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Regular Article |
a Department of Anatomy and Embryology, School of Veterinary Medicine, Autonomous University of Barcelona, Barcelona 08193, Spain
b Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California, Davis, California 95616
ABSTRACT
It is generally accepted that spermatozoa become functionally mature during epididymal transit. The objective of this study was to determine whether the cellular location of equine PH-20 is modified during epididymal transit and, if so, the mechanism for such modification. Sperm were isolated from caput and cauda epididymal regions from stallions undergoing castration (n = 7) and used as whole sperm cell or subjected to nitrogen cavitation for isolation of plasma membrane proteins. Both caput and cauda sperm and sperm protein extracts were subjected to N-deglycosylation, O-deglycosylation, or trypsinization. The SDS-PAGE and Western blot analysis using a polyclonal anti-equine PH-20 IgG were performed in sperm extracts, and indirect immunofluorescence on whole sperm was also performed to determine the cellular distribution of plasma membrane PH-20 following similar treatments (deglycosylation or trypsinization). Hyaluronan substrate gel electrophoresis was performed to detect hyaluronidase activity in SDS-PAGE proteins. Western blots revealed significant differences in electrophoretic migration of PH-20 proteins from caput and cauda epididymal sperm. No effect was seen from deglycosylation treatments on the Western blot pattern; caput protein extracts exposed to trypsin showed the same band pattern as extracts from the cauda epididymis. N-deglycosylation resulted in the loss of hyaluronidase activity of sperm from both epididymal regions, whereas O-deglycosylation or trypsinization did not affect hyaluronidase activity. In caput epididymal sperm, the PH-20 protein is distributed over the entire sperm head; in cauda epididymal sperm, it is restricted to the postacrosomal region. No effect from deglycosylation on the cellular distribution of PH-20 was observed; however, treatment with trypsin changed the cellular distribution of PH-20 in caput sperm similar to that of the distribution of cauda sperm. These results suggest that PH-20 distribution during epididymal maturation is dependent on proteolytic trypsin-like mechanisms and, possibly, on complementary membrane-associated factors.
epididymis, gamete biology, male reproductive tract, sperm maturation, testis
INTRODUCTION
The formation of a mature sperm cell capable of fertilizing an egg is a complex developmental process initiated in the testis and ultimately completed within the female reproductive tract. Spermatozoa leave the testis neither fully motile nor able to recognize or fertilize an oocyte. It is generally accepted that spermatozoa become functionally mature during epididymal transit (for review, see [1, 2]). After leaving the testis, the spermatozoon undergoes morphological and biochemical changes in the intraluminal environment of the epididymis; these changes are termed epididymal maturation. During this particular maturation, numerous molecules in the acrosome and plasma membrane undergo biochemical modifications that alter their immunostaining pattern, suggesting a redistribution to adjacent cellular domains [1, 2]. The molecules in the plasma membrane may play a variety of roles during fertilization, and this molecular rearrangement can help to accomplish these different functions [3]
PH-20 is a glycosyl phosphatidylinositol (GPI)-linked sperm surface protein with hyaluronidase- [4, 5] and zona-binding activity [6]. This protein has been described in the sperm plasma membrane of guinea pigs [7, 8], rats and mice [9–12], macaques [13, 14], humans [15], and recently, horses [16]. In the guinea pig, PH-20 is expressed at the spermatid stage during spermiogenesis, and it is localized to the plasma membrane overlying the whole head of the sperm [17]. After epididymal maturation, PH-20 is subsequently distributed to the posterior head via lateral movement within the plane of the lipid bilayer of the sperm membrane [18, 19]. Similarly, in the horse, PH-20 is localized to the anterior head in caput epididymis-derived sperm, but with a posterior head location in cauda and ejaculated sperm [20]. However, whether this is a general mechanism used by sperm head surface proteins to redistribute and to form new domains or is unique to certain GPI-anchored proteins is not known. Little information is available concerning maturation of equine sperm, particularly with regard to cell markers for sperm maturation. It is important to determine whether equine sperm have maturational mechanisms similar to those described for other species to identify potential intervention points for which diagnostic criteria and semen storage protocols may be optimized to improve the fertility of stallion sperm.
The objective of this study was to determine whether the cellular localization of equine PH-20 is modified during epididymal transit and, if so, the mechanism for such modification.
MATERIALS AND METHODS
Materials and Reagents
Fluorescein-conjugated Pisum sativum agglutinin was obtained from Vector Laboratories (Burlingame, CA). Hyaluronic acid was obtained from ICN Pharmaceuticals (Costa Mesa, CA). All other chemicals were obtained from Sigma Chemical Company (St. Louis, MO). The protease inhibitor cocktail consisted of 1 mg/ml of leupeptin, 1 mM PMSF, 2 mg/ml of antipain, 10 mg/ml of benzamidine, 1 mg/ml of chymostatin, 1 mg/ml of pepstatin A.
Sperm Collection and Preparation
Testes were collected from seven horses after routine surgical castration procedures at the University of Pennsylvania, New Bolton Center (Kennett Square, PA). Epididymides from each testicle were dissected, and sections of caput and cauda were incubated separately in Petri dishes with gentle agitation at room temperature in 1 ml of Dulbecco phosphate-buffered saline (DPBS) supplemented with a cocktail of protease inhibitors. Sperm were removed from both regions by extrusion from the epididymal tubule using a sterile plastic applicator stick. Sperm were washed twice by centrifugation [16] and resuspended in supplemented DPBS at 4°C.
Indirect Immunofluorescence Microscopy
Indirect immunofluorescence using rabbit polyclonal immunoglobulins (IgG) raised against equine PH-20 (UP849) was carried out as previously described [16]. Briefly, sperm suspensions (caput and caudal epididymal sperm) were fixed for 10 min in 2% (w/v) paraformaldehyde and washed by centrifugation and resuspension using DPBS in siliconized microcentrifuge tubes at 400 x g for 2 min [16]. The samples were incubated for 30 min in blocking solution (5% BSA in DPBS). Purified IgG (UP849) was added to sperm suspension at a concentration of 1 µg/ml, and the samples were incubated for 60 min. Samples were then washed twice by centrifugation, resuspended in 1 ml of DPBS, and incubated for 60 min in a 1:50 dilution of fluoroisothiocyanate-conjugated goat anti-rabbit IgG. Subsequently, samples were rinsed three times in DPBS, and an antifade solution was added to enhance and preserve cell fluorescence (Vectashield; Vector).
Preparation of Sperm Protein Extracts
The sperm plasma membrane fraction was isolated as described previously [16]. Briefly, sperm solution was centrifuged at 300 x g for 5 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 and adjusted to a final concentration of 106 sperm/ml. 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 suspension was then centrifuged twice at 6000 x g for 10 min at 4°C to remove the remaining sperm cells. The supernatant was centrifuged at 35 000 x g for 15 min and the pellet discarded, after which the supernatant was centrifuged again at 100 000 x g for 2 h at 4°C. The resulting pellet was resuspended in 2 ml of Hepes-PBS (pH 5.0) and solubilized with a solubilization buffer (0.2 M PBS, 10% glycerol, 1% NP-40, pH 6.8) at 4°C, and the suspensions were vigorously vortexed for 3 min, followed by centrifugation at 10 000 x g for 10 min. The pellets were discarded and the supernatants stored at -70°C until assayed using Western blot procedures.
Deglycosylation and Trypsinization
Aliquots of protein extracts were N- and O-deglycosylated separately according to modified techniques described previously by Deng et al. [12] and Anakwe et al. [21], respectively. Briefly, 50 µg of sperm protein were diluted in DPBS supplemented with protease inhibitor cocktail to a final volume of 100 µl. N-Glycosidase F (peptide-N4-[acetyl-ß-glucosaminyl] asparagine amidase; Boehringer Manheim, Indianapolis, IN) was added to the reaction mixture at a final concentration of 20 U/ml. O-Glycosidase (O-glycopeptide endo-D-galactosyl-N-acetyl-
-galactosamino hydrolase; Boehringer Manheim) was added at a final concentration 80 mU/ml plus 10 mM calcium acetate and 1% Triton X-100 according to the manufacturer's instructions. The reaction mixtures were incubated at 37°C for 24 h. Control samples were incubated under the same conditions, except that the enzyme was replaced by DPBS vehicle control. Aliquots of protein extracts were also incubated with trypsin (100 µg/ml) for 10 min at 37°C in absence of protease inhibitors.
Gel Substrate Electrophoresis for Hyaluronidase Activity
Hyaluronidase activity was determined from the detergent-extracted whole sperm using a gel substrate electrophoresis technique as described by Cherr et al. [14] and modified by Meyers and Rosenberger [16]. Briefly, 10% SDS polyacrylamide gels were prepared containing 170 µg/ml of hyaluronan (HA) in the running gel before polymerization. The stacking gel did not contain HA. Following electrophoresis, gels were incubated for 1 h at room temperature in TBS containing 3% Triton X-100. Then, gels were incubated at 37°C for 15–18 h in PBS at pH 7.4. To detect zones of digestion of HA in the gels, the following protocol was used: gels were stained with 0.5% Alcian blue in 3% acetic acid for 2 h, destained in 7% acetic acid for 1 h, then counterstained with Coomassie blue R-250 and again destained with 30% methanol and 10% acetic acid for 15 min. The relative molecular weights of polypeptides within a given lane were determined by scanning of gels and subsequent analysis using a gel scanning macro in the public domain NIH Image Program 1.60, developed at the NIH (http://rsb.info.nih.gov/nih-image/). Prestained molecular weight standards were used (New England Biolabs, Beverly, MA) to visualize them in the HA-containing gel.
SDS-PAGE and Western Blot Analyses
Nonreduced, detergent-extracted protein samples were subjected to SDS-PAGE using 10% polyacrylamide gels as previously described [14]. Gels were blotted to polyvinylidene fluoride membranes, incubated in blocking solution (9% nonfat dry milk, 0.1% Tween-20 in Tris-buffered saline [TTBS]) for 2 h, then probed with UP849 polyclonal IgG diluted 1:2000 in TTBS. Goat anti-rabbit IgG conjugated to horse radish peroxidase (1:5000) was used as the secondary antibody. Protein bands were detected using a chemiluminescence procedure (Renaissance; NEN Life Science Products, Boston, MA). Prestained molecular markers were used to assess the molecular weights of the bands. The relative molecular weights of polypeptides within a given lane were determined by optical scanning of gels and subsequent analysis of scans using a gel scanning macro for NIH Image 1.60 on a Power Macintosh computer (Apple Computer, Cupertino, CA).
Sperm Culture and Treatment
In a preliminary study, increased viability of sperm cells collected from the caput of the epididymis was seen when they were cocultured with bovine oviductal epithelial cells (BOEC). So, caput and cauda sperm recovered as described above were cocultured with subconfluent cultures of BOEC [22]. Bovine oviductal epithelial cells were collected from oviducts obtained at slaughter from abattoir cows in the follicular phase of their ovarian cycle (follicle > 15 mm and no corpus luteum) and kept in cold DPBS containing antibiotic-antimycotic (AB-AM) solution (10 000 IU/ml of penicillin, 10 000 µg/ml of streptomycin, and 25 µg/ml of amphotericin B) for transport to the lab. Once in the lab, samples were always manipulated in a laminar-flow tissue culture hood, and BOEC were recovered during luminal lavage of the oviduct with DBPS supplemented with 10% fetal calf serum (FCS) and 2% AB-AM solution. The BOEC from three or more animals were pooled together to minimize individual female effects. The recovered BOEC were washed twice by centrifugation at 300 x g, and the pellet was resuspended in culture medium (Dulbecco modified Eagle medium [DMEM]:F-12, 1:1) supplemented with 10% FCS, insulin (5 µg/ml; Collaborative Biomedical, Bedford, MA), transferrin (5 µg/ml; Collaborative Biomedical), selenium (5 ng/ml; Collaborative Biomedical), epidermal growth factor (10 ng/ml; Boehringer Mannheim), and AB-AM solution as described by Ellington et al. [22] and seeded at approximately 5 x 105 cells/cm2 in 24-well plates. Cells in primary culture were allowed to grow until 85–90% of confluence, and immunocytokeratin staining of monolayers was done to confirm the epithelial nature of the cells. Only cells derived from more than three passages were used for sperm coculture. Successful pools of cells were cryopreserved in media with 50% FCS and 10% dimethyl sulfoxide in an alcohol freezer (Nalgene, Inc., Rochester, NY) placed in -70°C and stored in liquid nitrogen (-196°C).
Caput and cauda sperm were cultured with homogeneous, subconfluent BOEC cultures at 37°C in 5% CO2 under the following conditions: control, sperm cultured with DMEM:F-12 media; N-deglycosylation, sperm cultured with 12 U/ml of N-glycosidase for 24 h; O-deglycosylation, sperm cultured with 80 mU/ml of O-glycosidase for 24 h; trypsin 1, sperm cultured with 20 µg/ml of trypsin-EDTA for 10 min; and trypsin 2, sperm cultured with 100 µg/ml of trypsin-EDTA for 10 min. Following treatments, total sperm motility was recorded, and sperm cells were recovered from BOEC by repeated gentle pipetting of 100-µl volumes of DPBS. Subsamples were processed to record viability by means of a vital staining kit (LIVE/DEAD Sperm Viability kit; Molecular Probes, Eugene, OR). Recovered sperm were immunolabeled for PH-20 immunofluorescence as described above.
Data Analysis
A minimum of 200 cells were counted for each treatment, and the percentage of cells with anterior sperm head, posterior sperm head, or whole-head immunofluorescence was tabulated for each treatment in addition to percentage viability and total and progressive sperm motility. Differences between treatment groups were assessed following normality testing using one-way ANOVA procedures with Stata software (Stata Corporation, College Station, TX) and a PC computer (Dell Computer, Inc., Austin, TX).
RESULTS
Localization of PH-20 in Caput and Cauda Equine Sperm
Indirect immunofluorescence of sperm isolated from epididymal regions showed that the distribution of PH-20 differed between sperm removed from caput and from cauda epididymis. In caput sperm (Fig. 1A), the protein was distributed over the entire sperm head in approximately 95% of cells, whereas in sperm recovered from the cauda epididymis, PH-20 remained restricted to the posterior head domain or postacrosomal region in 92% of cells (Fig. 1B). Approximately 8% of cauda sperm displayed fluorescence distributed over the sperm head in a patchy or mottled immunofluorescence pattern. Following BOEC coculture and deglycosylation treatment of caput epididymis-derived sperm, the anterior sperm head appeared completely immunolabeled, indicating a uniform distribution of PH-20 overlying the entire sperm head. The percentage of cells displaying this pattern was similar to that of control-treated sperm. No differences were observed in the immunolabeling pattern between control, O-deglycosylation, or N-deglycosylation of sperm.
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The PH-20 labeling pattern of caput sperm cells subjected to trypsinization (20 µg/ml) was similar to that observed in nontreated cauda sperm. A band of fluorescence localized to the posterior acrosomal region of the sperm was seen in approximately 74% of the trypsin-treated caput sperm cells (Fig. 2A). The remaining 26%, appeared with different patterns, ranging from whole-head labeling (8%) to the absence of fluorescence (3%). Incubation of caput sperm with a higher concentration of trypsin (100 µg/ml) revealed the same immunolabeling pattern as that with the lower trypsin concentration. However, a higher percentage of unlabeled sperm (13%) were seen following the higher-concentration treatment.
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For cauda epididymis-derived sperm coincubated with BOEC and O- or N-deglycosylated, no differences were observed from control-treated sperm. Following trypsin 1 treatment, cauda epididymis-derived sperm showed only faint posterior head immunolabeling, whereas trypsin 2 treatment resulted in sperm head labeling of the postacrosomal region in addition to a high number of cells (>45%) displaying irregular variations in immunolabeling patterns. Approximately 18% of cells had a pattern similar to caput-type, whole-head fluorescence labeling (Fig. 2B), whereas approximately 20% of the cells appeared without any fluorescence (Fig. 2C). Percentages of labeling patterns following different treatments are displayed in Figures 3 and 4 for caput and cauda epididymal sperm, respectively.
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Western Blot Analyses and Gel Substrate Electrophoresis for Hyaluronidase Activity
Under nonreducing conditions, Western blot analysis revealed different band patterns in detergent-extracted caput epididymal sperm than those of the cauda sperm. In sperm collected from the caput, the pattern consisted of three major protein bands with approximate molecular masses of 97, 64, and 62 kDa (Fig. 5, lane 1). The lower two bands (64 and 62 kDa) appeared to be less abundant than the major band (97 kDa). All samples collected from the cauda epididymis showed a wide protein band from approximately 97 to approximately 83 kDa, with two smaller bands appearing as a doublet at 64 and 62 kDa. An additional band appeared at 54 kDa, and two low-molecular-mass bands were observed as a doublet at 32 and 35 kDa (Fig. 5, lane 2).
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When sperm protein extracts from caput and cauda epididymides were subjected to N-deglycosylation and O-deglycosylation treatments, the protein migration patterns of PH-20 observed in Western blots were not different from that of the controls (nonexposed to enzymes) (Fig. 6). However, caput protein extracts exposed to trypsin showed the same band pattern as extracts from the cauda epididymis (Fig. 6). Samples from the caput epididymis treated with trypsin showed the 64- and 62-kDa bands more intensely than in samples from cauda (Fig. 6, lane 4).
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Hyaluronidase activity in caput sperm extracts was detectable in three bands corresponding to those seen in Western blots (Fig. 7, lane 1). However, the higher enzyme activity was seen in the doublets (
60 and 56 kDa) as compared to the 83- to 97-kDa band that was the major band detected with Western blot techniques. Hyaluronidase activity in cauda sperm was also detected in three major bands; however, the two lowest bands appeared to migrate further than those seen in the caput extracts and were located at 54 kDa (Fig. 7). In addition, although equivalent concentrations of total protein were loaded into each lane (protein per lane, 8 µg for Western blots and 4 µg for gel substrate electrophoresis for hyaluronidase activity) in the caput and cauda hyaluronan substrate gels, the hyaluronidase activity detected in cauda sperm was markedly greater than that in caput sperm. Whereas O-deglycosylation and trypsinization did not affect hyaluronidase activity in samples from caput and cauda epididymis, N-deglycosylation resulted in the loss of hyaluronidase activity of sperm from both epididymal regions (Fig. 8, lanes 2 and 6).
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DISCUSSION
Mammalian spermatozoa are highly differentiated cells when they leave the testes, but they lack forward motility and the capacity to fertilize oocytes. When sperm pass through the epididimydes, they acquire both functions [1, 3]. Changes in sperm plasma membrane proteins are caused by interaction with epididymal secretory proteins, either directly by adsorption onto the plasma membrane or indirectly by modification of existing membrane proteins [3]. Mechanisms that could account for the establishment of surface membrane protein domains are unknown, especially in light of the fact that mammalian sperm are biosynthetically quiescent. Consequently, new proteins cannot be synthesized for insertion into the plasma membrane [23]. For this reason, all observed changes in equine PH-20 protein must be posttranslational. With immunofluorescence microscopy, we have observed PH-20 molecules in different plasma membrane domains in relation to the epididymal regions. In the present study and in previous work from this laboratory, immature sperm from the caput epididymis demonstrated that PH-20 was distributed over the entire surface of the head, with no recognizable domains. In cauda epididymis-derived sperm, PH-20 was restricted to the postacrosomal region or posterior head domain [20, 24]. These findings are consistent with those observed in sperm from guinea pig [17, 18, 25–28] and mouse [12], in which the PH-20 cellular location changes during epididymal transit. Biochemical maturation of PH-20 was also revealed by changes in the band pattern on Western blots of sperm protein extracts obtained from caput and cauda epididymal regions.
Several different hypotheses need to be considered to explain this developmental restriction of PH-20 to the posterior head domain. The first is that the PH-20 molecules located in the anterior head domain in caput epididymal sperm are removed from the sperm, leaving only the posterior head domain in cauda sperm. Precedents for this mechanism exist in polarized epithelial cells, such as MDCK (Madin-Darby canine kidney) cells, in which cell surfaces create new protein domains by removing and degrading certain proteins [29, 30]; this has been suggested to be the result of endocytic processing [30]. In sperm, no evidence exists, to our knowledge, for a process of endocytosis of large molecules, so this mechanism seems unlikely with regard to equine PH-20. Similarly, an epididymal-secreted protease would not be likely to remove only anterior sperm head acrosomal PH-20, as we observed in this study.
An alternative hypothesis is that surface proteins, either membrane integrated or adsorbed, may alter or mask the specific PH-20 epitopes over the anterior sperm head domain during transit through the epididymal lumen. Collectively, these putative proteins are known as decapacitation factors [1], and some of these glycoproteins and peptides are believed to stabilize the plasma membrane and may prevent premature acrosome reactions or premature hyperactivation [31–34]. These decapacitation factors acquired during epididymal passage possibly could mask PH-20 epitopes from the anterior head, resulting in a different immunofluorescence pattern in sperm derived from the cauda epididymis. However, if this hypothesis is correct, it suggests a mechanism that could selectively prevent the binding of these masking factors in PH-20 protein located in the posterior head domain. Additionally, it would provide a possible explanation for the exclusive cellular redistribution of equine PH-20 under capacitation conditions and before acrosomal exocytosis [16] due to a partial unmasking of existing antibody-binding sites. However, we have not been able to unmask hidden PH-20 epitopes in vitro when live sperm recovered from the cauda epididymides were subjected to detergent washing or trypsinization. The effect of trypsin on sperm cells is not fully understood, but some negative effects were observed in the present study and were described when this enzyme was used experimentally to inactivate viruses in infected bull semen samples [35]. Another alternative explanation for the masking of pre-existing PH-20 epitopes in the anterior head domain could be the action of the carbohydrate chains that constitute surface glycoproteins. Structurally, glycoproteins on the outside of the sperm plasma membrane have carbohydrate chains that could extend several microns to form the so-called glycocalyx [3, 36]. This glycocalyx has, among other functions, a putative capacity to hinder the lateral movement of proteins, presumably via charge interactions, in such a way that an increase of protein diffusion coefficients has been frequently observed after digestion with sialidases [3]. No change in PH-20 cell localization after enzyme treatment was observed in our study, suggesting that carbohydrates were not primarily involved in masking PH-20 expression or in PH-20 migration processes.
The third hypothesis is that PH-20 located in the anterior head migrates and joins those located in the postacrosomal domain during transit through the epididymis. This mechanism for translocation of surface molecules involves a lateral movement in the outer lipid bilayer of the plasma membrane, as has been previously described for guinea pig PH-20 [18, 25, 26]; 2B1, the rat orthologue of guinea pig PH-20 [10]; and another sperm surface protein, fertilin [28]. Previous studies of guinea pig sperm showed that PH-20 molecules undergo two migrations, first from the anterior sperm head to the posterior head domain [27] and then a second, lateral translocation, following the acrosome reaction, from the posterior head plasma membrane to the inner acrosomal membrane [25, 26]. This hypothesis appears to be the most reasonable based on experiments in which migration of fluorescently labeled PH-20 was recorded in single sperm cells [26]. This migratory mechanism of the PH-20 protein is described to be an active process that serves to accumulate the protein into discrete membrane domains against a concentration gradient. For this active transport, an interaction with a putative translocating component has been suggested [26]. Interactions of PH-20 with extracellular components have also been proposed to account for the restricted mobility observed for PH-20 in posterior head plasma membrane after using photobleaching techniques [18].
In all three hypotheses, the idea of an unknown factor emerges that could interact with PH-20 to accomplish PH-20 restriction to the posterior sperm head domain. PH-20 is one of several sperm surface proteins (CE9 [37–40], fertilin, AH-40, AH-50 [28], and 2B1 [10]) that reportedly localize to different domains while the sperm is transiting the epididymis. The final destination for each of these molecules seems to be related to its physiological function, but a common mechanism could be present to initiate protein translocation. In the present work, as in others [27, 28], trypsin treatment seems to mimic in vitro this common pattern of protein redistribution.
The processes suggested to explain the restriction of equine PH-20 to the posterior sperm head domain would not necessarily involve a modification of the protein molecular weight, but minor changes have been detected. Our data suggest that the reduction in the molecular weight of equine PH-20 during epididymal passage is mainly due to structural modifications derived from proteolytic processes. We have been able to obtain the same pattern of immunoreactivity as that observed in nontreated cauda sperm from caput epididymis-derived sperm after a trypsin treatment. The major band of approximately 97 kDa observed in sperm recovered from caput epididymis appeared to be an inactive precursor form of the 83-kDa protein, because no enzymatic activity is detectable. The 83-kDa band observed must be a very active form of hyaluronidase, because it was strongly detected in caput sperm extracts with gel substrate for hyaluronidase activity but only weakly detected with Western blot techniques.
Little is known regarding the biochemical maturation of PH-20 during epididymal transit. Deng et al. [12] suggested that the reduction in molecular weight of mouse PH-20 during epididymal passage was due to structural modifications on its N-linked oligosaccharide side chains. In our study, no modifications in Western blot protein migration patterns were seen after subjecting sperm protein extracts from caput and cauda epididymides to N-deglycosylation and O-deglycosylation treatments. However, a definite loss of hyaluronidase activity was detected in samples from caput and cauda epididymis subjected to N-deglycosylation. This could indicate that, as in mouse PH-20, N-linked oligosaccharide side chains in equine PH-20 are regulating hyaluronidase activity from caput and cauda sperm. Sperm hyaluronidase has a high homology among mammalian species and with bee venom hyaluronidase [15]. Although its function as a hyaluronidase and as a sperm-zona binding molecule seems to be common among mammalian species, equine PH-20 exhibits certain unique properties.
Proteolysis is a common and important mechanism for regulating biological functions [41, 42], and our data suggest that the reduction in molecular weight of PH-20 during epididymal passage is due to structural modifications derived from proteolytic processes. These findings about equine PH-20, biochemical maturation, and creation of different domains may be intrinsically related by a common, proteolytic-sensitive mechanism. As suggested by Hunnicutt et al. [28], proteolysis of sperm surface proteins might occur between caput and corpus epididymal regions, and the responsible proteases could be in the epididymal fluid and/or on the sperm surface and be regulated by epididymal factors [43].
Hyaluronidase activity and Western blot band pattern in equine ejaculated sperm was detectable in three major bands with approximate molecular weights of 54, 59, and 83 kDa [16]. In the present study, some differences in the results have been observed between cauda sperm and those from ejaculated sperm. The main differences were the presence of a wide protein band, starting from approximately 97 to approximately 83 kDa; the appearance of a doublet of 64 and 62 kDa instead of a single band of 59 kDa; and the presence of a doublet of 35 and 32 kDa. All these differences could be due to a final process of maturation while sperm are stored in cauda epididymis, and even to a final modification of sperm proteins during contact with seminal plasma following emission and ejaculation. It has been suggested that modification of sperm to achieve their maturation requires preprogrammed cleavage of molecules and remodeling by the action of molecules found in the suspending fluids [42, 43]. Most of these biocatalysts are secreted by a series of specialized regions in the epididymal epithelium, but some are provided in seminal plasma [43]. Thus, it is not difficult to hypothesize that, when maturing sperm initially enter the cauda, they display a heterogeneous population of proteins until their maturation can be completed. This could include the proteolytic modification of the 97-kDa band to 83 kDa and the disappearance of the 32- and 35-kDa bands. Taken together, the data presented suggest that during epididymal passage, equine sperm hyaluronidase responds to a common proteolytic mechanism for regulating structural modifications and changes of localization pattern in different sperm head domains. These structural modifications of PH-20 are associated with the acquisition of new isoforms having increased hyaluronidase activity. The modification and localization of PH-20 into the posterior head plasma membrane likely could place the sperm cell's PH-20 in a favorable position to reach the inner acrosome membrane, as has been demonstrated following acrosomal exocytosis [16].
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ACKNOWLEDGMENTS
The authors would like to thank Betsy Rosenberger and Karen Orpneck for technical assistance and Dr. Xiangning Deng for assistance with the manuscript.
FOOTNOTES
First decision: 19 February 2001.
1 Supported by USDA grant 98-35203-6584. J.R. was supported by a postdoctoral grant from NATO. 
2 Correspondence. FAX: 530 752 690; smeyers{at}ucdavis.edu
Accepted: June 6, 2001.
Received: January 23, 2001.
REFERENCES
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