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Characterization of PH-20 in canine spermatozoa and testis
Theriogenology 57 (2002) 977±987
Characterization of PH-20 in canine spermatozoa and testis K. Sabeur, K. Foristall, B.A. Ball* Department of Population Health and Reproduction, University of California, Davis, CA 95616, USA Received 1 May 2001; accepted 18 July 2001
Abstract The purpose of this study was to characterize the sperm membrane protein PH-20 in the dog. Canine spermatozoa were extracted with Triton X-100 and the presence of PH-20 was determined by immunoblot with an antibody against recombinant macaque PH-20. The hyaluronidase activity of canine PH-20 was determined with substrate gel electrophoresis based upon digestion of hyaluronic acid (HA) incorporated into the separating gels. Hyaluronidase activity was also quanti®ed using a microplate assay. Sperm extracts were incubated at pH 4 or 7 in wells containing agarose and HA. For immunolabeling of PH-20 on canine sperm membranes, canine sperm were ®xed and incubated with R-10 primary antibody, and an anti-rabbit IgG-FITC secondary antibody. Samples were visualized by ¯uorescence microscopy. Non-reducing SDS-PAGE and Western blot of detergent-extracted canine sperm revealed a major band at 50 kDa, and three other bands at 42, 124, and >209 kDa. Substrate PAGE revealed translucent bands of hyaluronidase activity of similar size to bovine testicular hyaluronidase. These bands were markedly more pronounced at pH 4 than at pH 7. The microplate assay also demonstrated that hyaluronidase activity was over four times greater at the acidic pH. Immunolabeling of canine spermatozoa demonstrated that PH-20 is localized to the anterior head region and appeared in the Golgi area of round spermatids as detected by the immunohistochemical staining of the testis. This study provides evidence that PH-20 is present on the membrane of canine spermatozoa and in round spermatids. Canine PH-20 exhibits hyaluronidase activity that is markedly more pronounced at acidic pH. # 2002 Elsevier Science Inc. All rights reserved. Keywords: Hyaluronidase; Dog; Sperm; Immunohistochemistry; Substrate PAGE
* Corresponding author. Tel.: 1-530-752-1358; fax: 1-530-752-4278. E-mail address: [email protected] (B.A. Ball).
0093-691X/02/$ ± see front matter # 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 3 - 6 9 1 X ( 0 1 ) 0 0 6 9 7 - 5
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K. Sabeur et al. / Theriogenology 57 (2002) 977±987
1. Introduction The membrane protein PH-20, similar to various other cell adhesion proteins, is a glycosylphosphatidylinositol (GPI)-anchored sperm-speci®c protein [1]. Initially characterized by Primakoff et al. [2] in guinea pig sperm, PH-20 has been found in many other species including cynomologus macaques and humans [3,4]. The gene encoding PH-20 is highly conserved [5]. Protein PH-20 is now recognized as a bifunctional protein involved in mammalian fertilization. It has been shown to have hyaluronidase activity, a role related to cumulus penetration [6,7] as well as zona binding properties. PH-20 involvement in cumulus penetration was supported by the observation that incubation of acrosome-intact sperm with anti-PH-20 antibodies or hyaluronidase inhibitors prevented penetration of the cumulus layer in vitro [7,8]. Its second role may be related to secondary binding of sperm to the zona pellucida following the acrosome reaction [3,8]. The role of PH-20 in secondary binding is supported by the fact that incubation of acrosome-reacted sperm with anti-PH-20 antibodies prevents binding of sperm to zona pellucida proteins [2]. The aminoterminal 300 amino acids of PH-20 are responsible for its hyaluronidase activity while the C-terminus could be involved in sperm±zona pellucida interactions [9]. Protein PH-20 has been localized to the plasma membrane of acrosome-intact sperm of mice, macaques and humans [3,4,8]. It is also expressed on the inner acrosomal membranes [10]. Induction of the acrosome reaction appears to result in migration of this protein from the plasma membrane to the inner acrosomal membrane in macaque [10], guinea pig [2], and human [4] sperm. Recent work has provided promising results regarding the use of PH-20 as an immunocontraceptive agent. Primakoff et al. were successful in their attempt to induce infertility in both male and female guinea pigs immunized with the PH-20 protein [11,12]. Depending on vaccination protocol, immunization of males can result in long-term (>1 year) or even irreversible infertility. Thus, it appears that PH-20 is a candidate for further investigation into the use of sperm-speci®c antigens as immunocontraceptive agents in dogs. Before PH-20 can be examined as a potential contraceptive agent in the dog, however, its presence on dog sperm must be veri®ed, and basic structural and biochemical properties must be characterized. Thus, the current research involved the following objectives: (1) determination of PH-20 expression on canine sperm; (2) characterization of hyaluronidase activity of canine PH-20. The data represents a preliminary evaluation of PH-20 as a possible candidate for immunocontraception in dogs. 2. Materials and methods 2.1. Animals Four intact, mixed breed dogs were obtained from the local animal shelter to be used as semen donors for the current experiments. Semen was collected into 15 ml polystyrene conical centrifuge tubes by manual manipulation and use of an arti®cial vagina.
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After collection, semen was immediately placed on ice for transport to the laboratory. Canine testes were obtained with client permission and after castration from client animals presented to the Veterinary Medical Teaching Hospital of the University of California, Davis. Tissues were stored at 70 8C until used. 2.2. Extraction of sperm membranes Semen (1 ml) was resuspended in 10 ml modi®ed Tyrodes's medium containing 0.1% polyvinyl alcohol (TALP-PVA) and centrifuged (300 g for 10 min). The sperm pellet was resuspended in 8 ml TALP-PVA and recentrifuged at the same speed. Pellet was then diluted to a ®nal concentration of approximately 200 106 sperm/ml. A cocktail of protease inhibitors was added to each sperm suspension (50 mM Tris, pH 7.4, 20 mM EDTA, 1 mM p-hydroxy-mercurobenzoate, 5 mM N-ethylmaleimide, and 1 mM p-benzamidine hydrochloride, ®nal concentrations). Samples were solubilized in 2% Triton X-100, vortexed for 5 min, and then centrifuged for 15 min at 10,000 g. The supernatant (which contained the solubilized membrane proteins) was combined with protease inhibitors (as above), aliquoted, and measured for protein (Bradford assay, BioRad, Hercules, CA). Samples were stored at 20 8C until use in Western blot analysis, substrate gel electrophoresis or microplate assay for hyaluronidase activity. 2.3. Electrophoresis and Western blot analysis For SDS-PAGE analysis, sperm membrane extracts were mixed with either nonreducing (62.5 mM Tris, 1% SDS, 10% glycerol and 0.5% bromophenol blue) or reducing (above, plus 5 mM b-mercaptoethanol) solubilization buffer. Samples were then heated at 95 8C for 5 min prior to electrophoresis. SDS-PAGE was performed as previously described [4]. Western blot analysis was carried out according to [13]. Brie¯y, after transfer, nitrocellulose membranes were placed in blocking solution, 3% gel Tris buffered saline±Tween 20 (TBS-T) for 4 h, and washed three times in TBS-T (10 min each). Membranes were then probed with antisera (R-10, 1:5880 [6]) raised in rabbits against recombinant cynomologus macaque PH-20 [8] for 15±20 h at room temperature. After three washes in TBS-T, membranes were incubated with secondary antibody, an anti-rabbit IgG conjugated to horseradish peroxidase (1:5000), in 1% gel TBS-T. Blots were visualized using a chemiluminescence (ECL) kit (Amersham, Arlington Heights, IL). 2.4. Immunolocalization of PH-20 on canine sperm membranes Immunolabeling of sperm was carried out by the method of Overstreet et al. [3]. Brie¯y, raw semen was diluted 1:1 in TALP-PVA for transport to the lab. Semen was then subjected to a two-step Percoll gradient, and then centrifuged (20 min at 300 g). Sperm pellets were washed in 10 ml TALP-PVA, and centrifuged again for 10 min. Samples were then resuspended to a ®nal concentration of approximately 20 106 sperm/ml. Aliquots of 500 ml were ®xed in 1% paraformaldehyde for 8 min at room temperature, and washed twice in DPBS by centrifugation. Sperm pellets were then resuspended in blocking agent (DPBS with 1% BSA, Sigma, St. Louis, MO) and incubated at room temperature
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for 30 min. Aliquots were centrifuged at 300 g for 10 min and resuspended in 300 ml DPBS-BSA. Primary antibody (R-10) or preimmune rabbit sera (control) was added at 1:100 dilution. Following incubation at 37 8C for 60 min, samples were washed twice, and then resuspended in 100 ml DPBS-BSA. The secondary antibody, FITC labeled goat antirabbit IgG was added at 1:20 dilution (v/v), and samples were incubated for another 60 min at 37 8C in dark. Each sample was then washed twice in DPBS-BSA, resuspended, and examined by epi¯uorescence microscopy. 2.5. Hyaluronic acid-substrate gel electrophoresis Sperm extracts were subjected to substrate gel electrophoresis according to Guntenhoner et al. [14]. Brie¯y, 7.5% acrylamide separating gels were prepared with 170 mg/ml hyaluronic acid (HA). The 4.5% acrylamide stacking gel did not contain HA. A commercial bovine testicular hyaluronidase (Sigma, St. Louis, MO, USA) was used as the positive control, at concentrations of 2.5 and 50 TRU/ml. Sperm extracts were electrophoresed for 1 h, and then rinsed in TBS containing 3% Triton X-100 for 2 h at room temperature. Gels were incubated in either pH 4 or 7 sodium acetate buffer for 18±20 h at room temperature. Gels were then washed twice in distilled water, stained with 0.2% alcian blue in 3% acetic acid for 2 h, and ®nally destained in 7% acetic acid. 2.6. Microplate assay for hyaluronidase activity Hyaluronidase activity of sperm extracts was determined quantitatively using a microplate assay described by Tung et al. [15]. Brie¯y, agarose was dissolved in warm sodium formate buffer (0.1 M, pH 4) or sodium phosphate buffer (0.3 M, pH 7) to get a ®nal concentration of 0.8% (w/v). Then, a solution of HA was mixed with the agarose to give a ®nal concentration of 0.8 mg/ml HA, and maintained at 55 8C before use. Ninety-six well plates were prewashed with either the pH 4 or 7 buffer. The agarose±HA solutions were pipetted into the microtiter wells (100 ml per well) and allowed to solidify at room temperature. The standard curve was produced by serial dilutions of a commercial bovine testicular hyaluronidase. Activity was expressed in terms of turbidity reducing units/ml, and ranged from 30 to 0.23 TRU/ml. Sperm extracts were thawed and diluted in either pH 4 or 7 buffer. Standards and samples were assayed in triplicate and repeated at both pH 4 and 7. Each standard or unknown was pipetted into the plate (100 ml per well) and incubated for 7 h at 37 8C. Following incubation, plates were washed three times in the appropriate buffer. A solution of cetylpyridinium chloride (CPC, 10% w/v) was pipetted across each plate (100 ml per well) in order to precipitate any undigested HA. Optical density was then recorded at 590 nm with a spectrometer (PE HTS 7000, Norwalk, CT); a higher optical density re¯ected a lower hyaluronidase activity. 2.7. Immunohistochemical localization of PH-20 in canine testis The testes of adult dogs were sliced and ®xed in 4% paraformaldehyde. After a series of graded ethanol incubations, tissues were embedded in paraf®n and sectioned at 5 mm. The tissue sections were then deparaf®nized and hydrated through xylenes and graded
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ethanol series. Immunoperoxidase staining was performed with the vectastain ABC-Elite kit (Vector labs, Burlingame, CA). The sections were incubated for 30 min in 0.3% H2O2± methanol to quench endogenous peroxidase activity. After a wash in PBS 0.3% Triton X100, the sections were incubated for 20 min with diluted blocking serum, washed and incubated with the anti-PH-20 antibody (1:100) overnight at 4 8C. The sections were then incubated for 30 min with biotinylated HRP-linked secondary antibody (1:200) followed by incubation in ABC reagent and developed with 3-amino-9-ethylcarbazole (AEC kit, Vector labs, Burlingame, CA). The sections were quickly counterstained with hematoxylin. Normal rabbit serum was used as the negative control. 2.8. Statistical analysis Data (hyaluronidase activity) were analyzed by ANOVA (StatView, Cary, NC). Differences between treatments were assessed with Fisher's protected least signi®cant difference test (StatView). Means were considered different at P < 0:05. Results are expressed standard error. 3. Results 3.1. Electrophoresis and Western blot analysis Under reducing conditions, SDS-PAGE and subsequent Western blot analysis of canine sperm extracts showed three bands of apparent molecular weights at 52, 40 and 18 kDa. Under non-reducing conditions, the anti-PH-20 antibody recognized one major band at 50 kDa, as well as three other bands at 42, 124, and >209 kDa (Fig. 1). 3.2. Localization of PH-20 on canine sperm membranes Labeling of canine sperm with polyclonal antibody to recombinant macaque PH-20 revealed that most of the label was localized to the sperm head of approximately 50% of cells (Fig. 2A). There was a punctate appearance to the label that was most evident on the anterior sperm head. No label was observed in control samples (Fig. 2B). Some faint ¯uorescence was present in the post-acrosomal region of both control and treated cells, but this ®nding was inconsistent across repeated experiments. 3.3. Hyaluronic acid-substrate gel electrophoresis Hyaluronidase activity of PH-20 in detergent-extracted canine sperm suspensions was determined with HA-substrate PAGE. Staining of gels with alcian blue revealed translucent bands that corresponded to areas of HA digestion. At pH 4, a doublet appeared that was similar in molecular weight to the positive control, bovine testicular hyaluronidase (Fig. 3A). At pH 7, very little activity was detected. However, the positive control was more active at pH 7, as evidenced by large, diffuse bands (Fig. 3B) indicating the assay conditions were appropriate.
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Fig. 1. Immunoblot of SDS-PAGE of canine sperm (10 mg protein per lane) underreducing (R) and nonreducing (NR) conditions. Blots were probed with antibody (R-10) to recombinant macaque PH-20. Secondary antibody was a HRP-conjugated goat anti-rabbit IgG. Immunoreactive proteins were detected by enhanced chemiluminescence.
3.4. Microplate assay for hyaluronidase activity Quantitative data on hyaluronidase activity in canine sperm extracts was obtained by use of a microplate assay. The results of this experiment supported the observation that canine sperm hyaluronidase activity is greater at acidic, rather than neutral pH. Although
Fig. 2. (A) Immunolocalization of PH-20 on acrosome-intact canine sperm using a polyclonal antibody raised against recombinant cynomologus macaque PH-20 and a FITC-conjugated goat anti-rabbit secondary antibody. Note the presence of label localized to the head of approximately 50% of sperm. (B) Acrosome-intact canine sperm incubated with preimmune rabbit serum and FITC-conjugated goat anti-rabbit IgG. Note absence of fluorescent label.
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Fig. 3. (A) Hyaluronic acid substrate PAGE of detergent-extracted canine sperm samples performed at pH 4. Lane 1: positive control, bovine testicular hyaluronidase (50 TRU/ml); lane 2: positive control, bovine testicular hyaluronidase (2.5 TRU/ml); lane 3±5: samples from dogs 1, 2, and 3, respectively. Note doublet of activity in sample lanes. (B) Hyaluronic acid substrate PAGE of detergent-extracted canine sperm samples performed at pH 7. Lane 1: positive control, bovine testicular hyaluronidase (50 TRU/ml); lane 2: positive control, bovine testicular hyaluronidase (2.5 TRU/ml); lanes 3±5: samples from dogs 1, 2, and 3, respectively. Note lack of activity in sample lanes.
there was marked variation across individuals, in all cases there was greater activity at pH 4 (Fig. 4). 3.5. Immunohistochemistry of dog testis Abundant staining was observed in the adluminal region of the seminiferous tubules corresponding to the round spermatids (Fig. 5A). The staining was localized to the Golgi region in the cytoplasm in the late spermatids. Cells in the basal part of the seminiferous
Fig. 4. Hyaluronidase activity of detergent-extracted canine sperm as determined by microplate assay for four dogs at pH 4 and 7. Data expressed as mean values S:E.
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Fig. 5. Immunohistochemistry of dog testis. The testes of adult dogs (n 3) were fixed in 4% paraformaldehyde, deparaffinized and hydrated through xylenes and graded ethanol series. Immunoperoxidase staining was performed with the vectastain ABC-Elite kit. The sections were incubated with the anti-PH-20 antibody (A) or control preimmune rabbit serum (B) overnight, at 4 8C. The sections were then incubated for 30 min with diluted biotinylated HRP-linked secondary antibody followed by incubation in ABC reagent and developed with AEC. The sections were quickly counterstained with hematoxylin (400).
tubules (spermatogonia and Sertoli cells) did not stain for PH-20. There was no staining in the control sample (Fig. 5B). 4. Discussion 4.1. Characterization of canine PH-20 In the present study, several forms of PH-20 were recognized in immunoblots of canine sperm extracts. Under non-reducing conditions two higher molecular bands were identi®ed at 209 and 124 kDa in addition to the 50 and 42 kDa bands. Canine PH-20 appears slightly different than PH-20 from most other species. Several higher molecular weight hyaluronidase (PH-20) proteins have also been reported in ram sperm under non-reducing conditions at molecular weight above 300 kDa and form oligomers; with a major band at 89 kDa under reducing conditions [16]. The major band around 50 kDa in canine sperm, is of a lesser apparent molecular weight than PH-20 characterized in the guinea pig [11], macaque [10], or human [4]. In these species, the major form of PH-20 under non-reducing conditions was of 62±66 kDa. The heterologous bands that were present on immunoblots of both non-reducing and reducing SDS-PAGE may be due to the type of antibody used in these experiments. This antibody, R-10, was raised in rabbits against recombinant macaque PH-20. The inherent heterogeneity in this system may have resulted in recognition of multimers and/or cleavage products of PH-20. It may be simply due to variation between species. The differences in apparent molecular weight may be attributed to differences between species in the degree of glycosylation of PH-20, since PH-20 is a glycosylated protein with six sites of N-linked glycosylation [5]. Under reducing conditions, the smaller size of canine PH-20 proteins as compared to other species may also be related to a higher proteolytic activity in the canine sperm, although a cocktail of protease inhibitors was
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included in the sperm extracts to minimize proteolysis. Under non-reducing conditions however, canine sperm PH-20 showed a much higher molecular weight. These electrophoretic differences could re¯ect species differences in disul®de bonds and/or sensitivity to endoproteolytic cleavage. A minor lower molecular weight form (53 kDa) of PH-20 has been observed in the supernatant of acrosome-reacted macaque [10] and human [4] sperm. This may hold true for the dog as well. However, in the present study, we did not examine acrosome-reacted sperm. It is suggested that the lower form of PH-20 produced from the 64 kDa form is a result of the action of acrosomal enzymes such as proteases, glycosidases and phospholipases [10] as is the case for other GPI-anchored proteins. Immunocytochemical staining of PH-20 on canine acrosome-intact sperm demonstrated that PH-20 is localized on the plasma membrane over the entire head, showing a pattern similar to that observed in macaque, human and mouse sperm [3,8]. The presence of PH-20 on the anterior head of acrosome-intact sperm suggests a potential role in penetration of the extracellular matrix (ECM) surrounding the oocyte, as only acrosome-intact cells may pass through the cumulus matrix [8]. In this location, membrane-bound hyaluronidase could depolymerize the cumulus ECM in the immediate vicinity of the acrosome-intact fertilizing sperm. Additionally, label was present on only a fraction of cells, which suggests that the status of individual cells varied markedly in this population. Induction of the acrosome reaction in guinea pig, macaque, human and mouse sperm results in the migration of PH-20 from the plasma membrane to the inner acrosomal membrane, where it plays a role in secondary binding to the zona pellucida [3,4,8,11]. 4.2. Hyaluronidase activity of canine PH-20 Previous research has shown that the N-terminus of PH-20 has signi®cant hyaluronidase activity, which is required for penetration of the ECM surrounding the oocyte. This matrix is composed of cumulus cells and a rich supply of HA. Blocking the hyaluronidase activity of PH-20 with hyaluronidase inhibitors or antibodies to PH-20, results in an inability to penetrate the ECM and thus failure of fertilization [7,8]. Thus, it appears that PH-20 is the only protein responsible for hyaluronidase activity associated with mammalian sperm. Indeed, use of substrate PAGE and the microtiter plate assay for hyaluronidase activity in canine sperm extracts supports the presence of a similar function in the dog. Interestingly, however, PH-20 in the dog appears to have a greater activity at acidic, rather than neutral pH. Substrate PAGE of non-capacitated canine sperm extracts resulted in a diffuse band that was distinct at pH 4, and virtually undetectable at pH 7. This is con®rmed by an increased hyaluronidase activity of extracted canine sperm at acidic pH as determined by the microplate assay. Earlier studies have also suggested that sperm hyaluronidases are acid-active soluble enzymes, which are released at the time of the acrosome reaction [16,17]. In the macaque and human, on the other hand, acrosome-intact sperm exhibit more hyaluronidase activity at pH 7 but still showed some hyaluronidase activity at acidic pH [3,4]. The physiological signi®cance of this difference is as yet unclear. The immunohistochemical localization of PH-20 on canine testis revealed positive labeling in the perinuclear Golgi apparatus of the round spermatids. No staining of other cells was detected. This observation is similar to earlier studies done in guinea pigs [18].
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In these studies, the earliest detection of PH-20 in spermatogenic cells was in the Golgi complex and PH-20 next appeared in the membrane of the developing secretory granule, the acrosome. Data from the present study supports the observation that the PH-20 protein in canine sperm is localized to the head region of acrosome-intact sperm, and has a hyaluronidase activity that is most effective at acidic pH. The current research provides preliminary evidence that its role in fertilization is similar to that in other species, and provides a background necessary for further evaluation of PH-20 as a potential immunocontraceptive agent in the male dog. Acknowledgements This work was supported by the Kenneth A. Scott Charitable Trust, a Keybank Trust. K Foristall was supported by the Geraldine Dodge foundation. The authors thank the VMTH at UC, Davis for providing canine testes, Anthony Vo for technical assistance and Dr. Paul Primakoff for providing the a-PH-20 antibody. References [1] Phelps BM, Primakoff P, Chapel DE, Low MG, Myles DG. Restricted lateral diffusion of PH-20, a GPI-anchored sperm membrane protein. Science 1988;240:1780±2. [2] Primakoff P, Hyatt H, Myles DG. A role for the migrating sperm surface antigen PH-20 in guinea pig sperm binding to the egg zona pellucida. Cell Biol 1985;101:2239±44. [3] Overstreet JW, Lin Y, Yudin AI, Meyers SA, Primakoff P, Myles DG, et al. Location of the PH-20 protein on acrosome-intact and acrosome-reacted spermatozoa of cynomolgus macaques. Biol Reprod 1995;52:105±14. [4] Sabeur K, Cherr GN, Yudin AI, Primakoff P, Li MW, Overstreet JW. The PH-20 protein in human spermatozoa. J Andrology 1997;18:151±8. [5] Lathrop WF, Carmichael EP, Myles DG, Primakoff P. cDNA cloning reveals the molecular structure of a sperm surface protein, PH-20, involved in sperm±egg adhesion and the wide distribution of its gene among mammals. J Cell Biol 1990;111:2939±49. [6] Gmachl M, Sagan S, Ketter S, Kreil G. The human sperm protein PH-20 has hyaluronidase activity. FEBS Lett 1993;336:545±8. [7] Meyers SA, Yudin AI, Cherr GN, VandeVoort CA, Myles DG, Primakoff P, et al. Hyaluronidase activity of macaque sperm assessed by an in vitro cumulus penetration assay. Mol Reprod Dev 1997;46:392±400. [8] Lin Y, Mahan K, Lathrop WF, Myles DG, Primakoff P. A hyaluronidase activity of the sperm plasma membrane protein PH-20 enables sperm to penetrate the cumulus cell layer surrounding the egg. J Cell Biol 1994;125(5):1157±63. [9] Ramarao CS, Myles DG, Primakoff P. Multiple role for PH-20 and fertilin in sperm±egg interactions. Sem Dev Biol 1994;5:265±71. [10] Cherr GN, Meyers SA, Yudin AI, VandeVoort CA, Myles DG, Primakoff P, et al. The PH-20 protein in cynomologus macaque spermatozoa: identi®cation of two different forms exhibiting hyaluronidase activity. Dev Biol 1996;175:142±53. [11] Primakoff P, Cowan A, Hyatt H, Tredick-Kline J, Myles DG. Puri®cation of the guinea pig sperm PH-20 antigen and detection of a site-speci®c endoproteolytic activity in sperm preparations that cleaves PH-20 into two disul®de-linked fragments. Biol Reprod 1988;38:921±34. [12] Primakoff P, Woolman-Gamer L, Tung KSK, Myles DG. Reversible contraceptive effect of PH-20 immunization in male guinea pigs. Biol Reprod 1997;56:1142±6.
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[13] Towbin HT, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979;76:4350±4. [14] Guntenhoner MW, Pogrel MA, Stern R. A substrate gel assay for hyaluronidase activity. Matrix 1992;12:388±96. [15] Tung KSK, Primakoff P, Woolman-Gamer L, Myles DG. Mechanism of infertility in male guinea pigs immunized with sperm PH-20. Biol Reprod 1997;56:1133±41. [16] Harrison RAP. Hyaluronidase in ram semen: quantitative determination and isolation of multiple forms. Biochem J 1988;252:865±74. [17] Zaneveld LJD, Polakoski Kl, Schumacher GFB. Properties of acrosomal hyaluronidase from bull spermatozoa. J Biol Chem 1973;248:564±70. [18] Phelps BM, Myles DG. The guinea pig sperm plasma membrane protein, PH-20, reaches the surface via two transport pathways and becomes localized to a domain after an initial uniform distribution. Dev Biol 1987;123:63±72.
Characterization of PH-20 in canine spermatozoa and testis K. Sabeur, K. Foristall, B.A. Ball* Department of Population Health and Reproduction, University of California, Davis, CA 95616, USA Received 1 May 2001; accepted 18 July 2001
Abstract The purpose of this study was to characterize the sperm membrane protein PH-20 in the dog. Canine spermatozoa were extracted with Triton X-100 and the presence of PH-20 was determined by immunoblot with an antibody against recombinant macaque PH-20. The hyaluronidase activity of canine PH-20 was determined with substrate gel electrophoresis based upon digestion of hyaluronic acid (HA) incorporated into the separating gels. Hyaluronidase activity was also quanti®ed using a microplate assay. Sperm extracts were incubated at pH 4 or 7 in wells containing agarose and HA. For immunolabeling of PH-20 on canine sperm membranes, canine sperm were ®xed and incubated with R-10 primary antibody, and an anti-rabbit IgG-FITC secondary antibody. Samples were visualized by ¯uorescence microscopy. Non-reducing SDS-PAGE and Western blot of detergent-extracted canine sperm revealed a major band at 50 kDa, and three other bands at 42, 124, and >209 kDa. Substrate PAGE revealed translucent bands of hyaluronidase activity of similar size to bovine testicular hyaluronidase. These bands were markedly more pronounced at pH 4 than at pH 7. The microplate assay also demonstrated that hyaluronidase activity was over four times greater at the acidic pH. Immunolabeling of canine spermatozoa demonstrated that PH-20 is localized to the anterior head region and appeared in the Golgi area of round spermatids as detected by the immunohistochemical staining of the testis. This study provides evidence that PH-20 is present on the membrane of canine spermatozoa and in round spermatids. Canine PH-20 exhibits hyaluronidase activity that is markedly more pronounced at acidic pH. # 2002 Elsevier Science Inc. All rights reserved. Keywords: Hyaluronidase; Dog; Sperm; Immunohistochemistry; Substrate PAGE
* Corresponding author. Tel.: 1-530-752-1358; fax: 1-530-752-4278. E-mail address: [email protected] (B.A. Ball).
0093-691X/02/$ ± see front matter # 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 3 - 6 9 1 X ( 0 1 ) 0 0 6 9 7 - 5
978
K. Sabeur et al. / Theriogenology 57 (2002) 977±987
1. Introduction The membrane protein PH-20, similar to various other cell adhesion proteins, is a glycosylphosphatidylinositol (GPI)-anchored sperm-speci®c protein [1]. Initially characterized by Primakoff et al. [2] in guinea pig sperm, PH-20 has been found in many other species including cynomologus macaques and humans [3,4]. The gene encoding PH-20 is highly conserved [5]. Protein PH-20 is now recognized as a bifunctional protein involved in mammalian fertilization. It has been shown to have hyaluronidase activity, a role related to cumulus penetration [6,7] as well as zona binding properties. PH-20 involvement in cumulus penetration was supported by the observation that incubation of acrosome-intact sperm with anti-PH-20 antibodies or hyaluronidase inhibitors prevented penetration of the cumulus layer in vitro [7,8]. Its second role may be related to secondary binding of sperm to the zona pellucida following the acrosome reaction [3,8]. The role of PH-20 in secondary binding is supported by the fact that incubation of acrosome-reacted sperm with anti-PH-20 antibodies prevents binding of sperm to zona pellucida proteins [2]. The aminoterminal 300 amino acids of PH-20 are responsible for its hyaluronidase activity while the C-terminus could be involved in sperm±zona pellucida interactions [9]. Protein PH-20 has been localized to the plasma membrane of acrosome-intact sperm of mice, macaques and humans [3,4,8]. It is also expressed on the inner acrosomal membranes [10]. Induction of the acrosome reaction appears to result in migration of this protein from the plasma membrane to the inner acrosomal membrane in macaque [10], guinea pig [2], and human [4] sperm. Recent work has provided promising results regarding the use of PH-20 as an immunocontraceptive agent. Primakoff et al. were successful in their attempt to induce infertility in both male and female guinea pigs immunized with the PH-20 protein [11,12]. Depending on vaccination protocol, immunization of males can result in long-term (>1 year) or even irreversible infertility. Thus, it appears that PH-20 is a candidate for further investigation into the use of sperm-speci®c antigens as immunocontraceptive agents in dogs. Before PH-20 can be examined as a potential contraceptive agent in the dog, however, its presence on dog sperm must be veri®ed, and basic structural and biochemical properties must be characterized. Thus, the current research involved the following objectives: (1) determination of PH-20 expression on canine sperm; (2) characterization of hyaluronidase activity of canine PH-20. The data represents a preliminary evaluation of PH-20 as a possible candidate for immunocontraception in dogs. 2. Materials and methods 2.1. Animals Four intact, mixed breed dogs were obtained from the local animal shelter to be used as semen donors for the current experiments. Semen was collected into 15 ml polystyrene conical centrifuge tubes by manual manipulation and use of an arti®cial vagina.
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After collection, semen was immediately placed on ice for transport to the laboratory. Canine testes were obtained with client permission and after castration from client animals presented to the Veterinary Medical Teaching Hospital of the University of California, Davis. Tissues were stored at 70 8C until used. 2.2. Extraction of sperm membranes Semen (1 ml) was resuspended in 10 ml modi®ed Tyrodes's medium containing 0.1% polyvinyl alcohol (TALP-PVA) and centrifuged (300 g for 10 min). The sperm pellet was resuspended in 8 ml TALP-PVA and recentrifuged at the same speed. Pellet was then diluted to a ®nal concentration of approximately 200 106 sperm/ml. A cocktail of protease inhibitors was added to each sperm suspension (50 mM Tris, pH 7.4, 20 mM EDTA, 1 mM p-hydroxy-mercurobenzoate, 5 mM N-ethylmaleimide, and 1 mM p-benzamidine hydrochloride, ®nal concentrations). Samples were solubilized in 2% Triton X-100, vortexed for 5 min, and then centrifuged for 15 min at 10,000 g. The supernatant (which contained the solubilized membrane proteins) was combined with protease inhibitors (as above), aliquoted, and measured for protein (Bradford assay, BioRad, Hercules, CA). Samples were stored at 20 8C until use in Western blot analysis, substrate gel electrophoresis or microplate assay for hyaluronidase activity. 2.3. Electrophoresis and Western blot analysis For SDS-PAGE analysis, sperm membrane extracts were mixed with either nonreducing (62.5 mM Tris, 1% SDS, 10% glycerol and 0.5% bromophenol blue) or reducing (above, plus 5 mM b-mercaptoethanol) solubilization buffer. Samples were then heated at 95 8C for 5 min prior to electrophoresis. SDS-PAGE was performed as previously described [4]. Western blot analysis was carried out according to [13]. Brie¯y, after transfer, nitrocellulose membranes were placed in blocking solution, 3% gel Tris buffered saline±Tween 20 (TBS-T) for 4 h, and washed three times in TBS-T (10 min each). Membranes were then probed with antisera (R-10, 1:5880 [6]) raised in rabbits against recombinant cynomologus macaque PH-20 [8] for 15±20 h at room temperature. After three washes in TBS-T, membranes were incubated with secondary antibody, an anti-rabbit IgG conjugated to horseradish peroxidase (1:5000), in 1% gel TBS-T. Blots were visualized using a chemiluminescence (ECL) kit (Amersham, Arlington Heights, IL). 2.4. Immunolocalization of PH-20 on canine sperm membranes Immunolabeling of sperm was carried out by the method of Overstreet et al. [3]. Brie¯y, raw semen was diluted 1:1 in TALP-PVA for transport to the lab. Semen was then subjected to a two-step Percoll gradient, and then centrifuged (20 min at 300 g). Sperm pellets were washed in 10 ml TALP-PVA, and centrifuged again for 10 min. Samples were then resuspended to a ®nal concentration of approximately 20 106 sperm/ml. Aliquots of 500 ml were ®xed in 1% paraformaldehyde for 8 min at room temperature, and washed twice in DPBS by centrifugation. Sperm pellets were then resuspended in blocking agent (DPBS with 1% BSA, Sigma, St. Louis, MO) and incubated at room temperature
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for 30 min. Aliquots were centrifuged at 300 g for 10 min and resuspended in 300 ml DPBS-BSA. Primary antibody (R-10) or preimmune rabbit sera (control) was added at 1:100 dilution. Following incubation at 37 8C for 60 min, samples were washed twice, and then resuspended in 100 ml DPBS-BSA. The secondary antibody, FITC labeled goat antirabbit IgG was added at 1:20 dilution (v/v), and samples were incubated for another 60 min at 37 8C in dark. Each sample was then washed twice in DPBS-BSA, resuspended, and examined by epi¯uorescence microscopy. 2.5. Hyaluronic acid-substrate gel electrophoresis Sperm extracts were subjected to substrate gel electrophoresis according to Guntenhoner et al. [14]. Brie¯y, 7.5% acrylamide separating gels were prepared with 170 mg/ml hyaluronic acid (HA). The 4.5% acrylamide stacking gel did not contain HA. A commercial bovine testicular hyaluronidase (Sigma, St. Louis, MO, USA) was used as the positive control, at concentrations of 2.5 and 50 TRU/ml. Sperm extracts were electrophoresed for 1 h, and then rinsed in TBS containing 3% Triton X-100 for 2 h at room temperature. Gels were incubated in either pH 4 or 7 sodium acetate buffer for 18±20 h at room temperature. Gels were then washed twice in distilled water, stained with 0.2% alcian blue in 3% acetic acid for 2 h, and ®nally destained in 7% acetic acid. 2.6. Microplate assay for hyaluronidase activity Hyaluronidase activity of sperm extracts was determined quantitatively using a microplate assay described by Tung et al. [15]. Brie¯y, agarose was dissolved in warm sodium formate buffer (0.1 M, pH 4) or sodium phosphate buffer (0.3 M, pH 7) to get a ®nal concentration of 0.8% (w/v). Then, a solution of HA was mixed with the agarose to give a ®nal concentration of 0.8 mg/ml HA, and maintained at 55 8C before use. Ninety-six well plates were prewashed with either the pH 4 or 7 buffer. The agarose±HA solutions were pipetted into the microtiter wells (100 ml per well) and allowed to solidify at room temperature. The standard curve was produced by serial dilutions of a commercial bovine testicular hyaluronidase. Activity was expressed in terms of turbidity reducing units/ml, and ranged from 30 to 0.23 TRU/ml. Sperm extracts were thawed and diluted in either pH 4 or 7 buffer. Standards and samples were assayed in triplicate and repeated at both pH 4 and 7. Each standard or unknown was pipetted into the plate (100 ml per well) and incubated for 7 h at 37 8C. Following incubation, plates were washed three times in the appropriate buffer. A solution of cetylpyridinium chloride (CPC, 10% w/v) was pipetted across each plate (100 ml per well) in order to precipitate any undigested HA. Optical density was then recorded at 590 nm with a spectrometer (PE HTS 7000, Norwalk, CT); a higher optical density re¯ected a lower hyaluronidase activity. 2.7. Immunohistochemical localization of PH-20 in canine testis The testes of adult dogs were sliced and ®xed in 4% paraformaldehyde. After a series of graded ethanol incubations, tissues were embedded in paraf®n and sectioned at 5 mm. The tissue sections were then deparaf®nized and hydrated through xylenes and graded
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ethanol series. Immunoperoxidase staining was performed with the vectastain ABC-Elite kit (Vector labs, Burlingame, CA). The sections were incubated for 30 min in 0.3% H2O2± methanol to quench endogenous peroxidase activity. After a wash in PBS 0.3% Triton X100, the sections were incubated for 20 min with diluted blocking serum, washed and incubated with the anti-PH-20 antibody (1:100) overnight at 4 8C. The sections were then incubated for 30 min with biotinylated HRP-linked secondary antibody (1:200) followed by incubation in ABC reagent and developed with 3-amino-9-ethylcarbazole (AEC kit, Vector labs, Burlingame, CA). The sections were quickly counterstained with hematoxylin. Normal rabbit serum was used as the negative control. 2.8. Statistical analysis Data (hyaluronidase activity) were analyzed by ANOVA (StatView, Cary, NC). Differences between treatments were assessed with Fisher's protected least signi®cant difference test (StatView). Means were considered different at P < 0:05. Results are expressed standard error. 3. Results 3.1. Electrophoresis and Western blot analysis Under reducing conditions, SDS-PAGE and subsequent Western blot analysis of canine sperm extracts showed three bands of apparent molecular weights at 52, 40 and 18 kDa. Under non-reducing conditions, the anti-PH-20 antibody recognized one major band at 50 kDa, as well as three other bands at 42, 124, and >209 kDa (Fig. 1). 3.2. Localization of PH-20 on canine sperm membranes Labeling of canine sperm with polyclonal antibody to recombinant macaque PH-20 revealed that most of the label was localized to the sperm head of approximately 50% of cells (Fig. 2A). There was a punctate appearance to the label that was most evident on the anterior sperm head. No label was observed in control samples (Fig. 2B). Some faint ¯uorescence was present in the post-acrosomal region of both control and treated cells, but this ®nding was inconsistent across repeated experiments. 3.3. Hyaluronic acid-substrate gel electrophoresis Hyaluronidase activity of PH-20 in detergent-extracted canine sperm suspensions was determined with HA-substrate PAGE. Staining of gels with alcian blue revealed translucent bands that corresponded to areas of HA digestion. At pH 4, a doublet appeared that was similar in molecular weight to the positive control, bovine testicular hyaluronidase (Fig. 3A). At pH 7, very little activity was detected. However, the positive control was more active at pH 7, as evidenced by large, diffuse bands (Fig. 3B) indicating the assay conditions were appropriate.
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Fig. 1. Immunoblot of SDS-PAGE of canine sperm (10 mg protein per lane) underreducing (R) and nonreducing (NR) conditions. Blots were probed with antibody (R-10) to recombinant macaque PH-20. Secondary antibody was a HRP-conjugated goat anti-rabbit IgG. Immunoreactive proteins were detected by enhanced chemiluminescence.
3.4. Microplate assay for hyaluronidase activity Quantitative data on hyaluronidase activity in canine sperm extracts was obtained by use of a microplate assay. The results of this experiment supported the observation that canine sperm hyaluronidase activity is greater at acidic, rather than neutral pH. Although
Fig. 2. (A) Immunolocalization of PH-20 on acrosome-intact canine sperm using a polyclonal antibody raised against recombinant cynomologus macaque PH-20 and a FITC-conjugated goat anti-rabbit secondary antibody. Note the presence of label localized to the head of approximately 50% of sperm. (B) Acrosome-intact canine sperm incubated with preimmune rabbit serum and FITC-conjugated goat anti-rabbit IgG. Note absence of fluorescent label.
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Fig. 3. (A) Hyaluronic acid substrate PAGE of detergent-extracted canine sperm samples performed at pH 4. Lane 1: positive control, bovine testicular hyaluronidase (50 TRU/ml); lane 2: positive control, bovine testicular hyaluronidase (2.5 TRU/ml); lane 3±5: samples from dogs 1, 2, and 3, respectively. Note doublet of activity in sample lanes. (B) Hyaluronic acid substrate PAGE of detergent-extracted canine sperm samples performed at pH 7. Lane 1: positive control, bovine testicular hyaluronidase (50 TRU/ml); lane 2: positive control, bovine testicular hyaluronidase (2.5 TRU/ml); lanes 3±5: samples from dogs 1, 2, and 3, respectively. Note lack of activity in sample lanes.
there was marked variation across individuals, in all cases there was greater activity at pH 4 (Fig. 4). 3.5. Immunohistochemistry of dog testis Abundant staining was observed in the adluminal region of the seminiferous tubules corresponding to the round spermatids (Fig. 5A). The staining was localized to the Golgi region in the cytoplasm in the late spermatids. Cells in the basal part of the seminiferous
Fig. 4. Hyaluronidase activity of detergent-extracted canine sperm as determined by microplate assay for four dogs at pH 4 and 7. Data expressed as mean values S:E.
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Fig. 5. Immunohistochemistry of dog testis. The testes of adult dogs (n 3) were fixed in 4% paraformaldehyde, deparaffinized and hydrated through xylenes and graded ethanol series. Immunoperoxidase staining was performed with the vectastain ABC-Elite kit. The sections were incubated with the anti-PH-20 antibody (A) or control preimmune rabbit serum (B) overnight, at 4 8C. The sections were then incubated for 30 min with diluted biotinylated HRP-linked secondary antibody followed by incubation in ABC reagent and developed with AEC. The sections were quickly counterstained with hematoxylin (400).
tubules (spermatogonia and Sertoli cells) did not stain for PH-20. There was no staining in the control sample (Fig. 5B). 4. Discussion 4.1. Characterization of canine PH-20 In the present study, several forms of PH-20 were recognized in immunoblots of canine sperm extracts. Under non-reducing conditions two higher molecular bands were identi®ed at 209 and 124 kDa in addition to the 50 and 42 kDa bands. Canine PH-20 appears slightly different than PH-20 from most other species. Several higher molecular weight hyaluronidase (PH-20) proteins have also been reported in ram sperm under non-reducing conditions at molecular weight above 300 kDa and form oligomers; with a major band at 89 kDa under reducing conditions [16]. The major band around 50 kDa in canine sperm, is of a lesser apparent molecular weight than PH-20 characterized in the guinea pig [11], macaque [10], or human [4]. In these species, the major form of PH-20 under non-reducing conditions was of 62±66 kDa. The heterologous bands that were present on immunoblots of both non-reducing and reducing SDS-PAGE may be due to the type of antibody used in these experiments. This antibody, R-10, was raised in rabbits against recombinant macaque PH-20. The inherent heterogeneity in this system may have resulted in recognition of multimers and/or cleavage products of PH-20. It may be simply due to variation between species. The differences in apparent molecular weight may be attributed to differences between species in the degree of glycosylation of PH-20, since PH-20 is a glycosylated protein with six sites of N-linked glycosylation [5]. Under reducing conditions, the smaller size of canine PH-20 proteins as compared to other species may also be related to a higher proteolytic activity in the canine sperm, although a cocktail of protease inhibitors was
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included in the sperm extracts to minimize proteolysis. Under non-reducing conditions however, canine sperm PH-20 showed a much higher molecular weight. These electrophoretic differences could re¯ect species differences in disul®de bonds and/or sensitivity to endoproteolytic cleavage. A minor lower molecular weight form (53 kDa) of PH-20 has been observed in the supernatant of acrosome-reacted macaque [10] and human [4] sperm. This may hold true for the dog as well. However, in the present study, we did not examine acrosome-reacted sperm. It is suggested that the lower form of PH-20 produced from the 64 kDa form is a result of the action of acrosomal enzymes such as proteases, glycosidases and phospholipases [10] as is the case for other GPI-anchored proteins. Immunocytochemical staining of PH-20 on canine acrosome-intact sperm demonstrated that PH-20 is localized on the plasma membrane over the entire head, showing a pattern similar to that observed in macaque, human and mouse sperm [3,8]. The presence of PH-20 on the anterior head of acrosome-intact sperm suggests a potential role in penetration of the extracellular matrix (ECM) surrounding the oocyte, as only acrosome-intact cells may pass through the cumulus matrix [8]. In this location, membrane-bound hyaluronidase could depolymerize the cumulus ECM in the immediate vicinity of the acrosome-intact fertilizing sperm. Additionally, label was present on only a fraction of cells, which suggests that the status of individual cells varied markedly in this population. Induction of the acrosome reaction in guinea pig, macaque, human and mouse sperm results in the migration of PH-20 from the plasma membrane to the inner acrosomal membrane, where it plays a role in secondary binding to the zona pellucida [3,4,8,11]. 4.2. Hyaluronidase activity of canine PH-20 Previous research has shown that the N-terminus of PH-20 has signi®cant hyaluronidase activity, which is required for penetration of the ECM surrounding the oocyte. This matrix is composed of cumulus cells and a rich supply of HA. Blocking the hyaluronidase activity of PH-20 with hyaluronidase inhibitors or antibodies to PH-20, results in an inability to penetrate the ECM and thus failure of fertilization [7,8]. Thus, it appears that PH-20 is the only protein responsible for hyaluronidase activity associated with mammalian sperm. Indeed, use of substrate PAGE and the microtiter plate assay for hyaluronidase activity in canine sperm extracts supports the presence of a similar function in the dog. Interestingly, however, PH-20 in the dog appears to have a greater activity at acidic, rather than neutral pH. Substrate PAGE of non-capacitated canine sperm extracts resulted in a diffuse band that was distinct at pH 4, and virtually undetectable at pH 7. This is con®rmed by an increased hyaluronidase activity of extracted canine sperm at acidic pH as determined by the microplate assay. Earlier studies have also suggested that sperm hyaluronidases are acid-active soluble enzymes, which are released at the time of the acrosome reaction [16,17]. In the macaque and human, on the other hand, acrosome-intact sperm exhibit more hyaluronidase activity at pH 7 but still showed some hyaluronidase activity at acidic pH [3,4]. The physiological signi®cance of this difference is as yet unclear. The immunohistochemical localization of PH-20 on canine testis revealed positive labeling in the perinuclear Golgi apparatus of the round spermatids. No staining of other cells was detected. This observation is similar to earlier studies done in guinea pigs [18].
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In these studies, the earliest detection of PH-20 in spermatogenic cells was in the Golgi complex and PH-20 next appeared in the membrane of the developing secretory granule, the acrosome. Data from the present study supports the observation that the PH-20 protein in canine sperm is localized to the head region of acrosome-intact sperm, and has a hyaluronidase activity that is most effective at acidic pH. The current research provides preliminary evidence that its role in fertilization is similar to that in other species, and provides a background necessary for further evaluation of PH-20 as a potential immunocontraceptive agent in the male dog. Acknowledgements This work was supported by the Kenneth A. Scott Charitable Trust, a Keybank Trust. K Foristall was supported by the Geraldine Dodge foundation. The authors thank the VMTH at UC, Davis for providing canine testes, Anthony Vo for technical assistance and Dr. Paul Primakoff for providing the a-PH-20 antibody. References [1] Phelps BM, Primakoff P, Chapel DE, Low MG, Myles DG. Restricted lateral diffusion of PH-20, a GPI-anchored sperm membrane protein. Science 1988;240:1780±2. [2] Primakoff P, Hyatt H, Myles DG. A role for the migrating sperm surface antigen PH-20 in guinea pig sperm binding to the egg zona pellucida. Cell Biol 1985;101:2239±44. [3] Overstreet JW, Lin Y, Yudin AI, Meyers SA, Primakoff P, Myles DG, et al. Location of the PH-20 protein on acrosome-intact and acrosome-reacted spermatozoa of cynomolgus macaques. Biol Reprod 1995;52:105±14. [4] Sabeur K, Cherr GN, Yudin AI, Primakoff P, Li MW, Overstreet JW. The PH-20 protein in human spermatozoa. J Andrology 1997;18:151±8. [5] Lathrop WF, Carmichael EP, Myles DG, Primakoff P. cDNA cloning reveals the molecular structure of a sperm surface protein, PH-20, involved in sperm±egg adhesion and the wide distribution of its gene among mammals. J Cell Biol 1990;111:2939±49. [6] Gmachl M, Sagan S, Ketter S, Kreil G. The human sperm protein PH-20 has hyaluronidase activity. FEBS Lett 1993;336:545±8. [7] Meyers SA, Yudin AI, Cherr GN, VandeVoort CA, Myles DG, Primakoff P, et al. Hyaluronidase activity of macaque sperm assessed by an in vitro cumulus penetration assay. Mol Reprod Dev 1997;46:392±400. [8] Lin Y, Mahan K, Lathrop WF, Myles DG, Primakoff P. A hyaluronidase activity of the sperm plasma membrane protein PH-20 enables sperm to penetrate the cumulus cell layer surrounding the egg. J Cell Biol 1994;125(5):1157±63. [9] Ramarao CS, Myles DG, Primakoff P. Multiple role for PH-20 and fertilin in sperm±egg interactions. Sem Dev Biol 1994;5:265±71. [10] Cherr GN, Meyers SA, Yudin AI, VandeVoort CA, Myles DG, Primakoff P, et al. The PH-20 protein in cynomologus macaque spermatozoa: identi®cation of two different forms exhibiting hyaluronidase activity. Dev Biol 1996;175:142±53. [11] Primakoff P, Cowan A, Hyatt H, Tredick-Kline J, Myles DG. Puri®cation of the guinea pig sperm PH-20 antigen and detection of a site-speci®c endoproteolytic activity in sperm preparations that cleaves PH-20 into two disul®de-linked fragments. Biol Reprod 1988;38:921±34. [12] Primakoff P, Woolman-Gamer L, Tung KSK, Myles DG. Reversible contraceptive effect of PH-20 immunization in male guinea pigs. Biol Reprod 1997;56:1142±6.
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[13] Towbin HT, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979;76:4350±4. [14] Guntenhoner MW, Pogrel MA, Stern R. A substrate gel assay for hyaluronidase activity. Matrix 1992;12:388±96. [15] Tung KSK, Primakoff P, Woolman-Gamer L, Myles DG. Mechanism of infertility in male guinea pigs immunized with sperm PH-20. Biol Reprod 1997;56:1133±41. [16] Harrison RAP. Hyaluronidase in ram semen: quantitative determination and isolation of multiple forms. Biochem J 1988;252:865±74. [17] Zaneveld LJD, Polakoski Kl, Schumacher GFB. Properties of acrosomal hyaluronidase from bull spermatozoa. J Biol Chem 1973;248:564±70. [18] Phelps BM, Myles DG. The guinea pig sperm plasma membrane protein, PH-20, reaches the surface via two transport pathways and becomes localized to a domain after an initial uniform distribution. Dev Biol 1987;123:63±72.