by verapamil and diltiazem, two known antagonists of the L-type Ca2+ channel(s), allows us to suggest that these channel(s) regulate Ca2+ influx that triggers the AR.
It is important to mention that G proteins, particularly G1-like proteins, have been implicated in ZP-induced signaling pathway [18–20]. Pertussis toxin, a bacterial toxin that inactivates the G1-like proteins, blocks triggering of the AR by solubilized mouse ZP [19, 20]. The Ca2+ influx that triggers the AR is believed to be mediated by ion channels regulated by G1-proteins. The toxin blocks mZP3-induced Ca2+ influx and sperm cytoplasmic pH changes, leading to the suggestion that G1-like proteins may regulate Ca2+ influx and internal pH [19, 20]. In our experimental conditions, the pertussis toxin had no effect on the number of spermatozoa undergoing the AR in the presence of three neoglycoproteins (Table 1). These data seem to suggest that the neoglycoprotein-induced AR, unlike the ZP3-induced AR, may not involve the G1-like proteins.
Several lines of evidence discussed previously [8] suggest that the neoglycoprotein-induced AR is analogous to the ZP3-induced AR. The main feature of this similarity is the involvement of carbohydrate moiety(ies) as well as the protein portion of these molecules [21]. Thus, it is reasonable to suggest that the carbohydrate-mediated interaction of the sperm surface receptor(s) and the terminal sugar moiety of the ZP or neoglycoprotein precedes the peptide-peptide interaction leading to the induction of the AR. This comparison may, however, be overly simplified, since many details of sperm-zona (ZP3) and sperm-neoglycoprotein interaction and the induction of the AR are not yet known.
Our previous studies suggested that the N-acetylglucosaminyl residues covalently bound to BSA are recognized by sperm surface GT [8]. We confirmed this finding when we used lactosamine (Gal
GlcNAc)-BSA as an acceptor for GT-mediated transfer of [3H]galactose and inducer of the AR. The protein-conjugated disaccharide neither induced the AR in capacitated spermatozoa nor was a substrate for the sperm surface GT (data not included). This result is consistent with the proposed role for terminal N-acetylglucosaminyl residues on the mZP3 [22] and GT on mouse spermatozoa [23, 24] in sperm-egg interaction and the induction of the AR. Also, the potential role of mouse sperm surface -D-mannosidase in recognizing mannose-BSA preceding the AR has been discussed previously [8, 25]. Hence, we examined the effect of diltiazem and verapamil on mouse sperm surface GT and -D-mannosidase activities. The fact that the L-type Ca2+ channel blockers, even at a concentration of 50-fold higher than used for blocking the neoglycoprotein-induced AR (Figs. 1 and 2), had little or no effect on sperm GT (Fig. 3) and -D-mannosidase activities (Fig. 4) provided evidence suggesting that the prevention of the acrosomal exocytosis by these reagents is due to a factor(s) other than blocking of the initial binding of sperm receptor(s) to the sugar moiety of the neoglycoprotein. It is tempting to suggest that the reagents prevent the neoglycoprotein-induced (and perhaps ZP-induced) AR by altering the primary structure of the polypeptide backbone, thereby affecting the peptide-peptide interactions. It is also possible that the reagents inactivate the L-type ion channels responsible for the Ca2+ influx and thereby prevent the induction of the AR.
In summary, data presented in this report suggest that unlike ZP3-induced signal transduction pathway, which likely involves multiple mechanisms to regulate Ca2+ influx that triggers the AR, the neoglycoprotein-induced acrosomal exocytosis is regulated only by L-type Ca2+ channels. It would be interesting to identify and characterize various components of the sperm surface proteins that interact with neoglycoproteins. This information will enable us to devise new strategies to alter the sequence of events preceding the AR and regulate the penetration of the oocyte and the fertilization process.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 615 343 7797; daulat.tulsiani{at}mcmail.vanderbilt.edu
3 Current address: Zentrum für Dermatologie and Andrologie, Justus-Liebig-Universität, Gaffkystr. 14, 35385 Giessen, Germany.
Accepted: April 13, 1999.
Received: January 11, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-D-Mannosidase of rat liver Golgi membranes: mannosidase II is the GlcNAcMan5-cleaving enzyme in glycoprotein biosynthesis and mannosidases IA and IB are the enzymes converting Man9 precursors to Man5 intermediates. J Biol Chem 1982; 257:3660–3668.1E expression in spermatogenic cells, and their possible relevance to the sperm acrosome reaction. FEBS Lett 1996; 388:150–154.[CrossRef][Medline]This article has been cited by other articles:
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  W. F. Hanna, C. L. Kerr, J. H. Shaper, and W. W. Wright Lewis X-Containing Neoglycoproteins Mimic the Intrinsic Ability of Zona Pellucida Glycoprotein ZP3 to Induce the Acrosome Reaction in Capacitated Mouse Sperm Biol Reprod, September 1, 2004; 71(3): 778 - 789. [Abstract] [Full Text] [PDF]
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P.C.N. Chiu, H.Y. Tsang, R. Koistinen, H. Koistinen, M. Seppala, K.F. Lee, and W.S.B. Yeung The Contribution of D-Mannose, L-Fucose, N-Acetylglucosamine, and Selectin Residues on the Binding of Glycodelin Isoforms to Human Spermatozoa Biol Reprod, June 1, 2004; 70(6): 1710 - 1719. [Abstract] [Full Text] [PDF]
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 H.-T. Zeng and D. R. P. Tulsiani Calmodulin antagonists differentially affect capacitation-associated protein tyrosine phosphorylation of mouse sperm components J. Cell Sci., May 15, 2003; 116(10): 1981 - 1989. [Abstract] [Full Text] [PDF]
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 S. Jagannathan, E. L. Punt, Y. Gu, C. Arnoult, D. Sakkas, C. L. R. Barratt, and S. J. Publicover Identification and Localization of T-type Voltage-operated Calcium Channel Subunits in Human Male Germ Cells. EXPRESSION OF MULTIPLE ISOFORMS J. Biol. Chem., March 1, 2002; 277(10): 8449 - 8456. [Abstract] [Full Text] [PDF]
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 K. J. Mengerink and V. D. Vacquier Glycobiology of sperm-egg interactions in deuterostomes Glycobiology, April 1, 2001; 11(4): 37R - 43R. [Abstract] [Full Text] [PDF]
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