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a Gamete Biology Laboratory, Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, Pennsylvania 19348-0692
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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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The process of sperm capacitation has been described as a series of largely uncharacterized cellular and molecular events that occur within the female reproductive tract and are required for the acrosome reaction and fertilization to progress [1]. Capacitation includes sperm plasma membrane events that lead to the increased cellular calcium influx and fusogenicity of the plasma and outer acrosomal membranes overlying the sperm head, which are prerequisites for the acrosome reaction. Recent studies have suggested that only capacitated sperm are capable of traversing the cumulus extracellular matrix and that antibodies to sperm PH-20 and inhibitors of hyaluronidase block in vitro cumulus penetration of macaque sperm [14, 15]. In addition, hyaluronan (HA), the substrate for hyaluronidase, has been reported to enhance acrosomal exocytosis in capacitated macaque sperm [16] but not in uncapacitated sperm.
In equine sperm, capacitation-related events have not been characterized, and endpoints of capacitation (such as in vitro fertilization) that have been used to evaluate sperm function in other species have not been successfully developed for horses. The goal of our study was to determine whether equine sperm have a plasma membrane-associated hyaluronidase and whether cellular distribution of hyaluronidase is affected by capacitation-associated events.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Hyaluronic acid was obtained from ICN Pharmaceuticals (Costa Mesa, CA). The IgG fraction was purified from rabbit polyclonal antiserum using an Immunopure kit obtained from Pierce Chemical Company (Rockford, IL). Fluorescein- and rhodamine-conjugated Pisum sativum agglutinin (PSA) were obtained from Vector Laboratories (Burlingame, CA). All other chemicals were obtained from Sigma Chemical Company (St. Louis, MO).
Animals and Semen
Semen was obtained from 5 fertile stallions individually housed at the Georgia and Philip Hofmann Research Center for Animal Reproduction at New Bolton Center at the University of Pennsylvania (Kennett Square, PA). Stallions were maintained on a diet of mixed grass hay and supplemental grain, with fresh water ad libitum and daily exercise according to Institutional Animal Care and Use Committee protocols at the University of Pennsylvania. Semen was obtained using an artificial vagina and ovariectomized mount mare. Immediately after collection, the ejaculate was filtered through cotton mesh filters to separate the gel fraction from the sperm-rich fraction. For capacitation studies, gel-free semen was diluted into capacitation medium (Tyrode's albumin lactate pyruvate, TALP) [17] prewarmed to 39°C and transported to the laboratory for processing within 5 min of collection. For biochemical studies of hyaluronidase, a protease inhibitor cocktail [5] was added to raw semen, which was then immediately transported to the laboratory in 50-ml conical centrifuge tubes on ice.
Sperm Processing for Hyaluronidase Activity and Antibody Production
Upon arrival in the laboratory, the sperm plasma membrane fraction was isolated by a modification of a standard nitrogen cavitation method reported for boar sperm [18] and stallion sperm [19]. Briefly, raw semen (20 ml) was layered over 20 ml 1.0 M sucrose and centrifuged at 1200 x g for 25 min at ambient temperature. The sperm pellet was resuspended in PBS containing Hepes (Hepes-PBS; 5 mM Hepes, 2.7 mM potassium chloride, 1.5 mM potassium diphosphate, 0.137 M sodium chloride, 8 mM disodium phosphate, pH 7.4) including protease inhibitors [5] and adjusted to give a final concentration of 108 sperm/ml (30–40-ml total volume). The sperm suspension was placed into a cell disruption bomb (Parr Instrument Company, Moline, IL) to which nitrogen was added to 650 psi. The chamber was sealed and incubated on ice for 10 min. The solution was extruded slowly into 50-ml conical centrifuge tubes on ice and was agitated gently for 5 min. The extruded cavitate was evaluated for acrosomal status using fluorescence microscopy (described below), and with phase-contrast microscopy for fractionated sperm heads and tails. The suspension was then centrifuged twice at 6000 x g for 10 min at 4°C to remove remaining sperm cells. The supernatant was centrifuged at 35 000 x g for 15 min and the pellet discarded. The supernatant was centrifuged again at 100 000 x g for 2 h at 4°C. The resulting pellet was resuspended in 2 ml Hepes-PBS (pH 5.0) and solubilized in 5-strength Laemmli SDS sample buffer [20] containing protease inhibitors and then frozen (-70°C) until assayed for enzyme activity.
Additional freshly ejaculated and washed sperm were extracted with Triton X-100 detergent for comparison of whole sperm hyaluronidase activity. Ejaculates (108 total sperm) were washed twice in Dulbecco's PBS containing protease inhibitors and subsequently incubated with 2% Triton X-100 for 30 min at ambient temperature. Each suspension was then centrifuged at 10 000 x g for 10 min, the pellet discarded, and the supernatant solubilized in 5-strength Laemmli SDS sample buffer [20]. The solubilized samples were stored frozen at -70°C until assayed for hyaluronidase activity.
Gel Substrate Electrophoresis for Hyaluronidase Activity
Hyaluronidase activity was determined from the isolated plasma membrane fraction and detergent-extracted whole sperm using a gel substrate electrophoresis technique that incorporates hyaluronic acid (HA; ICN) into the 10% SDS-polyacrylamide gels, as described previously [5, 21]. Briefly, 10% SDS-polyacrylamide gels were prepared that contained 17 µg/ml HA in the separating gel prior to polymerization. The stacking gel did not contain HA. After electrophoresis of sperm plasma membrane extracts, gels were incubated for 2 h at 25°C in 0.025 M Tris-buffered saline containing 3% Triton X-100 to remove SDS. Gels were then incubated in PBS (pH 7.4) or glycine-HCl (0.2 M, pH 3.0) buffer at 37°C for 16 h. To visualize regions of digestion of HA in the gels, they were stained with 0.5% Alcian blue in 3% acetic acid for 2 h, destained in 7% acetic acid, and counterstained with Coomassie blue R-250. The molecular masses of polypeptides within a given lane were determined by optical scanning of gels and subsequent analysis of scans using NIH Image gel Scanning Macro (Bethesda, MD) on a Power Macintosh (Cupertino, CA) computer. Prestained molecular weight standards were used (New England Biolabs, Beverly, MA) for visualization in the HA-containing gel.
Anti-Equine Hyaluronidase Antibody Production and Purification
Polyclonal antiserum against the 54-kDa protein band of equine sperm hyaluronidase was raised in New Zealand White rabbits. The 54-kDa hyaluronidase protein was excised from two 8% SDS-HA substrate polyacrylamide gels after each of two sequential electrophoretic runs at 150 V for 2 h at 4°C. Antibodies were produced in adult male rabbits by administration of four s.c. injections at 2-wk intervals of approximately 50–100 µg of purified protein in incomplete Freund's adjuvant. On Day 56 after initial inoculation, the rabbits were anesthetized using ketamine hydrochloride (10 mg/lb, i.m.) in combination with xylazine (2 mg/lb) and exsanguinated by cardiac puncture for harvest of antiserum. Purified IgG was obtained using the Immunopure IgG purification kit (Pierce) and was used as indicated below for cellular fluorescence labeling and Western blotting.
Western Blots
For SDS-PAGE analyses, plasma membrane fraction and detergent-extracted whole sperm samples that were either nonreduced or reduced (addition of 5 mM ß-mercaptoethanol with heating to 95°C for 5 min) were subjected to SDS-PAGE using 10% polyacrylamide gels as previously described [5]. Gels were blotted to nitrocellulose membranes, incubated in blocking solution (9% nonfat dry milk, 0.1% Tween 20 in Tris-buffered saline) for 
2 h, and probed with purified IgG fraction (1:2000, 0.25 µg/ml) from polyclonal antisera raised in rabbits (UP849) against the isolated equine plasma membrane fraction or control rabbit serum. Goat anti-rabbit IgG conjugated to horseradish peroxidase (Sigma) was the secondary antibody (1:5000). Protein bands were detected using a chemiluminescence procedure (Renaissance; NEN Life Science Products, Boston, MA) [22]. Molecular weight standards were prestained broad-range molecular weight markers (New England Biolabs, Beverly, MA). The molecular masses of polypeptides within a given lane were determined by optical scanning of gels as described for gel substrate electrophoresis.
Determination of GPI Linkage of Sperm Hyaluronidase
Raw semen samples were washed twice by centrifugation and resuspension using DPBS at ambient temperature (400 x g, 2 min). The samples were incubated for 30 min at ambient temperature at 100 x 106 sperm/ml in DPBS. After incubation, 1 unit of phosphatidylinositol-specific phospholipase C (PI-PLC) was added to the 1-ml sperm suspensions, and they were incubated at 37°C for an additional 60 min. Control tubes contained vehicle only. After incubation, protease inhibitor cocktail was added and the samples were centrifuged at 400 x g for 5 min. The supernatant was centrifuged at 100 000 x g at 4°C for 60 min, aspirated, and stored frozen at -70°C until assayed for hyaluronidase activity and Western blotting, as described above.
Sperm Processing for Capacitation, Acrosome Reactions, and Immunofluorescence
Within 2 min of ejaculation, semen samples were filtered and diluted 1:1 (v:v) in TALP medium and transported to the laboratory. Upon arrival at the laboratory, semen samples were centrifuged at 50 x g for 5 min to sediment clumped sperm and debris. The supernatant (1 ml) was layered over a Percoll-TALP gradient consisting of 1 ml each of two solutions (42% upper layer and 84% lower layer). The samples were processed as previously described for equine sperm capacitation using TALP medium [17] and incubated for up to 5 h at 39°C in a humidified 95% air and 5% CO2 atmosphere [17]. Experiments were also performed in which HA (100 µg/ml) or UP849 polyclonal IgG (1 µg/ml) was included during the capacitation period to enhance or inhibit, respectively, hyaluronidase redistribution over the sperm head. Controls used in these studies received 10 µl DPBS vehicle only. Acrosome reactions were induced in subsamples using 3.0 µM progesterone, as previously described [17], to confirm that capacitation had been initiated. After in vitro capacitation, the sperm suspensions were fixed for 10 min in 2% paraformaldehyde and washed by centrifugation and resuspension using DPBS in siliconized microcentrifuge tubes at 400 x g for 2 min. Remaining steps were carried out at ambient temperature. The samples were incubated for 30 min in blocking solution (DPBS plus 50 mg/ml BSA). Purified IgG (UP849) or preimmune IgG was added to sperm samples at a concentration of 1.0 µg/ml, which had in preliminary experiments been determined to allow adequate immunostaining of equine sperm cells. The samples were incubated for 60 min, washed twice, and resuspended in 1 ml DPBS. Fluoresceinated goat anti-rabbit IgG was added as second antibody to the samples (1:50 dilution in DPBS), and they were incubated an additional 60 min. After secondary antibody incubation, the cell suspensions were washed twice using DPBS, and a fluorescence enhancer solution was added (Vectashield; Vector) to preserve cell fluorescence.
Acrosomal status was assessed to confirm capacitation in subsamples of the sperm suspensions using fluoresceinated PSA, as described previously [17]. Sperm viability was assessed using the nuclear exclusion dye Hoechst 33258 at a concentration of 50 µg/ml, added to sperm suspensions for 5 min immediately prior to fixation with 2% paraformaldehyde. In double-fluorescence labeling experiments (hyaluronidase and acrosomal status determination), sperm samples were fixed using 2% formaldehyde and washed using centrifugation and resuspension with DPBS. The samples were then labeled using fluoresceinated goat anti-rabbit IgG as second antibody, as described above. After incubation with the second antibody, the cells were washed twice in DPBS, resuspended in DPBS containing 100 µg/ml rhodamine-PSA (Vector), and incubated for 10 min. The cells were then washed two times and resuspended in 100 µl DPBS containing 1 drop of Vectashield.
Sperm cell samples were placed on glass microscope slides with coverslips, and indirect immunofluorescence was visualized using oil immersion at x1000 magnification with a Leitz (Leitz Wetzlar GBH, Wetzlar, Germany) Dialux fluorescence microscope using a fluorescein filter with excitation at wavelength 480/30 and emission at wavelength 535/40. In double-fluorescence studies, individual sperm were imaged twice, using first a fluorescein filter with excitation at wavelength 480/30 and emission at wavelength 535/40 and then a rhodamine fluorescence filter with excitation at wavelength 545/30 and emission at wavelength 610/75. Images were captured and analyzed using a Sony (Park Ridge, NJ) DKC-5000 digital CCD camera, ImagePro Plus software (Media Cybernetics, Silver Spring, MD) software, and a Windows 95-based personal computer.
Statistical Analysis
Percentages of fluorescence sperm head labeling patterns were compared for durations of in vitro capacitation. Data were arcsin transformed and analyzed using ANOVA techniques with a randomized block design for in vitro treatments and blocking for stallion effects and Stata statistical software (Stata Corp., College Station, TX). Post hoc comparisons were made using Tukey's method when appropriate.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Western blots revealed 3 major bands of immunoreactivity in detergent-extracted whole sperm, as well as in the isolated plasma membrane fractions, that corresponded to the hyaluronidase activity observed in HA substrate gels (Fig. 1, lanes 1 and 2). Hyaluronidase activity was also detectable in three major protein bands with approximate molecular masses of 54, 59, and 83 kDa (Fig. 1, lanes 4 and 5) in both detergent-extracted whole sperm and plasma membrane fractions. The lower two bands appeared to be enzyme doublets as detected by gel substrate electrophoresis. Enzyme activity of all the active hyaluronidases (54, 59, and 83 kDa) was greater when they were incubated in HA substrate gels at pH 7.4 than at pH 3.0 (Fig. 2). Incubation of the HA-containing gels overnight in the presence of polyclonal antibodies resulted in no detectable enzyme activity (not shown).
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Under reducing conditions, both detergent-extracted whole sperm and plasma membrane extracts displayed a single protein band with a molecular mass of approximately 62 kDa in Western blots (Fig. 1, lane 3). No hyaluronidase activity was observed in substrate gels under reducing conditions, indicating irreversible protein denaturation at acidic pH.
Detergent extraction of whole sperm after treatment with PI-PLC revealed the appearance of all three hyaluronidase proteins in supernatants after centrifugation of sperm (Fig. 3, lane 3). Immunofluorescence labeling of sperm after PI-PLC treatment and subsequent fixation revealed no hyaluronidase labeling. Immunofluorescence labeling of the sperm pellet after nitrogen cavitation revealed posterior sperm head labeling only, whereas detergent extraction of whole sperm revealed little or no detectable hyaluronidase on sperm cell surfaces (not shown).
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In freshly ejaculated stallion semen, approximately 98% of the spermatozoa were labeled in the posterior head or postacrosomal region, as detected using indirect immunofluorescence (Fig. 4B). When sperm were incubated under capacitating conditions, four distinct cell labeling patterns were observed. Type 1 cells displayed only postacrosomal labeling (Fig. 5A, left); type 2 cells demonstrated a halo-like peripheral rim in addition to posterior acrosomal labeling (Fig. 5A, right); type 3 cells demonstrated mottled fluorescence labeling over the acrosomal region in addition to posterior acrosomal labeling (Fig. 5B); and type 4 cells demonstrated smooth anterior head labeling associated with the acrosomal region and slight labeling in the postacrosomal region (Fig. 5C). Control samples were incubated at ambient temperature and remained labeled only in the posterior acrosomal region of sperm. Subsamples labeled with the nuclear exclusion dye H33258 revealed that over 84% of sperm were viable cells, indicating that the mottled appearance over the sperm heads was observed in viable cells (not shown).
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Induction of acrosomal exocytosis using progesterone (3.0 µM) following 4-h capacitating conditions resulted in approximately 25% acrosome-reacted viable cells as detected by fluoresceinated lectin staining. Immunofluorescence labeling for hyaluronidase after progesterone treatment revealed patchy anterior head fluorescence (type 3) in approximately 60% of cells; however, approximately 20% of cells displayed a more uniform labeling pattern over the anterior sperm head (type 4) rather than sporadic patchy fluorescence. Double-fluorescence labeling with rhodamine-conjugated PSA, in addition to UP849 immunofluorescence labeling, demonstrated that the population of cells showing uniform fluorescence was acrosome reacted (Fig. 6, A and B).
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A time-course study with ejaculates from 3 stallions revealed that migration over the anterior head of the sperm cell was not detectable until 4 h of incubation (p < 0.05) (Fig. 7). Treatment of sperm with 3.0 µM progesterone demonstrated increased percentages of type 4 cells (p < 0.05) after 4-h in vitro capacitation (Fig. 7).
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To further characterize the anterior sperm head labeling pattern of equine sperm hyaluronidase, sperm were incubated under capacitating conditions in the presence of HA (100 µg/ml), a substrate for the enzyme, or in the presence of UP849 polyclonal IgG, an antagonist of the hyaluronidase protein. After incubation under capacitating conditions, sperm cells exposed to HA demonstrated decreased postacrosomal labeling (cell type 1) and increased anterior sperm head labeling (cell types 2, 3, and 4) in comparison to controls not containing HA (p < 0.05) at 4-h incubation (Fig. 7). As compared to controls receiving no antibody, sperm incubated in the presence of UP849 IgG (1 µg/ml) during the entire duration of in vitro capacitation demonstrated similar percentages of postacrosomal labeling. Although there was a higher percentage of both types 2 and 3 cells compared to values in controls incubated without anti-hyaluronidase antibody (p < 0.05), the relative percentages of cell types were similar to those for 0-h controls (Fig. 7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Of the three equine protein bands observed under nonreducing conditions, the 54-kDa band was initially selected as an antibody target because it was possible that the 83-kDa band was an aggregate or parental molecule of the two lower molecular weight species, notably the 54-kDa and 59-kDa proteins. Our data suggest that equine sperm contains a potent hyaluronidase that is associated with the plasma membrane, since all three bands of enzyme activity were clearly present in the partially purified plasma membrane-containing sperm fraction. The data also indicate that an inner acrosomal membrane source of hyaluronidase may also be present, as sperm pellets after nitrogen cavitation contained significant hyaluronidase activity. The presence of the acrosomal matrix as detected using a fluoresceinated lectin may have interfered with inner acrosomal access by the UP849 antibody and could explain the lack of inner acrosomal fluorescence signal in nitrogen-treated sperm. Several authors have reported that nitrogen cavitation selectively removes plasma membrane from the anterior apical region of sperm in pigs [18] and horses [19], leaving the posterior segment intact. Treatment of ejaculated sperm using detergent extraction or PI-PLC resulted in the removal of potent hyaluronidase activity from sperm and resulted in the loss of sperm surface fluorescence labeling using indirect immunofluorescence coincident with recovery of hyaluronidase activity in the media supernatant. This indicates that plasma membrane-associated equine sperm hyaluronidase is a GPI-linked membrane protein, as has been reported for PH-20/hyaluronidase from mouse, guinea pig, monkey, and human sperm [3–5, 28].
Under reducing conditions, the immunoreactive hyaluronidase migrates as a single band at 62 kDa, which suggests that equine sperm hyaluronidase may be a single protein, a family of related proteins, or protein subunits joined by disulfide bonds. PH-20, reported to be the sole hyaluronidase, migrates as a 64-kDa protein in guinea pig, monkey, and human sperm [4, 5, 26]. In those species, active PH-20 appears as a single protein band even under nonreducing conditions. It is possible that equine sperm hyaluronidase is homologous to PH-20 of other species but that the protein and three-dimensional structure differs in stallion sperm. The amino acid sequence of equine hyaluronidase protein has not been reported. Harrison demonstrated that a family of hyaluronidase proteins (a "ladder" of immunologically related oligomers and monomers) was purified from bull and ram sperm that resolved to a pair of protein bands of approximately 81 and 89 kDa under reducing conditions [6, 29]. Recent studies using cloned cDNA and amino acid sequence analysis have characterized the soluble hyaluronidase from bull testes as a fragment of the membrane-bound PH-20 enzyme. Meyer and coworkers showed that bovine PH-20 was present as both membrane-bound and soluble fractions, but that the soluble fraction is likely to be a fragment of the larger PH-20 molecule [27].
Equine sperm hyaluronidase displays strong enzymatic activity at neutral pH, similar to that of primate, human, and guinea pig sperm PH-20, although a soluble fraction released at the time of acrosomal exocytosis in those species displays a more acidic pH optimum (pH 3.0–6.0) [4, 5, 26]. We observed no hyaluronidase activity from equine sperm at pH 3.0. This suggests that equine sperm hyaluronidase would function in the neutral microenvironment of the cumulus matrix surrounding the oocyte rather than the immediate postexocytotic environment, which is likely to be acidic [1]. At the time of acrosomal exocytosis immediately following zona pellucida-specific binding, the acrosomal contents are expelled and dispersed in the vicinity of the sperm head. As such, acid-active enzymes would be expected to function best near their respective pH optima in the acidic microenvironment surrounding the sperm head after the acrosome reaction. Neutral-active enzymes would, in contrast, function optimally either as plasma membrane components at neutral pH or as they became dispersed away from the sperm head after acrosomal exocytosis. Since the sperm plasma membrane in most species, including horses, appears to be the origin of PH-20/hyaluronidase, the enzymatic degradation of the HA-rich cumulus matrix would most likely occur prior to zona pellucida binding and acrosomal exocytosis. In the mouse and monkey, it appears that plasma membrane hyaluronidase is essential in order for sperm to traverse the cumulus ECM, since blockage of hyaluronidase activity by antibodies to PH-20 or flavonoid hyaluronidase inhibitors also reduces the rate of sperm penetration through the cumulus [8, 14, 15].
The cellular distribution of equine sperm hyaluronidase appears to be unique. Our data indicate that ejaculated sperm from stallions contains hyaluronidase associated with the plasma membrane of the postacrosomal region of the sperm head. This is consistent with findings on guinea pig sperm PH-20 [30, 31] but dissimilar from findings of anterior head localization of hyaluronidase in bovine sperm [6] and simultaneous anterior and posterior head localization of PH-20 in macaque and human sperm [2, 4]. However, species differences in cellular hyaluronidase localization could be due, at least in part, to differences in hyaluronidase epitopes and antibody variations among species. In all species studied, the surface-associated hyaluronidase appears to change its cellular distribution after acrosomal exocytosis. We have demonstrated that stallion sperm display an apparent cellular redistribution after incubation under capacitating conditions (cell types 2 and 3) and before acrosomal exocytosis. Alternatively, it is possible that the apparent rearrangement of anterior sperm head hyaluronidase distribution could be due to alterations or partial unmasking of existing antibody binding sites that has been hypothesized to occur during sperm capacitation (reviewed by Yanagimachi [1]), and we are presently investigating this possibility. It seems likely that the type 4 cellular pattern seen following progesterone treatment is due to hyaluronidase associated with the inner acrosomal membrane rather than a redistribution over the sperm head. In guinea pig and monkey sperm, the localization of PH-20 was observed to change to a pattern associated with the sperm cell's exposed inner acrosomal membrane after acrosomal exocytosis [2, 32, 33]. Using bovine and ovine sperm, Harrison and Gaunt [6] demonstrated that acrosome-intact sperm from these species displayed hyaluronidase over the apex and periphery of the anterior acrosomal segment, as labeled using monoclonal antibodies to ram sperm hyaluronidase. These authors have not reported hyaluronidase labeling patterns from acrosome-reacted bovine or ovine sperm.
In our study, in vitro-capacitated equine sperm displayed a time-dependent change in cellular distribution from posterior to the acrosomal segment to an acrosome-associated distribution in a significant population of sperm. This cellular migration pattern was enhanced when incubations included HA and was inhibited when incubations included the UP849 polyclonal antibody to equine hyaluronidase. We hypothesize that enhancement of labeling by HA could have occurred by enzyme-substrate interactions and subsequent accelerated random movement over the sperm head, whereas inhibition of hyaluronidase migration could have occurred by antibody cross-linking of hyaluronidase on the posterior sperm head. We cannot, however, rule out the possibility that rearrangement of hyaluronidase over the sperm head is under the influence of surface proteins that may alter or mask the enzyme-active site or specific epitopes. Several studies have shown that sperm surface changes occur in capacitating sperm and are consistent with masking factors or so-called decapacitation factors [1]. Studies on the rat sperm surface antigen, 2B1, have demonstrated that the cellular distribution of 2B1, a homologue of guinea pig PH-20, changes during epididymal maturation from the anterior head to a sperm tail location [9, 34].
After acrosomal exocytosis, we observed a more uniform labeling pattern over the entire sperm head in a population of sperm corresponding to acrosome-reacted cells. In monkey sperm, Overstreet and coworkers [2] reported that freshly ejaculated and chemically activated sperm displayed anterior sperm head immunofluorescence and immunogold labeling with anti-PH-20 antibodies. The region of sperm labeled included the equatorial and posterior segment of the head. Those cells experiencing acrosomal exocytosis demonstrated head labeling of the anterior head region only; PH-20 labeling of the posterior segment was no longer observable as the inner acrosomal membrane became exposed, suggesting a migration phenomenon. In addition, Yudin and coworkers [13] demonstrated that monkey sperm PH-20 can be rearranged, or aggregated, over the sperm surface when capacitated sperm are incubated in the presence of antibodies to recombinant PH-20, but not when Fab fragments are used. To our knowledge, stallion sperm appears to be unique in that capacitation-related migration of hyaluronidase over the anterior sperm head from a posterior head origin occurs prior to acrosomal exocytosis.
The implications of this anterior head migration of hyaluronidase are significant for several reasons. First, the equine sperm hyaluronidase appears to be mobile within the plane of the plasma membrane during in vitro capacitation, suggesting that this protein could be involved in sperm capacitation and gamete interaction in vivo. Secondly, the antibodies to the protein could be useful as molecular probes of sperm function in this species. This is significant because in vitro fertilization has not been refined in this species and is not available as a clinical evaluation method for spermatozoal competence [35, 36]. Thirdly, the antigenicity of PH-20/hyaluronidase makes it likely that anti-sperm antibodies may be directed against PH-20/hyaluronidase and could impair fertility. This characteristic also makes it a likely candidate antigen for an immunocontraceptive agent in animals and humans. Fully effective contraception was reported in both male and female guinea pigs immunized against homologous PH-20, suggesting the importance of this protein in fertilization [37–39].
Taken together, the data presented suggest that equine sperm hyaluronidase is similar in many ways to that of other species, although it demonstrates certain unique properties. The redistribution of this GPI-linked sperm hyaluronidase on the surface of equine sperm may be associated with capacitation and could play an important role in rendering the sperm competent for subsequent cumulus penetration. As such, the equine sperm hyaluronidase could be PH-20 in this species, and it may be useful as a novel monitor of sperm capacitation as well as a molecule essential to sperm function and fertility in this species.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Stuart A. Meyers, Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California, Davis, CA 95616. Tel: 530-752-1174; smeyers{at}ucdavis.edu
Accepted: March 30, 1999.
Received: May 8, 1998.
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REFERENCES |
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