Localization of a carbohydrate antigen associated with growing oocytes and ovarian surface epithelium.

We used a monoclonal antibody (PS1) to a carbohydrate antigen to study the development of the oocyte and follicle during early stages of differentiation in several mammalian species. This antigen has been shown to localize within the cytoplasm of oocytes in primordial follicles as well as in growing oocytes. It is also localized within distinct layers of the zona pellucida (ZP) of developing follicles. Although this antibody was made against a specific ZP glycoprotein, the antigen also appears to be abundant in cells of the ovarian surface epithelium (OSE). The localization of this carbohydrate moiety has been observed in ovaries of rabbits of different ages as well as in the ovarian surface epithelium of other mammalian species including cat, cynomolgus monkey, baboon, and human. These studies demonstrate that there is an abundant carbohydrate antigenic determinant which is associated with both the mammalian oocyte and the ovarian surface epithelium but which is not apparent in other ovarian cell types or in non-ovarian secretory epithelium. This antibody probe should provide a valuable tool for studying the development and differentiation of the ovary, since this antigen is associated with two highly differentiated but distinct cell types.     

Monoclonal antibodies to rabbit and pig zonae pellucidae distinguish species-specific and shared antigenic determinants

Monoclonal antibodies against rabbit or porcine zonae pellucidae (ZP) demonstrate species-specific and shared antigenic determinants. In addition, these antibodies are used to characterize the biochemical nature of these determinants. All of six monoclonal antibodies developed against porcine ZP react with porcine but not with rabbit ZP. Only one of seven monoclonal antibodies developed against rabbit ZP cross-reacts with porcine ZP. None of these antibodies recognized antigens associated with other tissues tested. High-resolution, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) followed by immunoblotting was used to demonstrate that the cross-reactive antibody recognizes an antigenic determinant which is associated with the major low molecular weight glycoprotein of both the pig and rabbit ZP. Since this antibody recognizes all charge species of this glycoprotein, it is apparent that the antigenic determinant recognized by this antibody involves protein. Further studies demonstrate that proteolytic digestion of ZP will destroy the antigenic determinant while glycosidic digestion of ZP has no effect on antibody binding. Although polyclonal antibodies to this glycoprotein inhibit sperm from binding to the zona pellucida, this monoclonal antibody does not affect sperm binding. None of the species-specific antibodies recognize ZP glycoproteins following 2D-PAGE. This is a property typical of antibodies directed against conformational antigenic determinants. The presence of common as well as unique zona antigenic determinants could explain why ZP proteins induce heteroantibodies which result in infertility while alloimmunization has no effect on fertility.     

Cryptodeterminant of a sperm maturation antigen on the mouse flagellar surface.

The determinant of a mouse sperm maturation antigen was examined morphologically and biochemically with monoclonal antibody T21 as a probe. The plasma membrane components of cauda epididymal spermatozoa were extracted with nonionic detergent Nonidet P-40 and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and by immunoblotting. Wheat germ agglutinin-lectin staining and immunoblotting indicated that the antigen recognized by T21 is a sialoglycoprotein of about 54,000 daltons (54 kDa). The antigenic determinant was more distinctly exposed after treatment with neuraminidase, as evaluated by immunohistochemistry, immunocytochemistry, and immunoblotting. The cryptic nature of the determinant was further confirmed by immunostaining nitrocellulose strips, subsequently digesting the strips with neuraminidase, and then reimmunostaining them. Results obtained by periodate oxidation treatment suggested that the epitope is a carbohydrate. Immunoperoxidase electron microscopy confirmed that the antigen is distributed on the flagellar plasma membrane of the sperm. This was demonstrated clearly when sperm were desialylated with neuraminidase. These results indicate that the 54 kDa sialoglycoprotein sperm maturation antigen has a cryptodeterminant which can be masked by a sialic acid residue, that is recognized by monoclonal antibody T21.     

Use of specific monoclonal and polyclonal antibodies to define distinct antigens of the porcine zonae pellucidae

Specific monoclonal and polyclonal antibodies to solubilized porcine and rabbit zonae pellucidae (ZP) and to purified ZP glycoprotein components have been used to define distinct ZP antigens. These studies demonstrate that the individual ZP glycoproteins contain both unique and shared determinants. One monoclonal antibody (R5) has been used to demonstrate that the major porcine ZP glycoprotein, which has multiple charge species ranging in molecular weight from 42,000 to 120,000, is composed of two distinct polypeptide antigens unique to this glycoprotein class. These distinct antigens can be differentiated by immunoblotting after high-resolution two-dimensional polyacrylamide gel electrophoretic separation of trypsin-treated or deglycosylated glycoproteins. The two polypeptides also differ in their staining properties with the silver-based color stain and in their susceptibility to proteolysis. A second monoclonal antibody (PSI) has been used to define a determinant shared by all three major porcine ZP glycoprotein classes. This determinant appears to involve either a carbohydrate moiety or some other molecular feature related to post-translational modification, since the antibody recognizes only the acidic species of each glycoprotein class, and does not recognize the deglycosylated forms of the proteins. This work demonstrates that there are both unique and shared antigenic determinants present in the individual components of the ZP, but that the immunodominant determinants appear to be unique to each glycoprotein.     

Sperm binding to the pig zona pellucida and inhibition of binding by solubilized components of the zona pellucida

An assay to determine the binding of pig spermatozoa to the zona pellucida (ZP) of pig oocytes was developed using conditions compatible with in-vitro fertilization of pig eggs and with pig sperm penetration of zona-free hamster ova. These conditions were used to define which of the pig oocyte ZP components were involved in sperm binding by a competitive inhibition approach. Assay variables that were optimized included: the method of sperm preparation; sperm preincubation time; sperm-oocyte coincubation time; sperm concentration and temperature; and methods for the separation of free from oocyte-bound spermatozoa. Inclusion of solubilized ZP in the sperm preincubation medium inhibited sperm binding approximately 50%. Both the 55K and 90K components inhibited sperm binding although the 55K component was more effective. The two polypeptides derived from chemical deglycosylation of the 55K families did not inhibit sperm binding. Of several monoclonal antibodies to the ZP components tested, only one directed against the 55K alpha glycoprotein family inhibited sperm binding. Sperm binding to pig oocyte ZP is therefore dependent on the carbohydrate moiety of the glycoproteins and appears to involve more than a single ZP glycoprotein.     

Electron-microscopic localization of baboon acrosomal antigens using antisperm monoclonal antibody.

A sperm antigen corresponding to baboon sperm monoclonal antibody 1A9 was localized in the testis and ejaculated sperm in this animal, using the immunofluorescence technique and immunogold labelling. Immunohistochemical studies of the baboon testis showed that the antigenic determinant was localized in the late spermatid cells and spermatozoa close to the seminiferous tubules. Immunofluorescence studies indicate that the protein was localized on the acrosome region of ejaculated baboon sperm. At the electron-microscopic level, gold particles indicative of the presence of this determinant recognized by 1A9 monoclonal antibody were detected on the inner acrosomal region of ejaculated baboon sperm.     

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.     

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.     

Immunolocalization of a guinea pig sperm surface antigen recognized by a monoclonal antibody E74

E74 is a mouse monoclonal antibody raised against the acrosome-reacted guinea pig spermatozoa. This study describes immunolocalization of the E74 antigen in guinea pig spermatozoa. Immunoelectron microscopy of guinea pig spermatozoa shows that the E74 antigen is localized on the equatorial segment plasma membrane following the acrosome reaction but not associated with the surface of the acrosome-intact spermatozoa. Immunoblot analysis of Triton X-100 extract of cauda epididymal guinea pig spermatozoa following one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis shows that E74 antibody recognizes a protein with an apparent molecular weight of 45,000 dalton. Immunoblot of sperm extracts separated by two dimensional gel electrophoresis indicates a broad spot of 45,000 dalton in the 5 to 7.5 isoelectric focusing range.     

Antigenic comparison of five species of mammalian zonae pellucidae

A comparison of glycoproteins of the zona pellucida (ZP) of five different mammalian species (cat, dog, rabbit, pig, and rat) has been made using polyclonal and monoclonal antibodies. In an enzyme-linked immunosorbent assay (ELISA), polyclonal antisera against total rabbit and pig ZP recognize their homologous ZP to the greatest extent but also detect crossreactive antigenic determinants in the ZP of all other species tested. Polyclonal antibodies against each of two purified rabbit ZP glycoproteins or one purified pig ZP glycoprotein also show some recognition of heterologous (pig, cat, and dog) ZP, but not rat ZP. Monoclonal antibodies (McR5-rabbit ZP protein determinant; McPSI-determinant associated with post-translational modifications of pig ZP proteins such as carbohydrates) further demonstrate that specific determinants are shared between some but not all of these mammalian species. For example, both of these antibodies recognize distinct determinants which are most abundant in pig and cat ZP. However, McR5 recognizes a determinant on all species of ZP except the rat, while McPSI does not recognize either the rabbit or rat ZP. Collectively, these studies suggest that the molecules of the pig, dog, and cat ZP are more closely related to each other than to those of the rabbit ZP, while there is little similarity with rat ZP molecules. Immunoblot analysis of ZP glycoproteins separated by two-dimensional polyacrylamide gel electrophoresis was used to identify antigenic relationships among four different species. The polyclonal antisera show that all of the major proteins of pig, rabbit, cat, and dog ZP are antigenically related.(ABSTRACT TRUNCATED AT 250 WORDS)     

Porcine zona pellucida: association of sperm receptor activity with the alpha-glycoprotein component of the Mr = 55,000 family

The immunogenicity and sperm receptor activity of five preparations of the major porcine zona pellucida glycoprotein family ZP3 (Mr = 55,000) were investigated. These included (1) ZP3, a chromatographically purified preparation of the 55,000 family; (2) ZP3 alpha, and (3) ZP3 beta, the two-component glycoproteins of the ZP3 family; (4) ZP3-EBGD, a partially deglycosylated preparation of ZP3 obtained by enzymatic treatment; and (5) ZP3-DG, a chemically deglycosylated preparation of ZP3. Titer studies using mouse and rabbit antisera prepared against each preparation yielded the following order of immunogenicity: ZP3 and ZP3 beta greater than ZP3-EBGD and ZP3 alpha greater than ZP3-DG, indicating that ZP3 becomes less immunogenic as more carbohydrate is removed. Pretreatment of intact zona with the various antisera prior to zona exposure to sperm resulted in an inhibition of sperm attachment to those zona treated with antibodies to ZP3, ZP3-EBGD, and ZP3 alpha. Pretreatment of zona with antibodies to ZP3 beta and ZP3-DG had no effect on sperm attachment. Studies involving pretreatment of boar sperm with the various ZP3 preparations prior to their use in a sperm-zona attachment assay and investigations involving displacement of the radiolabeled ZP3 preparations from sperm by unlabeled ZP3 preparations also yielded findings similar to the antibody studies. Collectively, these data indicate that ZP3 alpha probably functions as a zona receptor for boar sperm and that carbohydrate has an important role in maintaining the functional integrity of the ZP3 alpha glycoprotein.     

Enzymatic dissection of the functions of the mouse egg's receptor for sperm

During the course of sperm-egg interaction in mice, zona pellucida glycoprotein ZP3 (approximately equal to 80 kDa) serves as both receptor for sperm (J. D. Bleil and P. M. Wassarman, 1980c, Cell 20, 873-882) and inducer of the acrosome reaction (J. D. Bleil and P. M. Wassarman, 1983, Dev. Biol. 95, 317-324). In this investigation, small ZP3 glycopeptides (approximately equal to 1.5-6 kDa), obtained by extensive digestion of the purified glycoprotein with insoluble Pronase, were assayed for both sperm receptor and acrosome reaction-inducing activities. While ZP3 glycopeptides were virtually as effective as intact ZP3 in inhibiting binding of sperm to eggs in vitro ("receptor activity"), unlike intact ZP3, they failed to induce sperm to undergo the acrosome reaction. The latter was determined by indirect immunofluorescence using a monoclonal antibody directed against the acrosomal cap region of sperm. These results suggest that the sperm receptor activity of ZP3 is dependent only on its carbohydrate components, whereas acrosome reaction-inducing activity is dependent on the polypeptide chain of ZP3 as well.     

Role of sperm surface arylsulfatase A in mouse sperm-zona pellucida binding

We have previously described the zonae pellucidae (ZP) binding ability of a pig sperm surface protein, P68. Our recent results on peptide sequencing of 3 P68 tryptic peptides and molecular cloning of pig testis arylsulfatase A (AS-A) revealed the identity of P68 as AS-A. In this report, we demonstrate the presence of AS-A on the mouse sperm surface and its role in ZP binding. Using anti-AS-A antibody, we have shown by immunoblotting that AS-A was present in a Triton X-100 extract of mouse sperm. The presence of AS-A on the sperm plasma membrane was conclusively demonstrated by indirect immunofluorescence, immunogold electron microscopy, and AS-A's desulfation activity on live mouse sperm. The AS-A remained on the head surface of in vivo capacitated sperm, as revealed by positive immunofluorescent staining of oviductal/uterine sperm. Significantly, the role of mouse sperm surface AS-A on ZP binding was demonstrated by dose-dependent decreases of sperm-ZP binding on sperm pretreatment with anti-AS-A IgG/Fab. Furthermore, Alexa-430 conjugated AS-A bound to mouse ZP of unfertilized eggs but not to fertilized ones, and this level of binding increased and approached saturation with increasing Alexa-430 AS-A concentrations. Moreover, in vivo fertilization was markedly decreased when mouse sperm pretreated with anti-AS-A IgG were artificially inseminated into females. All of these results designated a new function for AS-A in mouse gamete interaction.     

Masking of sperm maturation antigen by sialic acid in the epididymis of the mouse. An immunohistochemical study

Sperm antigen expression during epididymal transit was examined in 4- to 16-week-old intact and castrated ICR mice, using the avidin-biotin complex (ABC) immunohistochemical method with monoclonal antibody T21 against a flagellar surface antigen. On untreated sections, the antigen was first expressed weakly on sperm in the proximal part of the corpus epididymis, and intraluminal components were stained in 4-week-old mice. Epididymal epithelial cells and their stereocilia, and cells in other reproductive organs were not stained. In contrast, on sections treated with neuraminidase, (1) the initial site of antigen appearance is a more proximal position in treated than in untreated sections, (2) stereocilia stained strongly, (3) the staining intensity of sperm and intraluminal components increased, and (4) some clear cells in the epithelium from the distal position of the caput to the corpus epididymis were stained. These results indicate that the antigen is produced by clear cells of the epididymal epithelium, that the antigenic determinant is masked initially by sialic acid residues, and that expression of the antigenic determinant on the sperm surface during epididymal maturation apparently involves desialylation.     

Analysis of a human sperm CD52 glycoform in primates: identification of an animal model for immunocontraceptive vaccine development

Sperm agglutination antigen-1 (SAGA-1) is a human male reproductive tract glycoform of CD52. Unique modification of CD52 N-linked oligosaccharide chains in the epididymis and vas deferens results in the appearance of a carbohydrate epitope that is localized over the entire surface of human spermatozoa. SAGA-1 was characterized by the sperm-inhibitory murine monoclonal antibody (mAb) S19, and it is the target antigen of a human mAb (H6-3C4) associated with antibody-mediated infertility. Collectively, sperm surface localization, antibody inhibition of sperm function, and potential reproductive-tissue specificity identify SAGA-1 as an attractive candidate contraceptive immunogen. To establish an animal model for the study of SAGA-1 in immunologic infertility and immunocontraceptive development, we investigated the appearance of the S19 carbohydrate epitope in nonhuman primates. The S19 mAb demonstrated little to no immunoreactivity by Western blot analysis with protein extracts of spermatozoa from the baboon, marmoset, bonnet, cynomolgus, and pigtailed macaques. Immunohistochemical analysis identified CD52 in the bonnet monkey epididymis; however, the N-linked carbohydrate moiety recognized by the S19 mAb, and unique to SAGA-1, was absent. In contrast, the S19 carbohydrate epitope was identified in chimpanzee sperm extracts by Western blot analysis and in chimpanzee epididymal tissue sections by immunohistochemical analysis, indicating that it is conserved in this close relative of the human. Chimpanzee testis, seminal vesicle, and prostate do not express the S19 epitope. Although anti-CD52 immunoreactivity was identified in the spleen, the carbohydrate moiety recognized by the S19 mAb was absent, corroborating data in the human that demonstrated tissue-specific glycosylation of sperm CD52. Immunofluorescent analysis indicated that the chimpanzee homologue of sperm CD52 was present over the entire spermatozoon. In addition, the S19 mAb agglutinated chimpanzee spermatozoa in a manner similar to the effect observed on human spermatozoa. These data indicate that the distinctive carbohydrate moiety of human sperm CD52 is present in the chimpanzee, and they identify the chimpanzee as the most appropriate primate model to study the potential of this unique CD52 glycoform as a contraceptive immunogen.     

Zona pellucida-induced acrosome reaction in boar sperm

Induction of the acrosome reaction in boar sperm by the zona pellucida (ZP) was investigated. A modified cytochemical staining method for measuring the acrosome reaction in boar sperm gave equivalent results to those obtained with transmission electron microscopy. Isolated heat-solubilized ZP effectively induced the acrosome reaction in boar sperm at a concentration of 25 micrograms/ml. Electrophoretically purified ZP components were also tested for acrosome reaction-inducing activity; both the 55,000 and 90,000 components of the ZP were effective. The carbohydrate moiety of the 55,000 component was necessary for activity because the polypeptides derived by chemical deglycosylation of the two glycoproteins did not induce the acrosome reaction.     

Localization and functional importance of a conserved zona pellucida 2 protein domain in the human and bovine ovary using monoclonal anti-ZP2 peptide antibodies

In mammals, gamete recognition and sperm binding to the oocyte are mediated by the zona pellucida (ZP), an acellular coat surrounding the plasma membrane of the oocyte that consists of particular ZP proteins. The ZP2 protein mediates secondary sperm binding to the ZP. Its primary structures are highly conserved as revealed by cDNA cloning. In the present study, we investigated the localization of ZP2 in human and bovine ovaries and oocytes and the influence of monoclonal anti-ZP2 peptide antibodies upon bovine sperm-egg interactions. We generated a monoclonal anti-ZP2 synthetic peptide antibody, mAb ZP2-20, against a sequence that is strongly conserved in the mammalian ZP2 amino acid sequence. Specificity of mAb ZP2-20 was determined by ELISA and immunoblotting, respectively. Our results show that mAb ZP2-20 specifically detected the peptide used as an antigen and reacted with its corresponding protein antigen in human and bovine ovaries. In order to elucidate effects of mAb ZP2-20 upon bovine sperm-ZP binding, we used the competitive hemizona assay (cHZA) and found that the antibodies clearly inhibit sperm binding to the ZP. We conclude that (i). monoclonal antibodies against ZP2 peptides react with ZP proteins present in bovine and human ovaries and can be used as a specific marker for ZP2; and that (ii). mAb ZP2-20 detects a ZP2 epitope that is of functional relevance for sperm-ZP interactions.     

Inhibition of bovine sperm‐oocyte fusion by a monoclonal antibody recognising the TEC‐2 epitope on bovine oocytes

The TEC‐2 antigenic determinant is a carbohydrate epitope located on a glycoprotein carrier molecule. In the mouse, this epitope is expressed on the zona pellucida and plasma membrane of the oocyte and is associated with the ZP2 glycoprotein and involved in the secondary sperm receptor mechanism. On the bovine oocyte expression is confined to the plasma membrane. The aim of this study was to determine the role the TEC‐2 epitope plays during fertilization in the bovine species using the monoclonal antibody TEC‐02. Incubating oocytes with the TEC‐02 antibody prior to fertilization inhibited cleavage in a dose‐dependent manner—the cleavage rate decreased as the concentration of the antibody increased. Significantly more sperm were bound to oocytes exposed to TEC‐02 (12 sperm/oocyte) compared to oocytes that were not incubated with the antibody (4 sperm/oocyte). Oocytes treated with the TEC‐02 antibody had a 7.5 ± 3.2% fusion rate and no cortical granule exocytosis compared with oocytes not exposed to the antibody, with 86.5 ± 5.8% of sperm‐oocyte fusions and release of cortical granules. The block to sperm‐oocyte fertilization observed in the pretreated group was overcome using intracytoplasmic sperm injection as the method of fertilization that bypassed the fusion process. Although sperm were binding to the oolemma these results suggest that fusion was not occurring and this may be due to the antibody occupying TEC‐2 epitope sites involved in the fusion process. In conclusion, the TEC‐2 epitope seems to be involved in sperm‐oocyte interaction in the bovine species and appears to be involved specifically during the fusion events of fertilization. Mol. Reprod. Dev. 54:173–178, 1999. © 1999 Wiley‐Liss, Inc.     

Porcine zona pellucida glycoprotein ZP4 is responsible for the sperm-binding activity of the ZP3/ZP4 complex

Summary The zona pellucida (ZP) is a transparent envelope that surrounds the mammalian oocyte and mediates species-selective sperm-egg interactions. Porcine and bovine ZPs consist of glycoproteins ZP2, ZP3, and ZP4. In both pig and bovine a heterocomplex consisting of ZP3 and ZP4 binds to sperm, however it is not clarified whether ZP3 or ZP4 in the complex is responsible for the sperm binding. Previously, we have established a baculovirus-Sf9 cell expression system for porcine ZP glycoproteins. A mixture of recombinant ZP3 (rZP3) and rZP4 displayed sperm-binding activity toward bovine sperm but not porcine sperm, probably due to differences in carbohydrate structure between the native and recombinant ZP glycoproteins. In this study, a mixture of porcine rZP3 and native ZP4 (nZP4) inhibited the binding of porcine sperm to the ZP. In contrast, a mixture of porcine nZP3 and rZP4 did not inhibit the binding of porcine sperm, although the mixture inhibited the binding of bovine sperm. The porcine rZP3/nZP4 mixture bound to the acrosomal region of porcine sperm, in a manner similar to that of the nZP3/nZP4 mixture. nZP3 was precipitated with rZP4, and nZP4 was precipitated with rZP3 by utilising the N-terminal tags on the recombinant proteins. These results indicated that nZP4, but not rZP4, is necessary for binding activity of porcine ZP3/ZP4 complex towards porcine sperm and further suggested that the carbohydrate structures of ZP4 in the porcine ZP3/ZP4 complex are responsible for porcine sperm-binding activity of the complex.     

Role of sialic acid in bovine sperm-zona pellucida binding

Sperm binding activity has been detected in zona pellucida (ZP) glycoproteins and it is generally accepted that this activity resides in the carbohydrate moieties. In the present study we aim to identify some of the specific carbohydrate molecules involved in the bovine sperm-ZP interaction. We performed sperm binding competition assays, in vitro fecundation (IVF) in combination with different lectins, antibodies and neuraminidase digestion, and chemical and cytochemical analysis of the bovine ZP. Both MAA lectin recognising alpha-2,3-linked sialic acid and neuraminidase from Salmonella typhimurium with catalytic activity for alpha-2,3-linked sialic acid, demonstrated a high inhibitory effect on the sperm-ZP binding and oocyte penetration. These results suggest that bovine sperm-ZP binding is mediated by alpha-2,3-linked sialic acid. Experiments with trisaccharides (sialyllactose, 3'-sialyllactosamine and 6'-sialyllactosamine) and glycoproteins (fetuin and asialofetuin) corroborated this and suggest that at least the sequence Neu5Ac(alpha2-3)Gal(beta1-4)GlcNAc is involved in the sperm-ZP interaction. Moreover, these results indicate the presence of a sperm plasma membrane specific protein for the sialic acid. Chemical analysis revealed that bovine ZP glycoproteins contain mainly Neu5Ac (84.5%) and Neu5GC (15.5%). These two types of sialic acid residues are probably linked to Galbeta1,4GlcNAc and GalNAc by alpha-2,3- and alpha-2,6-linkages, respectively, as demonstrated by lectin cytochemical analysis. The use of a neuraminidase inhibitor resulted in an increased number of spermatozoa bound to the ZP and penetrating the oocyte. From this last result we hypothesize that a neuraminidase from cortical granules would probably participate in the block to polyspermy by removing sialic acid from the ZP.