The carbohydrate structure of DEFB126, the major component of the cynomolgus Macaque sperm plasma membrane glycocalyx.

Based on the amino-acid sequence of the macaque epididymal secretory protein, ESP 13.2 (Q9BEE3/AJ236909), it has now been classified as beta-defensin DEFB126. DEFB126 is one of the five beta-defensins with genes that are clustered along chromosome 20pl3, and all five proteins have an extended carboxy terminus that continues beyond the 6-cysteine beta-defensin core region. This 60-amino acid carboxyl tail extension of the DEFB126 molecule is extraordinarily rich in threonine and serine (40%), many of which appear to be likely candidates for having O-glycosylation. DEFB126 has been shown to coat the entire surface of cynomolgus macaque sperm as they move through the corpus/caudal region of the epididymis. It is a major glycocalyx barrier to the external environment and is retained until the completion of capacitation. Sperm exposed to fluorescein-conjugated poly-L-lysine or Alexa488-histone showed a very uniform fluorescent labeling pattern over the entire sperm surface, almost identical to that observed with anti-DEFB126 Ig label. Sperm surface components that were released following treatment with caffeine/cAMP (in vitro capacitation) were blotted and probed with three different lectins which are known to recognize terminal sialic acid residues, and all three recognized the 35 kDa DEFB126 band. Neuraminidase treatment of sperm shifted the molecular weight of DEFB126 from 34-36 kDa to approximately 38-40 kDa and removed or greatly inhibited sialic acid-specific lectin recognition. O-Glycanase treatment alone was ineffective at removal of the oligosaccharides, but prior treatment with neuraminidase was sufficient to enable the O-glycanase treatment to effectively change the apparent molecular weight to 10 kDa, confirming that a major portion of the molecular mass is associated with the carbohydrate portion. Western blots of neuraminidase-treated DEFB126 showed strong recognition with a number of lectins that identify beta-galactose and also lectins that recognize the N-acetylgalactosamine-serine/threonine, the proposed connection site of O-glycosylation. All of the lectins that recognized DEFB126 on Western blots were used to fluorescently probe sperm. The fluorescent patterns that were observed with poly-L-lysine, Alexa488-histone, sialic acid-specific lectins, and galactose-specific lectins showed even distributions over the entire sperm surface and the patterns were identical to sperm labeled with anti-DEFB126 Ig, and all but the antibody did not recognize neuraminidase-treated sperm.

Identification of a novel GPI-anchored CRISP glycoprotein, MAK248, located on the posterior head and equatorial segment of cynomolgus macaque sperm.

To identify a sperm-surface component that is highly antigenic, we immunized female cynomolgus macaques with glycosylphosphatidylinositol (GPI)-anchored sperm surface proteins that were released following treatment with phosphatidylinositol-specific phospholipase C (PI-PLC). Five different adjuvants were used in combination with the PI-PLC-released proteins, and three of these proteins (24, 48, and 53 kDa) were shown to be potent antigens for immunization of female monkeys. The 53 kDa protein was found to be a surface coating protein and not a GPI-anchored protein. Polyclonal antibodies to the 24 kDa protein and the 48 kDa protein were produced in rabbits. The two antibodies recognized both proteins on Western blots. The same rabbit antibodies recognized 28, 18, and 10 kDa bands on a Western blot of chemically reduced PI-PLC-released proteins, suggesting that the 48 kDa protein is a dimer of the 24 kDa protein, which we refer to as MAK248. Rabbit polyclonal antibodies developed to reduced fragments of the 24 kDa protein showed that the 18 and 10 kDa bands are proteolytic peptide fragments of the 24 kDa protein. Screening of tissues from male macaques showed that MAK248 is expressed only in the epididymis. Microsequencing of two proteolytic fragments of the 18 kDa component showed 100% amino acid homology to a 233 deduced amino acid sequence previously identified in human testes genome. Antibodies to MAK248 recognized a 24 kDa protein released from human sperm exposed to PI-PLC. Antibodies to MAK248 recognized the equatorial segment and posterior head regions of capacitated cynomolgus macaque sperm. Structural analysis suggests that MAK248 is a novel CRISP protein and a member of the CAP (CRISP, Ag 5, PR-1) family of proteins. Based on amino acid sequence homology, it is possible that MAK248 functions as a protease inhibitor.

Immunological response of female macaques to the PH-20 sperm protein following injection of recombinant proteins or synthesized peptides.

Because of its location on the sperm surface and its multiple functions during fertilization, the PH-20 protein is a potential target for contraceptive vaccines. Cynomolgus macaques were immunized using four different adjuvants together with synthesized peptides or recombinant proteins representing selected regions of macaque PH-20. The synthesized peptide (amino acids 387-412, designated Peptide 4) was used as a linear molecule in a 1:1 ratio with a peptide sequence of tetanus toxoid, as well as a multiple antigenic peptide (MAP) matrix held together by scaffolding lysine residues. In the MAP construct, the ratio of Peptide 4 to tetanus peptide was 4:1. To circumvent the poor production of recombinant PH-20 in bacterial cells, two truncated forms of the molecule were expressed in Escherichia coli, G18 (encoding amino acids 143-510) and E10 (encoding amino acids 291-510). The adjuvants were Montanide ISA 51, Titermax Gold, Syntex adjuvant formulation (SAF), and QS-21. All of the antigen/adjuvant combinations produced significant immune responses as measured by ELISA. The circulating antibodies from immunized animals recognized macaque sperm surface PH-20 on Western blots and were shown by indirect immunofluorescence to bind to the surface of macaque sperm. Montanide and Titermax were associated with higher titers of anti-PH-20 antibodies than QS-21 and SAF adjuvants. Immunization with Titermax, however, resulted in sterile abscesses in 4 of 8 animals injected. We conclude that antigens derived from synthesized peptides and recombinant proteins representing selected regions of the PH-20 molecule can be used as vaccine components in combination with the adjuvant Montanide to elicit a significant sperm-directed antibody response in immunized macaques.

The dual functions of GPI-anchored PH-20: hyaluronidase and intracellular signaling.

The ovulated mammalian oocyte is surrounded by the "cumulus ECM", composed of cells embedded in an extracellular matrix that is rich in hyaluronic acid (HA). The cumulus ECM is a viscoelastic gel that sperm must traverse prior to fertilization. Mammalian sperm have a GPI-anchored hyaluronidase which is known as PH-20 and also as SPAM 1. PH-20 is located on the sperm surface, and in the lysosome-derived acrosome, where it is bound to the inner acrosomal membrane. PH-20 appears to be a multifunctional protein; it is a hyaluronidase, a receptor for HA-induced cell signaling, and a receptor for the zona pellucida surrounding the oocyte. The zona pellucida recognition function of PH-20 was discovered first. This function is ascribed to the inner acrosomal membrane PH-20, which appears to differ biochemically from the PH-20 on the sperm surface. Later, when bee venom hyaluronidase was cloned, a marked cDNA sequence homology with PH-20 was recognized, and it is now apparent that PH-20 is the hyaluronidase of mammalian sperm. PH-20 is unique among the hyaluronidases in that it has enzyme activity at both acid and neutral pH, and these activities appear to involve two different domains in the protein. The neutral enzyme activity of plasma membrane PH-20 is responsible for local degradation of the cumulus ECM during sperm penetration. Plasma membrane PH-20 mediates HA-induced sperm signaling via a HA binding domain that is separate from the hyaluronidase domains. This signaling is associated with an increase in intracellular calcium and as a consequence, the responsiveness of sperm to induction of the acrosome reaction by the zona pellucida is increased. There is extensive evidence that GPI-anchored proteins are involved in signal transduction initiated by a diverse group of cell surface receptors. GPI-anchored proteins involved in signaling are often associated with signaling proteins bound to the cytoplasmic leaflet of the plasma membrane, typically Src family, non-receptor protein tyrosine kinases. PH-20 appears to initiate intracellular signaling by aggregating in the plasma membrane, and a 92-kDa protein may be the cell signaling molecule linked to PH-20.

Identification of a hyaluronic acid (HA) binding domain in the PH-20 protein that may function in cell signaling.

The macaque sperm surface protein PH-20 is a hyaluronidase, but it also interacts with hyaluronic acid (HA) to increase internal calcium ( [Ca(2+)](i) ) in the sperm cell. A region of the PH-20 molecule, termed Peptide 2 (aa 205-235), has amino acid charge homology with other HA binding proteins. The Peptide 2 sequence was synthesized and two recombinant PH-20 proteins were developed, one containing the Peptide 2 region (G3, aa 143-510) and one without it (E12, aa 291-510). On Western blots, affinity-purified anti-Peptide 2 IgG recognized the 64 kDa band corresponding to PH-20 in acrosome intact sperm and, under reducing conditions, recognized the whole 67 kDa PH-20 and the endoproteolyzed N-terminal fragment of PH-20. HA conjugated to a photoaffinity substrate specifically bound to sperm surface PH-20. Indirect immunofluorescence demonstrated that Fab fragments of anti-Peptide 2 IgG bound to the head of live sperm. Biotinylated HA was bound by Peptide 2 and by sperm extracts in a microplate binding assay, and this binding was inhibited by Fab fragments of anti-Peptide 2 IgG. Biotinylated HA bound to the G3 protein and this binding was inhibited by anti-Peptide 2 Fab, but HA did not bind to the E12 protein. Fab fragments of anti-Peptide 2 IgG inhibited the increase in [Ca(2+)](i) induced in macaque sperm by HA. Our results suggest that the Peptide 2 region of PH-20 is involved in binding HA, which results in the cell signaling events related to the elevation of [Ca(2+)](i) during sperm penetration of the cumulus.

Diffusion of macromolecules and virus-like particles in human cervical mucus.

To determine whether or not large macromolecules and viruses can diffuse through mucus, we observed the motion of proteins, microspheres, and viruses in fresh samples of human cervical mucus using fluorescent recovery after photobleaching and multiple image photography. Two capsid virus-like particles, human papilloma virus (55 nm, approximately 20,000 kDa) and Norwalk virus (38 nm, approximately 10,000 kDa), as well as most of the globular proteins tested (15-650 kDa) diffused as rapidly in mucus as in saline. Electron microscopy of cervical mucus confirmed that the mesh spacing between mucin fibers is large enough (20-200 nm) for small viruses to diffuse essentially unhindered through mucus. In contrast, herpes simplex virus (180 nm) colocalized with strands of thick mucus, suggesting that herpes simplex virus, unlike the capsid virus particles, makes low-affinity bonds with mucins. Polystyrene microspheres (59-1000 nm) bound more tightly to mucins, bundling them into thick cables. Although immunoglobulins are too small to be slowed by the mesh spacing between mucins, diffusion by IgM was slowed by mucus. Diffusion by IgM-Fc(5 mu), the Fc pentamer core of an IgM with all 10 Fab moieties removed, was comparably slowed by mucus. This suggests that the Fc moieties of antibodies make low-affinity bonds with mucins.

Characterization of the active site of monkey sperm hyaluronidase.

The mammalian sperm hyaluronidase, PH-20, is active in macaque spermatozoa at neutral and acid pH. Antibodies were produced to synthesized peptides representing regions of PH-20 that may be involved in hyaluronidase activity and designated peptide 1 (amino acid sequence 142-172) and peptide 3 (amino acid sequence 277-297). Western blotting of proteins extracted from the surface of acrosome-intact spermatozoa showed that the two peptide-specific, affinity-purified IgGs label a 64 kDa band corresponding to the PH-20 molecule. Western blots of acrosome-reacted spermatozoa showed that, under reducing conditions, the two anti-peptide IgGs label the 44 kDa band only, which represents the N-terminal fragment of PH-20. Anti-peptide 3 IgG also labels the 53 kDa form of PH-20 in extracts of acrosome-reacted spermatozoa. Peptide-specific, affinity-purified Fab fragments from both IgGs were shown by fluorescence microscopy and transmission electron microscopy to label the sperm plasma membrane, fused acrosomal vesicles, acrosomal matrix and inner acrosomal membrane. Fab fragments of anti-peptide 1 IgG, but not anti-peptide 3 IgG, inhibited hyaluronidase activity of PH-20 from the sperm surface and from extracts of acrosome-reacted spermatozoa at pH 7.0. Fab fragments of both anti-peptide IgGs inhibited sperm hyaluronidase activity at pH 5.0. It is concluded that the region of PH-20 encompassed by the amino acid sequence 142-172 is essential for hyaluronidase activity at neutral pH, whereas the region of amino acid sequence 277-297 may be more important at a lower pH. It is likely that these two regions are the acid/base catalyst site and the nucleophilic site, respectively, of PH-20 hyaluronidases.

Calpain and calpastatin are located between the plasma membrane and outer acrosomal membrane of cynomolgus macaque spermatozoa.

Mammalian sperm must undergo an acrosome reaction prior to penetration of the zona pellucida and subsequent fusion with an oocyte. Sperm gain the capability to acrosome react after a period of capacitation, which primarily involves biochemical changes in the sperm membranes. The morphological events of the acrosome reaction have been well-documented, but the underlying cellular mechanisms that regulate capacitation and the acrosome reaction remain unclear. Antibodies to the 2 ubiquitous calpains, mu and m, as well as the small subunit, which associates with both calpains, were localized at the ultrastructural level to the region between the plasma membrane and the outer acrosomal membrane of cynomolgus macaque sperm. After the acrosome reaction, all of the anti-calpain antibodies labeled the acrosomal shroud, suggesting that calpains are located throughout the cytoplasmic area between the 2 outer sperm membranes. Calpastatin is an endogenous modulator of calpain activity and is also localized within the same cytoplasmic region as calpains. The antibodies used for ultrastructural localization were also used to probe Western blots of sperm extracts. Antibodies to either the mu- or m-calpain recognized an 80-kd protein, which is similar to the molecular weights of other ubiquitous calpains described. The small subunit (30 kd) was also recognized with a specific monoclonal antibody. An antibody to calpastatin recognized a major band at 78 kd and a lighter band at 45 kd, while the antibody to the testis-specific isoform of calpastatin (TCAST) recognized a 110-kd protein. We hypothesize that this cysteine protease system may be functional in cynomolgus macaque sperm during capacitation, the acrosome reaction, or both.

Acrosomal status and motility of guinea pig spermatozoa during in vitro penetration of the cumulus oophorus.

Previous studies have suggested that both acrosome-intact and acrosome-reacted guinea pig sperm are capable of binding to the zona pellucida of cumulus-free oocytes, but the acrosomal status of guinea pig sperm during penetration of the cumulus has not been reported. We made video recordings of the interaction between capacitated guinea pig sperm and cumulus-invested guinea pig oocytes. The videotapes were analysed to identify sperm with hyperactivated motility and to classify the acrosomal status of sperm during penetration of the cumulus and after binding to the zona pellucida. The resolution of the video recordings was not sufficient to recognise sperm with swollen acrosomes. However, sperm that had completed the acrosome reaction were easily identified. Acrosome-reacted sperm were found adherent to the outer boundary of the cumulus, but were never observed to penetrate the cumulus. The percentage of acrosome-intact, hyperactivated sperm was higher in the cumulus oophorus than in culture medium, suggesting that changes in motility were elicited in response to contact with the cumulus. Fully acrosome-reacted sperm were found adherent to the zona pellucida, and solubilised guinea pig zona pellucida was capable of inducing acrosome reactions in capacitated guinea pig sperm. Acrosome-intact sperm were also observed on the zona, but they were not tightly bound and did not have hyperactivated motility, suggesting that these sperm were not functionally capacitated. Our observations demonstrate that guinea pig sperm penetrate the cumulus matrix in an acrosome-intact state. Although we did not observe sperm undergoing the acrosome reaction, our observations and experimental data suggest that the acrosome reaction of guinea pig sperm is completed on or near the surface of the zona pellucida.

Soybean trypsin inhibitor as a probe for the acrosome reaction in motile cynomolgus macaque sperm.

Soybean trypsin inhibitor (SBTI) inhibits the catalytic activity of serine proteases, and has been shown to bind to acrosin, an acrosomal hydrolase which is not exposed on the surface of macaque sperm until after the acrosome reaction. Following activation with caffeine and dibutyryl cAMP, cynomolgus macaque sperm were induced to acrosome react with calcium ionophore A23187 in the presence of SBTI and were fixed for ultrastructural observation. Transmission electron microscopy (TEM) revealed secondary labelling of anti-SBTI-IgG with colloidal gold in association with the acrosomal matrix and fused membranes of sperm undergoing the acrosome reaction, but gold labelling was not observed on acrosome-intact sperm. When SBTI was conjugated with the fluorochrome Alexa 488, labelled (acrosome-reacted) sperm showed bright fluorescence that ranged from a patchy or punctate appearance to solid labelling over the region of the acrosomal cap. Following treatment with ionophore, the percentages of total acrosome-reacted sperm (motile and non-motile) as assessed with Alexa-SBTI, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA), and TEM were 54.6%, 51.6% and 61.5%, respectively. Measures of acrosomal status with FITC-PSA and Alexa-SBTI were highly correlated (r = 0.94; n = 3). Macaque zonae pellucidae were co-incubated with activated sperm for 1 min and then rinsed in medium containing Alexa-SBTI and immediately observed with epifluorescence microscopy. The mean percentage of Alexa-SBTI-labelled (acrosome-reacted) motile sperm bound to the zona was 45.7 +/- 14 (range: 22-80.4%; n = 4). Fewer than 1% of the motile sperm in suspension surrounding the zonae were acrosome-reacted. Alexa-SBTI had no effect on sperm motility, survival, or zona binding capability.

Hyaluronic acid and the cumulus extracellular matrix induce increases in intracellular calcium in macaque sperm via the plasma membrane protein PH-20.

The hyaluronic acid (HA)-rich extracellular matrix (ECM) of the cumulus oophorus is known to facilitate fertilization. It has been suggested that HA may enhance fertilisation in a number of species, and in macaque sperm, HA has been shown to increase the number of acrosome reactions that follow sperm binding to the zona pellucida. In this study, we investigated the effects of HA on intracellular Ca2+ in capacitated cynomolgus macaque sperm. Fluorometry studies using the intracellular Ca2+ indicator Fluo-3 showed that addition of 100 micrograms/ml of HA induced a rapid increase in intracellular Ca2+. This Ca2+ increase (approximately 2-3 times above basal levels) was inhibited by preincubation of sperm with Fab fragments of anti-recombinant PH-20 IgG. The frequency of acrosome reactions in sperm exposed to HA was not above control levels. A synthetic gel was prepared with similar viscosity to the cumulus and with HA trapped in its matrix. Video imaging of individual sperm was used to demonstrate that capacitated sperm swimming into the HA gel had increased intracellular Ca2+ levels. Preincubation of sperm with Fab fragments of anti-PH-20 IgG inhibited the increased intracellular Ca2+ levels induced by the HA gel. Sperm in control gel (no HA) did not show increased intracellular Ca2+, while sperm in gel containing anti-PH-20 IgG showed increased Ca2+ (positive control). Sperm loaded with Fluo-3 were allowed to interact with cynomolgus macaque cumulus masses, and sperm within the cumulus ECM clearly showed increased intracellular Ca2+ that was inhibited when sperm were preincubated in anti-PH-20 Fab. Fluorescein isothiocyanate (FITC)-HA was found to bind to sperm over the acrosomal region, corresponding to PH-20 localisation, and this binding could be inhibited by preincubation of sperm with anti-PH-20 fragments. The results of this study show that HA increases intracellular Ca2+ in macaque sperm through interaction with plasma membrane PH-20. We propose that HA binding to plasma membrane PH-20 induces an aggregation of receptors that in turn results in intracellular signalling. As a result, sperm have higher basal CA2+ levels and are more responsive to induction of the acrosome reaction after binding to the zona pellucida.

Identification of bladder tumor-derived hyaluronidase: its similarity to HYAL1.

The glycosaminoglycan hyaluronic acid (HA) and its degrading enzyme, hyaluronidase, are intricately associated with tumor metastasis and angiogenesis. HA promotes tumor cell adhesion and migration, whereas its small fragments stimulate angiogenesis. Such small HA fragments are generated from the degradation of HA by hyaluronidase. We have previously shown (V. B. Lokeshwar et al., Cancer Res., 57: 773-777, 1997) that the HA levels are elevated in the urine and tumor tissues of bladder cancer patients regardless of the tumor grade (G). The hyaluronidase levels were found to be elevated in the urine and tumor tissues of G2 and G3 bladder cancer patients. Furthermore, angiogenic HA fragments were isolated from the urine of G2/G3 bladder cancer patients, which stimulated endothelial cell proliferation, a key event in angiogenesis. In this study, we characterized the bladder tumor-derived hyaluronidase. Analysis of hyaluronidase activity in the culture-conditioned media (CM) of 11 bladder cancer cell lines, using an ELISA-like assay and a substrate (HA)-gel technique, showed that the invasive bladder cancer cell lines secrete elevated levels of a Mr approximately 60,000 hyaluronidase. Reverse transcription-polymerase chain reaction, cloning, and sequence analyses revealed the expression of an HYAL1 transcript in bladder cancer lines. HYAL1 encodes for a hyaluronidase that is present in serum. Immunoblot analysis using an anti-HYAL1 peptide IgG confirmed the presence of a Mr approximately 60,000 HYAL1-related protein in the CM of bladder cancer cell lines, in the urine specimens from G2 and G3 bladder cancer patients, and in the partially purified preparations of bladder tumor-derived hyaluronidase. No HYAL1-related protein was detected in urine specimens from normal individuals, G1 bladder cancer patients, and patients with a history of bladder cancer but no disease at the time of testing. The bladder tumor-derived hyaluronidase present in CM and partially purified preparations was found to have maximum activity at a pH range of 4.1-4.3. The identification of bladder tumor-derived hyaluronidase should help in elucidating its role in bladder tumor progression.

PH-20 but not acrosin is involved in sperm penetration of the macaque zona pellucida.

In this study, we investigated the functions of PH-20 and acrosin during the interaction of macaque sperm with the zona pellucida. Both of these sperm enzymes have been reported to be present on the inner acrosomal membrane of acrosome reacted sperm, and have been suggested to play a role during secondary sperm-zona binding in other species. Anti-macaque PH-20 IgG, anti-pig acrosin IgG and soybean trypsin inhibitor (SBTI) were used as probes for immunolocalization of the two proteins at the ultrastructural level, and as reagents for blocking sperm penetration of the macaque zona pellucida in vitro. As a control, we performed similar studies with antibodies to CD-46, which is also located on the inner acrosomal membrane, but has no known function in sperm-zona pellucida interaction. After labeling with anti-acrosin IgG, gold label was not present on the sperm surface before the acrosome reaction, but was detected over the entire head of sperm that were induced to acrosome react with calcium ionophore A23187. In contrast, when sperm were induced to acrosome react by binding to intact zona pellucida, acrosin was present in the acrosomal shroud but not on the inner acrosomal membrane. Similar results were obtained when SBTI was used as a probe for enzyme localization. PH-20 and CD-46 were demonstrated on the inner acrosomal membrane of sperm induced to acrosome react by ionophore treatment and by zona binding. Neither anti-acrosin IgG nor anti-CD-46 IgG affected sperm penetration of the zona at concentrations up to 300 microg/ml, but zona penetration was blocked completely when anti-PH-20 IgG (100 microg/ml) was present during sperm-oocyte interaction. Ultrastructural observations of oocytes incubated with anti-PH-20 IgG showed that acrosomal shrouds were present on the zona surface but no sperm had begun to penetrate into the zona substance. We conclude that anti-PH-20 IgG prevented sperm penetration of the macaque zona pellucida by interference with secondary sperm-zona binding, rather than primary sperm-zona binding or the zona-induced acrosome reaction. Acrosin was not detected on the inner acrosomal membrane of sperm that are induced to acrosome react after zona binding, and acrosin does not appear to be critical for sperm penetration of the macaque zona pellucida.

Hyaluronic acid enhances induction of the acrosome reaction of human sperm through interaction with the PH-20 protein.

When capacitated human sperm were treated with hyaluronic acid (HA) for 30 min prior to the addition of progesterone or solubilised human zonae pellucidae, there was a significant increase in the percentage of acrosome reactions. Progesterone treatment alone increased acrosome reactions from 10.5% to 21.8% and pretreatment with 100 micrograms/ml HA resulted in 33.0% acrosome reactions. With zonae pellucidae treatment alone the increase was from 9.0% to 23.5% and with HA pretreatment it was 48.8%. HA treatment alone had no direct effect on acrosome reactions, and the enhancing effect of HA was not removed when sperm were washed prior to the addition of either acrosome reaction agonist. Experiments with sperm 5 min after HA treatment demonstrated that enhancement of acrosome reactions was apparent as early as 1 min after addition of zonae and within 5 min after addition of progesterone. When sperm were pretreated with Fab fragments of anti-PH-20 IgG, then with HA and then with progesterone or zonae pellucidae, there was no enhancement of the acrosome reaction. Fab treatment did not induce acrosome reactions and did not interfere with the action of either agonist in the absence of HA. Sperm that were treated with HA had significantly higher intracellular calcium levels, and pretreatment with Fab reduced this increase to 42.7%. Addition of progesterone to HA-treated sperm was followed by another large increase in intracellular calcium, which was lower when sperm were pretreated with Fab. These results suggest that HA interacts with the PH-20 protein to increase basal levels of intracellular calcium and thereby potentiates the acrosome reaction. The data support the hypothesis that HA in the cumulus matrix may act to prime the fertilising sperm for induction of the acrosome reaction by constituents of the cumulus and/or zona pellucida.

Rearrangement of the PH-20 protein on the surface of macaque spermatozoa following exposure to anti-PH-20 antibodies or binding to zona pellucida.

Capacitated cynomolgus macaque sperm have a surface hyaluronidase (PH-20) that is evenly distributed over the entire head and can be visualized at the ultrastructural level using a secondary antibody labeled with colloidal gold . Exposure of sperm to mono-specific, bivalent polyclonal antibodies to PH-20 causes a rapid clustering of PH-20. The predominant morphological consequence of PH-20 redistribution is its aggregation along the lateral edge of the sperm head. Monovalent Fab fragments of the anti-PH-20 antibody bound to the sperm head but did not induce a change in PH-20 distribution. PH-20 aggregation was observed in almost all sperm following treatment with the polyclonal antibody, but only about 20% of the sperm had morphological acrosome reactions, regardless of the time of exposure or the concentration of antibody. There was morphological evidence of swelling of the acrosomal matrix in over 50% of the sperm following exposure to anti-PH-20 antibodies. Anti-PH-20 Fab fragments did not induce the acrosome reaction or acrosomal matrix swelling. Sperm bound to macaque zona pellucida also showed aggregation of the PH-20 protein as soon as 30 sec after sperm-zona interaction. This aggregation was not observed when macaque sperm were bound to hamster zona pellucida. When macaque sperm were surface-labeled with biotin and then incubated with anti-PH-20 antibodies or macaque zona pellucida, there was no evidence of a global surface protein rearrangement, although PH-20 protein was aggregated on the surface of the same sperm cells. An increase in levels of internal sperm Ca++ was measured in association with the antibody-induced PH-20 aggregation. Fab fragments did not increase Ca++ levels, but when they were crosslinked with anti-Fab antibody there was a significant Ca++ increase and induction of acrosome reactions. Anti-PH-20 Fab fragments did not block macaque sperm binding to macaque zona pellucida or the zona-induced acrosome reaction. We conclude that PH-20 on the sperm surface is involved in sperm-zona pellucida interaction and the zona-induced acrosome reaction.

Biochemical characterization of the PH-20 protein on the plasma membrane and inner acrosomal membrane of cynomolgus macaque spermatozoa.

Preparations of sperm membranes (plasma membranes and outer acrosomal membranes) and denuded sperm heads were isolated from macaque sperm, and the PH-20 proteins present were characterized by Western blotting, hyaluronic acid substrate gel analysis, and a microplate assay for hyaluronidase activity. Because we have shown previously that PH-20 is located on the plasma membrane and not on the outer acrosomal membrane, the PH-20 in the membrane preparations was presumed to be plasma membrane PH-20 (PM-PH-20). PM-PH-20 had an apparent molecular weight of 64 kDa and the optimum pH for its hyaluronidase activity was 6.5. The PH-20 associated with denuded sperm heads was localized by immunogold label to the persistent inner acrosomal membrane (IAM) and was presumed to be IAM-PH-20, which included a major 64 kDa form and a minor 53 kDa form. The 53 kDa form was not detected in extracts of denuded sperm heads from acrosome intact sperm that were boiled in nonreducing sample buffer, but was present in extracts of sperm heads from acrosome reacted sperm and in the soluble material released during the acrosome reaction, whether or not the samples were boiled. Substrate gel analysis showed that the hyaluronidase activity of the 53 kDa form of PH-20 was greatest at acid pH, and this activity was probably responsible for the broader and lower optimum pH of IAM hyaluronidase activity. When hypotonic treatment was used to disrupt the sperm acrosome and release the acrosomal contents, less than 0.05% of the total hyaluronidase activity was released. The PH-20 protein released by hypotonic treatment was the 64 kDa form and not the 53 kDa form, suggesting that its source might be the disrupted plasma membranes. Our experiments suggest that the soluble form of hyaluronidase, which is released at the time of the acrosome reaction, is derived from the IAM. This soluble hyaluronidase is composed of both the 64 kDa form and 53 kDa form of PH-20. The 53 kDa form appears to be processed from the 64 kDa form at the time of the acrosome reaction.

Inhibition of monkey sperm hyaluronidase activity and heterologous cumulus penetration by flavonoids.

A microplate assay for hyaluronidase and a heterologous cumulus penetration assay were used to determine the effects of four flavonoids (tannic acid, kaempferol, quercetin, and apigenin) on the function of cynomolgus monkey sperm. All four flavonoids inhibited the activity of hyaluronidase extracted from monkey sperm in a concentration-dependent manner over the range of 50-200 microM. Tannic acid and apigenin had lower inhibitory effects than kaempferol and quercetin. Kaempferol, quercetin, and apigenin at 100 microM were shown to significantly inhibit monkey sperm penetration into hamster cumulus. There was a significant linear relationship between the capacity of the flavonoids to inhibit monkey sperm hyaluronidase activity and their inhibitory effects on hamster cumulus penetration (r = 0.97). Tannic acid was observed to reduce sperm motility, and it was not used in the cumulus penetration assay. The other three flavonoids tested in the cumulus penetration assay did not affect sperm motility, nor did they induce acrosome reactions. The results demonstrate that the flavonoids are useful tools for assessing the involvement of hyaluronidase in the functions of monkey sperm that are involved in fertilization.

Interaction of acrosome-reacted macaque sperm with the macaque zona pellucida.

In this study we investigated the ultrastructure of macaque sperm induced to acrosome-react with calcium ionophore A23187, and the interaction between these acrosome-reacted sperm and the macaque zona pellucida. Transmission electron microscopy revealed that the majority of ionophore-treated sperm retained the vesiculated acrosomal cap or "shroud." Untreated, acrosome-reacted sperm on the zona had a similar ultrastructural appearance. In sperm-zona binding experiments, a mean of 4.5 ionophore-treated sperm were bound per zona after 1 min of coincubation compared with 41 sperm per zona in the solvent control. Vigorous pipetting was used to remove the acrosomal shrouds from approximately 50% of acrosome-reacted sperm before incubation with oocytes. Significantly more of these mechanically treated sperm were bound to the zona after a 4-min coincubation compared with acrosome-reacted sperm that were not pipetted. The number of mechanically treated sperm bound to the zona was the same whether the sperm and oocytes were coincubated in calcium-free medium or in control medium. The percentage of mechanically treated sperm that were acrosome-reacted on the zona also was not different in the two media. We conclude that macaque sperm that undergo the acrosome reaction on the zona surface are bound by the acrosomal shroud before zona penetration. When sperm acrosome-react before interaction with the oocyte, their zona binding capacity is significantly reduced. Removal of the acrosomal shroud and exposure of the inner acrosomal membrane increases the affinity of sperm for the zona. This sequence occurs naturally during the transition from primary binding to secondary binding on the zona surface.

Hyaluronidase activity of macaque sperm assessed by an in vitro cumulus penetration assay.

A model system consisting of cynomolgus macaque sperm and ovulated hamster ova-cumulus complexes (OCCs) was utilized to study the role of the sperm protein PH-20 in cumulus penetration. The hyaluronidase activity of solubilized macaque sperm PH-20 was evaluated using an ELISA-like microplate assay prior to and following the addition of the hyaluronidase inhibitors heparin (0-100 microg/ml) and apigenin (250 microM), as well as the Ig fraction of a polyclonal antibody raised against purified recombinant macaque PH-20 (R10; 10-400 microg/ml). Sperm motility following exposure to enzyme inhibitors was evaluated using computer-aided sperm motility analysis. Macaque sperm were labeled with the permeant fluorescent nuclear dye, Hoechst 33342, and were coincubated with ovulated hamster OCCs for 30 min at 37 degrees C. The addition of heparin, apigenin, or R10 antibody to solubilized sperm extracts resulted in a linear dose-dependent decrease in hyaluronidase activity (P < .01). In the heterologous cumulus penetration assay, fluorescently labeled macaque sperm that were pretreated with heparin (1-100 microg/ml), apigenin (250 microM), or R10 antibody (Ig fraction, 10-400 microg/ml) demonstrated a dose-dependent decrease in the ability to penetrate hamster OCCs (P < 0.01), in the absence of effects on sperm motility. In the homologous assay, experiments using macaque OCCs and fluorescently labeled macaque sperm confirmed that the same concentrations of heparin and R10 antibody similarly suppressed spermatozoal cumulus penetration (P < .01). These results suggest that macaque sperm PH-20-derived hyaluronidase participates in cumulus penetration in this species, and that this model system is useful for further studies into primate gamete interaction.

The PH-20 protein in human spermatozoa.

PH-20 is a sperm plasma-membrane protein that has been shown to have hyaluronidase activity in several mammalian species including nonhuman primates. In this investigation, the PH-20 protein was characterized in noncapacitated human sperm and in capacitated human sperm. Two forms of PH-20 were observed in immunoblots of sodium dodecylsulfate polyacrylamide-gel electrophoresis (SDS PAGE) using a polyclonal antibody to recombinant PH-20: a major band of 64 kDa appeared in noncapacitated and capacitated sperm extracts and a 53-kDa band that appeared only in the acrosome-reaction supernatant of acrosome-reacted sperm. Using hyaluronic acid substrate gel analysis, we demonstrated that noncapacitated sperm extracts, capacitated sperm extracts, and the acrosome-reaction supernatant had hyaluronidase activity at neutral pH (pH 7) and acid pH (pH 4). The 64-kDa form in all samples had hyaluronidase activity at both neutral and acid pH, but the 53-kDa form was only active at acid pH. Total hyaluronidase activity, as measured by a microplate assay, was higher at pH 7 than at pH 4. Very low hyaluronidase activity was detected in the acrosome-reaction supernatant. Transmission electron microscopy and immunogold labeling showed that PH-20 of acrosome-intact human sperm was located on the plasma membrane over the entire head but not on the sperm midpiece and tail. After the acrosome reaction, PH-20 was also located on the inner acrosomal membrane. The biochemical characteristics and the ultrastructural localization of PH-20 in human sperm suggest that this protein is the human sperm hyaluronidase and, therefore, has an important function during fertilization.