Role of sperm sulfogalactosylglycerolipid in mouse sperm-zona pellucida binding

Sulfogalactosylglycerolipid (SGG) is the major sulfoglycolipid of mammalian male germ cells. Like other sulfoglycolipids, SGG is believed to be involved in cell-cell/extracellular matrix adhesion. Specifically, we investigated whether sperm SGG played a role in sperm-egg interaction. Initially, we produced an affinity-purified, rabbit polyclonal immunoglobulin (Ig) G antibody that specifically recognized SGG (anti-SGG). Indirect immunofluorescence using anti-SGG IgG localized SGG to the convex and concave ridges and the postacrosome of the mouse sperm head. Pretreatment of sperm with anti-SGG IgG/Fab inhibited sperm-zona pellucida (ZP) binding in vitro in a concentration-dependent manner (to a maximum of 62%). This inhibition was observed at the level of primary binding. Sperm treated with anti-SGG IgG underwent the spontaneous and ZP-induced acrosome reaction at the same rate as control sperm treated with preimmune rabbit serum IgG. Fluorescently labeled SGG liposomes were shown to associate specifically with the egg ZP, whereas fluorescently labeled liposomes of galactosylglycerolipid (SGG's parental lipid) and phosphatidylserine (negatively charged like SGG) did not. Furthermore, coincubation of SGG liposomes with sperm and isolated ZP inhibited sperm-ZP binding in a concentration-dependent manner. These results strongly suggest an involvement of sperm SGG in direct binding to the ZP.     

Sperm capacitation induces an increase in lipid rafts having zona pellucida binding ability and containing sulfogalactosylglycerolipid

Sperm gain full ability to bind to the zona(e) pellucida(e) (ZP) during capacitation. Since lipid rafts are implicated in cell adhesion, we determined whether capacitated sperm lipid rafts had affinity for the ZP. We demonstrated that lipid rafts, isolated as low-density detergent resistant membranes (DRMs), from capacitated pig sperm had ability to bind to homologous ZP. This binding was dependent on pig ZPB glycoprotein, a major participant in sperm binding. Capacitated sperm DRMs were also enriched in the male germ cell specific sulfogalactosylglycerolipid (SGG), which contributed to DRMs-ZP binding. Furthermore, SGG may participate in the formation of sperm DRMs due to its interaction with cholesterol, an integral component of lipid rafts, as shown by infrared spectroscopic studies. Since sperm capacitation is associated with cholesterol efflux from the sperm membrane, we questioned whether the formation of DRMs was compromised in capacitated sperm. Our studies indeed revealed that capacitation induced increased levels of sperm DRMs, with an enhanced ZP affinity. These results corroborated the implication of lipid rafts and SGG in cell adhesion and strongly suggested that the enhanced ZP binding ability of capacitated sperm may be attributed to increased levels and a greater ZP affinity of lipid rafts in the sperm plasma membrane.     

Percoll gradient-centrifuged capacitated mouse sperm have increased fertilizing ability and higher contents of sulfogalactosylglycerolipid and docosahexaenoic acid-containing phosphatidylcholine compared to washed capacitated mouse sperm

Although Percoll gradient centrifugation has been used routinely to prepare motile human sperm, its use in preparing motile mouse sperm has been limited. Here, we showed that Percoll gradient-centrifuged (PGC) capacitated mouse sperm had markedly higher fertilizing ability (sperm-zona pellucida [ZP] binding and in vitro fertilization) than washed capacitated mouse sperm. We also showed that the lipid profiles of PGC capacitated sperm and washed capacitated sperm differed significantly. The PGC sperm had much lower contents of cholesterol and phospholipids. This resulted in relative enrichment of male germ cell-specific sulfogalactosylglycerolipid (SGG), a ZP-binding ligand, in PGC capacitated sperm, and this would explain, in part, their increased ZP-binding ability compared with that of washed capacitated sperm. Analyses of phospholipid fatty acyl chains revealed that PGC capacitated sperm were enriched in phosphatidylcholine (PC) molecular species containing highly unsaturated fatty acids (HUFAs), with docosahexaenoic acid (DHA; C22: 6n-3) being the predominant HUFA (42% of total hydrocarbon chains of PC). In contrast, the level of PC-HUFAs comprising arachidonic acid (20:4n-6), docosapentaenoic acid (C22:5n-6), and DHA in washed capacitated sperm was only 27%. Having the highest unsaturation degree among all HUFAs in PC, DHA would enhance membrane fluidity to the uppermost. Therefore, membranes of PGC capacitated sperm would undergo fertilization-related fusion events at higher rates than washed capacitated sperm. These results suggested that PGC mouse sperm should be used in fertilization experiments and that SGG and DHA should be considered to be important biomarkers for sperm fertilizing ability.     

Isolation of antiSLIP1-reactive boar sperm P68/62 and its binding to mammalian zona pellucida

Single-step purification of boar sperm P68/62 that is cross-reactive with a polyclonal antibody against sulfolipidimmobilizing protein 1 (SLIP1) was achieved by chromatofocusing. This method is useful for obtaining P68/62 in quantity. The two proteins, P68 and P62, were antigenically related, since the antibody generated specifically against the 68-kDa band reacted with both the 68- and 62-kDa bands. Like rat testis SLIP1, purified boar sperm P68/62 bound to sulfogalactosylglycerolipid (SGG) and inhibited sperm-egg binding in a dose-dependent manner when added exogenously to sperm-egg coincubates. This inhibitory effect occurred at the level of the zona pellucida (ZP), and further studies showed that biotinylated boar sperm P68/62 bound to the ZP of unfertilized mouse eggs. Furthermore, biotinylated boar sperm P68/62 bound to isolated ZP of unfertilized eggs from other species, including pig, rat, cat, dog, and human, as well as to ZP of intact fertilized mouse eggs and preimplantation embryos of various developmental stages, although the degree of its binding to the ZP of intact eight-cell embryos, morulae, and blastocysts was much lower than that of fertilized eggs and two-cell embryos. These results suggest that P68/62 of capacitated sperm must act together with other sperm surface proteins/molecules that regulate zona binding specificity within homologous species and in unfertilized eggs. Together with our previous findings, we suggest that rather than being a true ZP receptor, sperm P68/62 may be involved in the initial step of sperm-ZP binding that is adhesive in nature. Mol. Reprod. Dev. 49:203–216, 1998 © 1998 Wiley-Liss, Inc.     

Precambrian animal life: probable developmental and adult cnidarian forms from Southwest China

We have previously described the affinity of a pig sperm surface protein, P68, to mammalian zonae pellucidae (ZP). In this report, we identified P68 as arylsulfatase A (AS-A) based on the presence of P68 tryptic peptide sequences in the pig testis AS-A cDNA sequence. Our objective was to demonstrate the presence of AS-A on the sperm surface and to elucidate its role in ZP binding. Immunogold electron microscopy revealed the presence of AS-A on the sperm surface. Furthermore, live pig sperm and the extract of peripheral sperm plasma membrane proteins exhibited AS-A's desulfation activity. Significantly, the role of pig sperm surface AS-A in ZP binding was demonstrated by dose-dependent decreases of sperm-ZP binding upon sperm pretreatment with anti-AS-A IgG/Fab, and by the binding of Alexa-430-conjugated sperm surface AS-A to homologous ZP. ZP pretreatment with anti-pig-ZP3 antibody abolished AS-A binding, suggesting that ZP3, recognized as the pig sperm receptor, was AS-A's binding ligand. This was further confirmed by the ability of exogenous ZP3 to competitively inhibit AS-A-ZP binding. Similarly, purified ZP3alpha, a major sperm receptor component of ZP3, exhibited great inhibitory effect on AS-A-ZP binding. All of these results designated a new function of AS-A in gamete interaction.     

Role of egg sulfolipidimmobilizing protein 1 on mouse sperm-egg plasma membrane binding.

We have shown that sperm sulfolipidimmobilizing protein 1 (SLIP1, molecular mass of 68 kDa), a sulfogalactosylglycerolipid (SGG)-binding protein, is significant in sperm-zona pellucida (ZP) interaction. The objective of this study was to localize SLIP1 on the egg and determine its role in gamete interaction. Immunofluorescence and immunoprotein A gold electron microscopy localized SLIP1 to the egg plasma membrane. In vitro gamete binding, using zona-free eggs preincubated with antiSLIP1 Fab before coincubation with sperm, showed a significant, dose-dependent decrease in sperm-egg plasma membrane binding. Similar results were obtained when affinity-purified antiSLIP1 IgG was used for egg pretreatment. The significance of egg SLIP1 in sperm-egg plasma membrane binding was further demonstrated by a decrease (36-52%) in in vitro fertilization when zona-intact eggs were pretreated with antiSLIP1 IgG. Since SLIP1 has been shown to bind SGG in vitro, we investigated the possibility that sperm SGG may participate in sperm-egg plasma membrane binding through egg SLIP1. Pretreatment of sperm with antiSGG Fab prior to coincubation with zona-free eggs resulted in a dose-dependent decrease in sperm-egg plasma membrane binding. Collectively, these findings strongly suggest a role for egg SLIP1 in sperm-egg plasma membrane interaction, which may be through its binding to sperm SGG.     

猪精子凝集素在精卵结合中的作用机制

猪精子凝集素（ＢＳＬ）位于精子头部,既与精子蛋白结合,亦与透明带糖蛋白ＺＰ３结合。并且ＢＳＬ自身分子间亦会聚合。与ＺＰ３结合的片段亦结合于岩藻素－Ｓｅｐｈａｒｏｓｅ亲和柱,但不与胎球蛋白－Ｓｅｐｈａｒｏｓｅ柱结合。该片段具有较强的抑制精卵结合的活性。ＢＳＬ经ＮＢＳ修饰后,血凝活力大大下降,同时推动与精子结合的活性,但不影响它与ＺＰ３的结合。修饰后的ＢＳＬ亦显著抑制精卵结合。表明ＢＳＬ以两个不同的部     

Evaluating zona pellucida structure and function using antibodies to rabbit 55 kDa ZP protein expressed in baculovirus expression system

A cDNA encoding the rabbit 55 kDa ZP protein was expressed using a baculovirus expression system and was evaluated for its ability to elicit antibodies which may interfere with sperm-ZP interaction. The expressed glycosylated protein, BV55, was purified by wheat germ agglutinin lectin affinity chromatography. Antisera made in guinea pigs immunized with BV55 (GP-α-BV55) is specific for the 55 kDa rabbit ZP protein. Indirect immunofluorescence studies indicate that GP-α-BV55 localizes to a filamentous meshwork on the surface of the ZP of isolated rabbit eggs. Immunohistochemical analysis of rabbit ovaries demonstrated that this antigen is localized within the ZP of primary and more advanced stage ovarian follicles but is not detected in primordial follicles. In addition, the 55 kDa antigen was detected in the granulosa cells of secondary stage follicles but not in the oocyte. GP-α-BV55 effectively blocked the binding of rabbit sperm to rabbit eggs in vitro. However, Fab fragments generated from GP-α-BV55 failed to block sperm binding, suggesting that the inhibitory effect of GP-α-BV55 was due to stearic hindrance rather than specific blocking of a sperm receptor site. Although the Fab fragment did not inhibit sperm binding, additional studies demonstrated that biotinylated BV55 protein bound to rabbit sperm in the acrosomal region in a manner consistent with ligand activity in the sperm-ZP interaction, and that BV55 bound to rabbit sperm in a dose-dependent manner. These studies therefore demonstrate that antibodies against recombinant ZP proteins recognize the native intact ZP and inhibit sperm-ZP interaction. They also provide evidence that the rabbit 55 kDa ZP protein, which is the homolog of the pig ZP3α sperm receptor protein, has sperm receptor activity. © 1996 Wiley-Liss, Inc.     

Vitelline envelope gps 63 and 75 specifically bind sperm in "in vitro" assays in Discoglossus pictus

In Xenopus, conflicting data related to sperm-vitelline envelope (VE) binding suggest that further experiments should be performed to study the role of VE glycoproteins in sperm binding. In this article, we studied the VE of Discoglossus pictus, where gp63, the product of the Dp ZP2 gene, has high molecular identity to Xenopus gp69/64 and to mouse ZP2 and only A23187-treated sperm bind to VE. Sperm bind to VE all over the egg, yet a sperm tuft was found only in the animal half of the egg, where the dimple, the site of fertilization, is located and an intense immunostain was detected in VE by an antiserum directed against gp69/64. The same antiserum inhibited sperm binding to VE. Sperm binding to beads coated with gp63, gp40, or gp75 was in the range of 62-70% for gp63-beads, 67-75% for 75 beads, and about 20% for BSA beads and gp40-coated beads. Soluble purified gp63 and gp75 competitively inhibited binding of sperm to gp63-coated beads. Similarly, the same glycoproteins inhibited sperm binding to gp75-coated beads. SDS-polyacrylamide gels (PAGE) of FE and comparison of VE and FE peptide maps showed that gp63 undergoes a minor shift to about 62 kDa in FE. In sperm binding assays with beads coated with FEs gp62, there was no binding. Following fertilization, in the region of the dimple, an F-layer is formed as well as an alteration of the VE structure. Lectin blots of the FE showed that the FE and in particular gp62 acquires a stronger affinity to Maackia amurensis agglutinin (MAA) with respect to VEs gp63. These results indicate that gps 63 and 75 are the sperm binding glycoproteins of D. pictus VE, where major post-fertilization changes occur as in other anuran species.     

Sperm from the calmegin-deficient mouse have normal abilities for binding and fusion to the egg plasma membrane

Calmegin is a putative testis-specific molecular chaperone required for the heterodimerization of fertilin alpha/beta and the appearance of fertilin beta on the sperm surface. Calmegin-deficient mice are almost completely sterile. The cause of the sterility initially was considered to be impaired abilities in sperm/zona pellucida (ZP) and sperm/egg plasma membrane (EPM) binding, and in the ascension of sperm to the oviduct, phenotypes similar to those seen in sperm from fertilin beta-deficient animals. We have developed a new method in which eggs were prepared without any detectable ZP3 on their surfaces by using a piezo-driven micromanipulator. Using these eggs and sperm containing the green fluorescent protein in their acrosomes, which can distinguish acrosome-intact from acrosome-reacted sperm, the binding and fusing abilities of calmegin-deficient sperm were reexamined. Under these conditions, acrosome-reacted sperm retained their ability to bind to and fuse with the EPM. The reduction in EPM binding of sperm from the calmegin(-/-) animals was apparently due to the artifactual binding of large numbers of acrosome-intact sperm from calmegin(+/-) mice to ZP remnants remaining on the EPM prepared with acidic Tyrode's solution. Thus, the sperm defect in calmegin-null animals is not at the level of sperm-EPM binding but rather may involve either sperm-ZP binding and/or sperm transit to the oviduct. Because fertilin beta is absent from calmegin-deficient mice, these results also suggest that the role of fertilin beta in sperm-EPM interaction needs to be reevaluated.     

Binding of arylsulfatase A to mouse sperm inhibits gamete interaction and induces the acrosome reaction

We have shown previously that male germ cell-specific sulfoglycolipid, sulfogalactosylglycerolipid (SGG), is involved in sperm-zona pellucida binding, and that SGG and its desulfating enzyme, arylsulfatase A (AS-A), coexist in the same sperm head area. However, AS-A exists at a markedly low level in sperm as compared to SGG (i.e., 1/400 of SGG molar concentration). In the present study, we investigated whether perturbation of this molar ratio would interfere with sperm-egg interaction. We demonstrated that purified AS-A bound to the mouse sperm surface through its high affinity with SGG. When capacitated, Percoll gradient-centrifuged mouse sperm were treated for 1 h with various concentrations of AS-A, their binding to zona-intact eggs was inhibited in a dose-dependent manner and reached the background level with 63 nM AS-A. This inhibition could be partially explained by an increase in premature acrosome reaction. The acrosome-reacted sperm population of the 63 nM AS-A-treated sperm sample was twice that of the control sample (treated with 63 nM ovalbumin) at 1 h (i.e., 32% vs. 15%) and rose to 53% at 2 h. This induction was presumably due to SGG aggregation attributed to AS-A, existing as a dimer at neutral pH, and could be mimicked by anti-SGG immunoglobulin (Ig) G/IgM + secondary IgG antibody. Drastic inhibition (75%) of in vivo fertilization was also observed in females inseminated with sperm suspension containing 630 nM AS-A as compared to the rate in females inseminated with sperm suspension included with 630 nM ovalbumin. Our results demonstrate a promising potential for AS-A as a nonhormonal, vaginal contraceptive.     

Distinct membrane fractions from mouse sperm bind different zona pellucida glycoproteins.

Interactions between sperm and zona pellucida (ZP) during mammalian fertilization are not well characterized at the molecular level. To identify sperm proteins that recognize ligand ZP3, we used sonicated sperm membrane fractions as competitors in a quantitative binding assay. Sonicated membranes were density fractionated into 4 fractions. Bands 1-3 contained membrane vesicles, and band 4 contained axonemal and midpiece fragments. In competitive binding assays, bands 1, 2, and 3 but not band 4 were able to compete with live, capacitated, intact sperm for soluble 125I-ZP binding. Affinity-purified ZP fractions consisting of a ZP3-enriched fraction (125I-ZP3) and a fraction enriched for ligands ZP1 and ZP2 and depleted of ZP3 (125I-ZP1/2) were obtained by antibody affinity purification of ZP3. In competitive binding assays, bands 2 and 3 competed for 125I-ZP3 binding, but band 1 did not interact with enriched 125I-ZP3. None of the membrane fractions competed for 125I-ZP1/2 binding. These results demonstrate that band 2 and band 3 contain sperm components that interact with ZP3 alone and that components in band 1 interact with ZP3 in conjunction with either ZP1 or ZP2. These data indicate that there must be at least 2 unique sperm plasma membrane components that mediate intact sperm interactions with ZP glycoproteins in mouse. Bands 2 and 3 are likely to contain a primary ZP-binding protein because they interacted directly with ZP3, whereas band 1 may contain sperm proteins involved in later interactions with the ZP, perhaps transitional interactions to maintain sperm contact with the ZP during acrosomal exocytosis.     

Male germ cell specific sulfogalactoglycerolipid is recognized and degraded by mycoplasmas associated with male infertility

Sulfogalactosylglycerolipid (SGG) is the major mammalian male germ cell glycolipid and has been implicated in sperm/egg binding. Mycoplasma pulmonis, a species of Mollicutes, is associated with male infertility in rodents. Purified SGG incubated in the presence of M. pulmonis was enzymatically degraded by both desulfation and deacylation. Desulfation occurred primarily at alkaline pH, and deacylation also increased with increased pH, indicating that these represent novel enzymatic activities. Digestion was facilitated, but not dependent on, the presence of detergent. Rat spermatozoa exposed to M. pulmonis showed a reduction in SGG content which was particularly marked for cauda (mature) spermatozoa. With the aid of tlc overlay binding procedure, intact M. pulmonis were found to bind specifically to sulfated glycolipids and thus SGG may provide the cell membrane receptor for this organism. The topology of mycoplasma binding to rat sperm was consistent with the known topology of sperm SGG. The reduced binding (and subsequent digestion) of caput spermatozoan SGG correlates with the membrane colocalization of SGG and its endogenous binding protein at this stage. Separation of SGG and its binding protein during epididymal sperm maturation appears to facilitate M. pulmonis binding to and digestion of cauda sperm SGG. The binding and degradation of the sperm SGG by M. pulmonis may play a role in the induction of infertility which follows infection with these organisms by interfering in sperm/egg receptor recognition.     

Evidence that P36, a human sperm acrosomal antigen involved in the fertilization process is triosephosphate isomerase

P36 is one of the immunodominant sperm antigens identified by antibodies eluted from the spermatozoa of infertile men. In a previous study, we isolated and characterized this auto-antigen as a glycoprotein with several isoforms. Specific rabbit antibodies were produced to investigate sperm topography and the role of P36 in the fertilization process and we showed that P36 is present on the equatorial segment of acrosome-reacted spermatozoa and is involved in sperm-binding and the penetration of zona-free hamster oocytes. In the present study, we demonstrated, by means of immunofluorescence and electron microscopy, that P36 is present all over the acrosomal membranes of non-reacted spermatozoa. We also investigated the role of P36 in the acrosome reaction and sperm binding to the zona pellucida (ZP). The exposure of capacitated spermatozoa to rabbit anti-P36 antibodies had no effect on primary fixation to the ZP, but inhibited secondary binding to the ZP and the Ca2+ ionophore-induced acrosome reaction. These results suggest that P36, an acrosomal antigen, is involved in several steps of the fertilization process. On two-dimensional Western blots, human anti-sperm antibodies (ASA) and rabbit anti-P36 antibodies recognized five to six isoforms of P36, all 36/37 kDa in size, with a pI between 5.1 and 5.7. Two major spots were identified as human triosephosphate isomerase (TPI) by MALDI-TOF mass spectrometry. Anti-TPI antibodies were shown to react with the isoforms recognized by human and rabbit anti-P36 antibodies. We also demonstrated the presence of TPI in human sperm heads. Further studies are underway to establish whether there is a sperm-specific isoform of TPI and its role in sperm function.     

Relevance of glycosylation of human zona pellucida glycoproteins for their binding to capacitated human spermatozoa and subsequent induction of acrosomal exocytosis

To delineate the functional aspects of zona pellucida (ZP) glycoproteins during fertilization in human, in the present study, fluorochrome-conjugated Escherichia coli (E. coli)- and baculovirus-expressed recombinant human ZP glycoprotein-2 (ZP2), -3 (ZP3), and -4 (ZP4) were employed. In an immunofluorescence assay, capacitated human sperm exhibited binding of the baculovirus-expressed recombinant ZP3 as well as ZP4 to either acrosomal cap or equatorial region whereas acrosome-reacted sperm failed to show any binding to the acrosomal cap. Using double labeling experiments, simultaneous binding of ZP3 and ZP4 to the acrosomal cap was observed suggesting the possibility of different binding sites of these proteins on the sperm surface. No binding of ZP2 was observed to the capacitated sperm. However, acrosome-reacted sperm (20.00 +/- 1.93%) showed binding of ZP2 that was restricted to only equatorial region. Interestingly, E. coli-expressed recombinant human zona proteins also showed very similar binding profiles. Competitive inhibition studies with unlabeled recombinant human zona proteins revealed the specificity of the above binding characteristics. Binding characteristics have been further validated by an indirect immunofluorescence assay using native human heat solubilized isolated zona pellucida. Employing baculovirus-expressed recombinant ZP3 and ZP4 with reduced N-linked glycosylation and respective E. coli-expressed recombinant proteins, it was observed that glycosylation is required for induction of acrosomal exocytosis but its absence may not compromise on their binding ability. These studies have revealed the binding profile of individual human zona protein to spermatozoa and further strengthened the importance of glycosylation of zona proteins for acrosomal exocytosis in spermatozoa.     

Expression of recombinant human zona pellucida protein 2 and its binding capacity to spermatozoa

The human zona pellucida (ZP) is composed of three major glycoproteins: ZP1, ZP2, and ZP3. The aim of this study was to clarify the role of ZP2 by focusing on the polypeptide structure. We produced in Escherichia coli a recombinant human ZP2 protein (rec-hZP2) corresponding to amino acid sequence 1-206 of the mature protein. The final yield of rec-hZP2 protein was 80 microg/ml Luria Broth medium. After 2-h incubation of human spermatozoa with rec-hZP2 in vitro, an immunofluorescent study indicated that rec-hZP2 bound only to acrosome-reacted spermatozoa. The binding site migrated from the acrosome to the midpiece of the spermatozoa. Rabbit and mouse antisera produced against rec-hZP2 stained native human ZP in the immunofluorescent study, and significantly blocked human sperm binding and penetration into human ZP as compared to control values. The N-terminal polypeptide portion of human ZP2 was shown to contain a binding site for acrosome-reacted spermatozoa and to play an important role in secondary sperm binding and penetration into the ZP.     

Mammalian zona pellucida glycoproteins: structure and function during fertilization

Zona pellucida (ZP) is a glycoproteinaceous translucent matrix that surrounds the mammalian oocyte and plays a critical role in the accomplishment of fertilization. In humans, it is composed of 4 glycoproteins designated as ZP1, ZP2, ZP3 and ZP4, whereas mouse ZP is composed of ZP1, ZP2 and ZP3 (Zp4 being a pseudogene). In addition to a variable sequence identity of a given zona protein among various species, human ZP1 and ZP4 are paralogs and mature polypeptide chains share an identity of 47%. Employing either affinity purified native or recombinant human zona proteins, it has been demonstrated that ZP1, ZP3 and ZP4 bind to the capacitated human spermatozoa and induce an acrosome reaction, whereas in mice, ZP3 acts as the putative primary sperm receptor. Human ZP2 only binds to acrosome-reacted spermatozoa and thus may be acting as a secondary sperm receptor. In contrast to O-linked glycans of ZP3 in mice, N-linked glycans of human ZP3 and ZP4 are more relevant for induction of the acrosome reaction. Recent studies suggest that Sialyl-Lewis^x sequence present on both N- and O-glycans of human ZP play an important role in human sperm?Cegg binding. There are subtle differences in the downstream signaling events associated with ZP3 versus ZP1/ZP4-mediated induction of the acrosome reaction. For example, ZP3 but not ZP1/ZP4-mediated induction of the acrosome reaction is dependent on the activation of the G_i protein-coupled receptor. Thus, various studies suggest that, in contrast to mice, in humans more than one zona protein binds to spermatozoa and induces an acrosome reaction.     

Porcine zona pellucida ZP3α glycoprotein mediates binding of the biotin-labeled Mr 55,000 family (ZP3) to boar sperm membrane vesicles

The two M_r 55,000 glycoproteins, ZP3α and ZP3b?, of porcine zona pellucida copurify as a preparation designated ZP3. Gamete binding assays have implicated ZP3α, but not ZP3b?, as participating in sperm-zona recognition events. We now report that boar sperm contain membrane-associated binding sites with specificity for ZP3α. Biotin-labeled (b-) preparations of ZP3 bind to intact boar sperm in a saturable manner, with localization on the anterior head region. Membrane vesicles obtained from capacitated sperm by nitrogen cavitation retain b-ZP3 binding sites as determined by an enzyme-linked method employing alkaline phosphatase-conjugated strepavidin. In competitive binding assays using b-ZP3 (0.1μg/ml) as probe, heat-solubilized zonae and ZP3 were effective competitors, whereas the nonzona molecules fetuin and fucoidin were not. Digestion of ZP3 with endo-b?-galactosidase, an enzyme that trims polylactosamines, enhanced its affinity for membrane receptors. In contrast treatments such as chemical deglycosylation, pronase digestion, or disruption of disulfide bonds abolished the ligand activity of ZP3. Finally, purified ZP3α was an at least 100-fold better antagonist than purified ZP3b?. The results demonstrate that binding of b-ZP3 to isolated boar sperm membranes is mediated by sperm receptors with specificity for the ZP3α macromolecular component and reveal a complex contribution of both carbohydrate and protein moieties toward the ligand activity of this sperm adhesive zona molecule. © 1993 Wiley-Liss, Inc.     

Human sperm bind to the N-terminal domain of ZP2 in humanized zonae pellucidae in transgenic mice

Fertilization requires taxon-specific gamete recognition, and human sperm do not bind to zonae pellucidae (ZP1-3) surrounding mouse eggs. Using transgenesis to replace endogenous mouse proteins with human homologues, gain-of-function sperm-binding assays were established to evaluate human gamete recognition. Human sperm bound only to zonae pellucidae containing human ZP2, either alone or coexpressed with other human zona proteins. Binding to the humanized matrix was a dominant effect that resulted in human sperm penetration of the zona pellucida and accumulation in the perivitelline space, where they were unable to fuse with mouse eggs. Using recombinant peptides, the site of gamete recognition was located to a defined domain in the N terminus of ZP2. These results provide experimental evidence for the role of ZP2 in mediating sperm binding to the zona pellucida and support a model in which human sperm-egg recognition is dependent on an N-terminal domain of ZP2, which is degraded after fertilization to provide a definitive block to polyspermy.     

Nitric oxide synthase inhibition in human sperm affects sperm-oocyte fusion but not zona pellucida binding

There is recent evidence that mouse and human spermatozoa contain constitutive nitric oxide synthase (cNOS) and can synthesize nitric oxide. The aim of this study was to investigate whether the inhibition of human sperm cNOS could affect sperm-oocyte fusion and sperm binding to the zona pellucida (ZP). N(G)-nitro-L-arginine methyl ester (L-NAME) was used as cNOS inhibitor. Sperm-oocyte fusion was evaluated using the hamster egg penetration test (HEPT). The ZP binding was evaluated using the hemizona assay. L-NAME added from the onset of capacitation strongly inhibited sperm-oocyte fusion. This inhibitory effect was dose dependent, stereospecific, and suppressed by L-arginine in a dose-dependent manner. L-NAME also inhibited sperm-oocyte fusion in the HEPT enhanced with progesterone (P), where P (5 microM) was added for 15 min to capacitated sperm. A lesser but significant inhibition was also observed when sperm suspensions were exposed to L-NAME following capacitation in both versions of HEPT. On the contrary, L-NAME did not affect ZP binding. In conclusion, the present study provides the evidence that cNOS plays a role in the human sperm's capacity to fuse with oocyte but not in the ZP binding.