Monoclonal antibodies to the major protein of the murine zona pellucida: effects on fertilization and early development

The growing murine oocyte is surrounded by an extracellular zona pellucida consisting of three sulfated glycoproteins, ZP-1, ZP-2, and ZP-3. The smallest of these, ZP-3, has been reported to be the species-specific sperm receptor. Monoclonal antibodies have been recently characterized to three different antigenic determinants, two found exclusively on ZP-2, and one found on both ZP-2 and ZP-3. The in vivo effect of these antibodies on the three known functions of the zona pellucida were examined. The most dramatic effect was the prevention of fertilization. After administration, the monoclonal antibodies were located in the ovary on the zona pellucida of growing oocytes. Eggs ovulated subsequently were coated with the monoclonal antibodies and failed to develop into 2-cell embryos after mating. Eighty days later, the monoclonal antibodies could no longer be detected on the zona of ovarian oocytes, and this loss coincided with the resumption of fertility. These findings provide molecular evidence for the hypothesis that the immunological block to sperm-egg binding need not involve antibody specific for the sperm receptor, and that antibodies to the zona pellucida block sperm access by steric hinderance. Other known functions of the zona were unaffected. The antibodies were unable to induce the biochemical changes in the zona associated with the postfertilization block to polyspermy and had no detectable effect on preimplantation development.     

Biosynthesis of the mouse zona pellucida and the effect of anti-zona monoclonal antibodies on fertilization and early development

The mouse zona pellucida is comprised of three sulfated glycoproteins designated ZP-1, ZP-2 and ZP-3. They are synthesized during oogenesis and secreted to form an extracellular matrix which mediates sperm-egg interactions and appears to protect the growing embryo as it passes down the oviduct. Parenterally administered anti-ZP-2 and anti-ZP-3 monoclonal antibodies localize uniquely to the ovary and coat the zona pellucida surrounding growing ooctyes. When ovulated, these eggs can bind sperm but the presence of the antibody effectively prevents sperm penetration of the zona pellucida and, thus, inhibits fertilization. This contraceptive effect continues until all of the antibody coated oocytes are ovulated (−60 days) at which time the treated animals are again fertile. The anti-zona antibodies do not otherwise perturb early development.     

Monoclonal antibodies as probes of the distribution of ZP-2, the major sulfated glycoprotein of the murine zona pellucida

Three sulfated glycoproteins (ZP-1, ZP-2, and ZP-3) make up the zona pellucida, an extracellular glycocalyx that surrounds mouse oocytes. We have produced five monoclonal antibodies specific to the zona. All five immunoprecipitated ZP-2, and in addition, two of the antibodies immunoprecipitated ZP-3. This suggests the presence of either a common antigenic site or one made up in part by each of the two glycoproteins. The monoclonal antibodies bound to approximately 1.3 X 10(8) binding sites per ovulated mouse egg which represents 2% of the total number of ZP-2 molecules present in the zona. ZP-2 appeared to be present throughout the zona and indirect immunofluorescence revealed a fibrous pattern with no evidence of localization. Furthermore, this pattern of distribution, which was identical for all five monoclones, remained constant after fertilization at the two-cell embryo stage. Laser photobleaching demonstrated that ZP-2 is stably integrated in the extracellular matrix of the zona pellucida. No mouse tissue other than the ovary contained ZP-2 and ZP-2 is antigenically distinct from other previously described extracellular matrix proteins.     

Role of intra-acrosomal antigenic molecules acrin 1 (MN7) and acrin 2 (MC41) in penetration of the zona pellucida in fertilization in mice

In this study the role of two intra-acrosomal molecules, acrin 1 (MN7) and acrin 2 (MC41), during in vitro fertilization (IVF) was examined. The pertinent monoclonal antibodies mMN7 and mMC41 specifically recognize a 90 kDa protein (acrin 1) localized to the entire acrosome and a 200 kDa protein (acrin 2) localized to the cortex region of the anterior acrosome, respectively. Experiments were designed to assess the effects of mMN7 and mMC41 on fertilization in mice using TYH medium containing mMN7 or mMC41 at 0.0, 0.025, 0.05 and 0.1 mg ml-1. Under these conditions, capacitated spermatozoa inseminated the cumulus-invested oocytes. Acrosome-reacted spermatozoa inseminated the zona pellucida-free oocytes. The antibodies had no effect on sperm motility and primary binding to the zona pellucida, but significantly inhibited the rate of fertilization of zona pellucida-intact oocytes in a dose-dependent manner. A significantly small number of spermatozoa remained attached to the zona pellucida at 5 h after insemination in the presence of mMC41. mMC41 and mMN7 antibodies did not affect the fertilization rate of zona pellucida-free oocytes. Confocal laser scanning microscopy with indirect immunofluorescence traced the effect of the monoclonal antibodies on the zona pellucida-induced acrosome reaction, and revealed that mMN7 prevented completion of acrosomal matrix dispersal, whereas mMC41 did not affect the acrosome reaction. mMC41 appeared to inhibit secondary binding or some biochemical steps on the zona pellucida after the acrosome reaction but before penetration of the zona pellucida. Thus, the intra-acrosomal antigenic molecules acrin 1 and acrin 2 are essential for distinct events before sperm penetration of the zona pellucida in mice.     

Fertilization of zona-drilled mouse oocytes treated with a monoclonal antibody to the zona glycoprotein, ZP3

Opening a small aperture in the zona pellucida of mouse oocytes by using micromanipulation and a stream of acidified Tyrode's solution (zona drilling) improved the efficiency of in vitro fertilization at low sperm concentrations without adversely affecting development to the blastocyst stage. Zona drilling also permitted in vitro fertilization and development when sperm penetration through the zona was blocked by a monoclonal antibody to the protein core of the zona glycoprotein, ZP3. These results provide a direct demonstration that sperm entry occurs through the aperture and also suggest that zona drilling of human oocytes may offer a therapeutic approach when autoantibodies to the zona pellucida are suspected as a cause of infertility.     

Identification of a secondary sperm receptor in the mouse egg zona pellucida: role in maintenance of binding of acrosome-reacted sperm to eggs

During fertilization in mice, acrosome-intact sperm bind via plasma membrane overlying their head to a glycoprotein, called ZP3, present in the egg extracellular coat or zona pellucida. Bound sperm then undergo the acrosome reaction, which results in exposure of inner acrosomal membrane, penetrate through the zona pellucida, and fuse with egg plasma membrane. Thus, in the normal course of events, acrosome-reacted sperm must remain bound to eggs, despite loss of plasma membrane from the anterior region of the head and exposure of inner acrosomal membrane. Here, we examined maintenance of binding of sperm to the zona pellucida following the acrosome reaction. We found that polyclonal antisera and monoclonal antibodies directed against ZP2, another zona pellucida glycoprotein, did not affect initial binding of sperm to eggs, but inhibited maintenance of binding of sperm that had undergone the acrosome reaction on the zona pellucida. On the other hand, polyclonal antisera and monoclonal antibodies directed against ZP3 did not affect either initial binding of acrosome-intact sperm to eggs or maintenance of binding following the acrosome reaction. We also found that soybean trypsin inhibitor, a protein reported to prevent binding of mouse sperm to eggs, did not affect initial binding of sperm to eggs, but, like antibodies directed against ZP2, inhibited maintenance of binding of sperm that had undergone the acrosome reaction on the zona pellucida. These and other observations suggest that ZP2 serves as a secondary receptor for sperm during the fertilization process in mice and that maintenance of binding of acrosome-reacted sperm to eggs may involve a sperm, trypsin-like proteinase.     

Localization by monoclonal antibodies of various surface antigens of hamster spermatozoa and the effect of antibody on fertilization in vitro

To determine the importance during fertilization of various plasma membrane components of the hamster spermatozoon, monoclonal antibodies were generated in the mouse against specific sperm surface antigens. BALB/C mice were immunized with washed hamster spermatozoa from the cauda epididymidis and immune splenocytes fused with myeloma cells (P3 X 63 Ag8). The sperm-specific immunoglobulins were detected in hybridoma cultures by a solid-phase assay (ELISA). Five monoclonal antibodies bound specifically to the surface of intact hamster spermatozoa, three immunoglobulins to restricted regions of the head and tail plasmalemma as detected by immunofluorescence. In two cases, the affinity of the membrane antigen was modified during passage through the epididymis. Monoclonal antibodies to the sperm head or to the head and tail inhibited fertilization in vitro by blocking sperm attachment to the zona pellucida and the oolemma.     

Status of the rat acrosome during sperm-zona pellucida interactions

Over the past 40 years evidence from many sources has indicated that the mammalian acrosome reaction occurs within or near the cumulus oophorus. Recently, however, workers investigating in vitro fertilization in the mouse have concluded that in this system the acrosome reaction takes place on the surface of the zona pellucida. We have investigated the interaction of rat spermatozoa and the zona pellucida by using the scanning electron microscope (SEM) and two monoclonal antibodies which are directed to antigens of the rat sperm acrosome. When in vitro inseminated eggs from which the cumulus has been removed are viewed with the SEM some sperm heads on the surface of the zona pellucida appear unaltered whereas others appear to be undergoing changes. In vivo, all displayed altered head morphology. Using immunogold labeling we found that the two antibodies employed, 2C4 and 5B1, were directed to acrosomal content and vesiculating acrosomal membranes. Immunofluoresence staining of zonae pellucidae in in vitro fertilization studies revealed numerous small positive regions. These were presumably acrosomal content and membranes which had been left on the zona surface by spermatozoa which had been associated with the zona surface. Our results suggest that the rat acrosome interacts with the zona pellucida. During this interaction some acrosomal content and membranes detach from the spermatozoon and remain on the surface of the zona pellucida.     

Localization of tyrosine phosphorylated proteins in human sperm and relation to capacitation and zona pellucida binding

Mammalian sperm must undergo a process known as capacitation before fertilization can take place. A key intracellular event that occurs during capacitation is protein tyrosine phosphorylation. The objective of this study was to investigate and visualize protein tyrosine phosphorylation patterns in human sperm during capacitation and interaction with the zona pellucida. The presence of specific patterns was also assessed in relation to the fertilizing capacity of the spermatozoa after in vitro fertilization. Protein tyrosine phosphorylation was investigated by immunofluorescence. Phosphorylation increased significantly with capacitation and was localized mainly to the principal piece of human sperm. Following binding to the zona pellucida, the percentage of sperm with phosphotyrosine residues localized to both the neck and the principal piece was significantly higher in bound sperm than in capacitated sperm in suspension. When the percentage of principal piece-positive sperm present after capacitation was <7%, fertilization rates after in vitro fertilization were reduced. Different compartments of human spermatozoa undergo a specific sequence of phosphorylation during both capacitation and upon binding to the zona pellucida. Tyrosine phosphorylation in the principal and neck piece may be considered a prerequisite for fertilization in humans.     

Fertility and taxon-specific sperm binding persist after replacement of mouse sperm receptors with human homologs

The zona pellucida surrounding ovulated mouse eggs contains three glycoproteins, two of which (ZP2 and ZP3) are reported sperm receptors. After fertilization, the zona pellucida is modified ad minimus by cleavage of ZP2, and sperm no longer bind. Crosstaxa sperm binding is limited among mammals, and human sperm do not bind to mouse eggs. Using transgenesis to replace mouse ZP2 and/or ZP3 with human homologs, mouse lines with human-mouse chimeric zonae pellucidae have been established. Unexpectedly, mouse, but not human, sperm bind to huZP2 and huZP2/huZP3 rescue eggs, eggs fertilized in vitro with mouse sperm progress to two-cell embryos, and rescue mice are fertile. Also unanticipated, human ZP2 remains uncleaved after fertilization, and mouse sperm continue to bind early rescue embryos. These observations are consistent with a model in which the supramolecular structure of the zona pellucida necessary for sperm binding is modulated by the cleavage status of ZP2.     

Egg cortical granule N-acetylglucosaminidase is required for the mouse zona block to polyspermy

The mammalian egg must be fertilized by only one sperm to prevent polyploidy. In most mammals studied to date, the primary block to polyspermy occurs at the zona pellucida, the mammalian egg coat, after exocytosis of the contents of the cortical granules into the perivitelline space. The exudate acts on the zona, causing it to lose its ability to bind sperm and to be penetrated by sperm previously bound to the zona. However, the cortical granule components responsible for the zona block have not been identified. Studies described herein demonstrate that N-acetylglucosaminidase is localized in cortical granules and is responsible for the loss in sperm-binding activity leading to the zona block to polyspermy. Before fertilization, sperm initially bind to the zona by an interaction between sperm surface GalTase and terminal N-acetylglucosamine residues on specific oligosaccharides of the zona glycoprotein ZP3 (Miller, D. J., M. B. Macek, and B. D. Shur. 1992. Nature (Lond.). 357:589-593). These GalTase-binding sites are lost from ZP3 after fertilization, an effect that can be duplicated by N-acetylglucosaminidase treatment. Therefore, N-acetylglucosaminidase, or a related glycosidase, may be present in cortical granules and be responsible for ZP3's loss of sperm-binding activity at fertilization. Of eight glycosidases assayed in exudates of ionophore-activated eggs, N-acetylglucosaminidase was 10-fold higher than any other activity. The enzyme was localized to cortical granules using immunoelectron microscopy. Approximately 70 or 90% of the enzyme was released from cortical granules after ionophore activation or in vivo fertilization, respectively. The isoform of N- acetylglucosaminidase found in cortical granules was identified as beta- hexosaminidase B, the beta, beta homodimer. Inhibition of N- acetylglucosaminidase released from activated eggs, with either competitive inhibitors or with specific antibodies, resulted in polyspermic binding to the zona pellucida. Another glycosidase inhibitor or nonimmune antibodies had no effect on sperm binding to activated eggs. Therefore, egg cortical granule N-acetylglucosaminidase is released at fertilization, where it inactivates the sperm GalTase- binding site, accounting for the block in sperm binding to the zona pellucida.     

Reassessing the molecular biology of sperm-egg recognition with mouse genetics

The zona pellucida is an extracellular coat that surrounds mammalian eggs and early embryos. This insoluble matrix separates germ from somatic cells during folliculogenesis and plays critical roles during fertilization and early development. The mouse and human zona pellucida contain three glycoproteins (ZP1 or ZPB, ZP2, ZP3), the primary structures of which have been deduced by molecular cloning. Targeted mutagenesis of endogenous mouse genes and transgenesis with human homologues provide models to investigate the roles of individual zona components. Collectively, the genetic data indicate that no single mouse zona pellucida protein is obligatory for taxon-specific sperm binding and that two human proteins are not sufficient to support human sperm binding. An observed post-fertilization persistence of mouse sperm binding to "humanized" zona pellucida correlates with uncleaved ZP2. These observations are consistent with a model for sperm binding in which the supramolecular structure of the zona pellucida necessary for sperm binding is modulated by the cleavage status of ZP2.     

EARLY STEPS OF SPERM—EGG INTERACTIONS DURING MAMMALIAN FERTILIZATION

Mammalian eggs are surrounded by two egg coats: the cumulus oophorus and the zona pellucida, which is an extracellular matrix composed of sulfated glycoproteins. The first association of the spermatozoon with the zona pellucida occurs between the zona glycoprotein, ZP3 and sperm receptors, located at the sperm plasma membrane, such as the 95kDa tyrosine kinase-protein. This association induces the acrosome reaction and exposes the proacrosin/acrosin system. Proacrosin transforms itself, by autoactivation, into the proteolytical active form: acrosin. This is a serine protease that has been shown to be involved in secondary binding of spermatozoa to the zona pellucida and in the penetration of mammalian spermatozoa through it. The zona pellucida is a specific and natural substrate for acrosin and its hydrolysis and fertilization can be inhibited by antiacrosin monoclonal antibodies. Moreover, inin vitrofertilization experiments, trypsin inhibitors significantly inhibits fertilization. The use of the silver-enhanced immunogold technique has allowed immunolocalization of the proacrosin/acrosin system in spermatozoa after the occurrence of the acrosome reaction. This system remains associated to the surface of the inner acrosomal membrane for several hours in human, rabbit and guinea-pig spermatozoa while in the hamster it is rapidly lost. In the hamster, the loss of acrosin parallels the capability of the sperm to cross the zona pellucida. Rabbit perivitelline spermatozoa can fertilize freshly ovulated rabbit eggs and retain acrosin in the equatorial and postacrosomal region. These spermatozoa also show digestion halos on gelatin plates that can be inhibited by trypsin inhibitors. This evidence strongly suggests the involvement of acrosin in sperm penetration through the mammalian zona. Recently it was shown, however, that acrosin would not be essential for fertilization. It is likely, then, that such an important phenomenon in the mammalian reproductive cycle would be ensured though several alternative mechanisms.     

Behaviour and role of an intra-acrosomal antigenic molecule, acrin 3, during mouse fertilisation in vitro

In this study we examined the behaviour and role of an intra-acrosomal antigenic molecule, acrin 3, during mouse fertilisation in vitro by assessing the effect of its pertinent monoclonal antibody mMC101. Experiments were designed to assess the effect of mMC101 on sperm-zona pellucida binding, the acrosome reaction, zona pellucida penetration, sperm-egg fusion, and fertilisation in vitro. mMC101 did not affect sperm motility or primary and secondary binding to the zona pellucida, but significantly inhibited fertilisation of zona-pellucida-intact oocytes in a dose-dependent manner. In the presence of mMC101 at 100 microg/ml concentration in TYH medium, none of the oocytes developed to pronuclear stage by 5 h after co-incubation of the gametes, but the pronucleus formation rate recovered to some extent (45.3%) after 8 h, indicating a delay of early embryonic development. mMC101 also delayed and significantly suppressed zona pellucida penetration by sperm. Acrin 3 dispersed and did not remain on completely acrosome-reacted sperm. Although mMC101 did not influence the zona-pellucida-induced acrosome reaction, it significantly inhibited fertilisation when acrosome-reacted sperm in the presence of mMC101 inseminated zona-pellucida-free oocytes. However, fertilisation remained unaffected when acrosome-reacted sperm in the absence of mMC101 inseminated zona-pellucida-free oocytes even in its presence. Thus, acrin 3 appears to facilitate zona pellucida penetration and is also likely to be involved in sperm-oocyte fusion by modifying the sperm plasma membrane during the acrosome reaction.     

Cytochemical localization of GalNAc and GalNAcβ1,4Galβ1,4 disaccharide in mouse zona pellucida

Carbohydrate residues contained in the zona pellucida play a key role in the process of sperm-egg interaction. In vitro fertilization experiments have shown that a specific monoclonal antibody against GalNAcş,4Galş,4 disaccharide inhibits fertilization in mice. In the present study, the ultrastructural cytochemical localization of GalNAc residues and the GalNAcş,4Galş,4 disaccharide was carried out in ovarian and postovulatory oocytes by using lectin-gold cytochemistry and immunocytochemistry. Plant lectins SBA and DBA showed an affinity for the entire zona pellucida matrix of ovarian oocytes throughout the follicular maturation; however, immunoreactivity for GalNAcş,4Galş,4 disaccharide was not detected in ovarian oocytes at the earliest stages of follicular development but was found to be associated with the inner region of the zona matrix at the trilaminar primary follicle stage. The Golgi apparatus, vesicular aggregates, and cortical granules of the oocyte were intensely labeled by SBA and DBA throughout follicular development. Immunoreactivity to GalNAcş,4Galş,4 disaccharide was first observed in the Golgi apparatus and vesicular aggregates in trilaminar primary follicles. No immunoreactivity was observed in the cortical granules. In postovulatory oocytes, results were similar to those observed in ovarian oocytes. Our results thus suggest that (1) GalNAcş,4Galş,4 disaccharide residues are present only in the inner region of the zona pellucida and, therefore, might be involved in sperm penetration through the zona pellucida, (2) the inner and outer regions of the zona pellucida contain different oligosaccharide chains, (3) the vesicular aggregates detected in the oocyte could represent an intermediate step in the secretory pathway of zona pellucida glycoproteins and might be involved in the formation of cortical granules.     

Capacitation dependent activation of tyrosine phosphorylation generates two sperm head plasma membrane proteins with high primary binding affinity for the zona pellucida

The recognition and binding of sperm cells to the zona pellucida (the extracellular matrix of the oocyte) are essential for fertilization and are believed to be species specific. Freshly ejaculated sperm cells do not bind to the zona pellucida. Physiologically this interaction is initiated after sperm activation in the female genital tract (capacitation) via a yet unknown mechanism, resulting in the binding of a receptor in the apical sperm plasma membrane to the zona pellucida. In order to mimic this biochemically, we isolated zona pellucida fragments from gilt ovaries to prepare an affinity column with the intact zona pellucida structure and loaded this column with solubilized apical plasma membranes of boar sperm cells before and after in vitro capacitation. With this technique we demonstrated that two plasma membrane proteins of capacitated boar sperm cells showed high affinity for zona pellucida fragments. Further analysis showed that these proteins were tyrosine phosphorylated. Plasma membrane proteins from freshly ejaculated sperm cells did not exhibit any zona pellucida binding proteins, likely because these proteins were not tyrosine phosphorylated.     

Sperm binding to the zona pellucida is not sufficient to induce acrosome exocytosis

At fertilization, spermatozoa bind to the zona pellucida (ZP1, ZP2, ZP3) surrounding ovulated mouse eggs, undergo acrosome exocytosis and penetrate the zona matrix before gamete fusion. Following fertilization, ZP2 is proteolytically cleaved and sperm no longer bind to embryos. We assessed Acr3-EGFP sperm binding to wild-type and huZP2 rescue eggs in which human ZP2 replaces mouse ZP2 but remains uncleaved after fertilization. The observed de novo binding of Acr3-EGFP sperm to embryos derived from huZP2 rescue mice supports a ;zona scaffold' model of sperm-egg recognition in which intact ZP2 dictates a three-dimensional structure supportive of sperm binding, independent of fertilization and cortical granule exocytosis. Surprisingly, the acrosomes of the bound sperm remain intact for at least 24 hours in the presence of uncleaved human ZP2 regardless of whether sperm are added before or after fertilization. The persistence of intact acrosomes indicates that sperm binding to the zona pellucida is not sufficient to induce acrosome exocytosis. A filter penetration assay suggests an alternative mechanism in which penetration into the zona matrix initiates a mechanosensory signal transduction necessary to trigger the acrosome reaction.     

Monoclonal antibodies to rabbit sperm autoantigens. I. Inhibition of in vitro fertilization and localization on the egg

Biochemical and antigenic similarities exist among members of what can be considered a family of low molecular weight rabbit sperm autoantigens. These autoantigens are intrinsic plasma membrane glycoproteins specific to spermatogenic cells and spermatozoa. The amino acid and carbohydrate compositions of rabbit sperm autoantigen-1 (RSA-1) and RSA-2 were compared and monoclonal antibodies (mAb) were raised in mice against rabbit sperm autoantigens. The epitopes recognized by the antibodies were present on RSA-1, 2 and 3. A monoclonal anti-RSA-1, 2 and 3 (designated A.F. 1) was used to localize the antigen on spermatozoa and testis cells and investigate the epitope's tissue specificity. This mAb inhibited in vitro fertilization but did not block the sperm from dispersing the cumulus cells surrounding the egg. The mAb also demonstrated the presence of RSA-1, 2 and 3 on the plasma membrane of the egg after fertilization. It is concluded that the RSA family plays a central role in zona penetration.     

Role of the sperm proteasome during fertilization and gamete interaction in the mouse

In this work, we have investigated the role of the sperm proteasome during in vitro fertilization (IVF) and gamete interaction in the mouse. Proteasome activity was measured in extract and intact sperm using a specific substrate. In addition, sperm were treated with specific proteasome inhibitors and evaluated during IVF, binding to the zona pellucida, and progesterone- and zona pellucida-induced acrosome reactions. In other experiments, sperm membrane proteins were obtained resuspending them in Triton X-114, shaking vigorously and let standing by 4 hr. Soluble sperm proteins were partitioned in the aqueous phase and sperm membrane proteins in the detergent phase. In both phases, proteasome activity was measured. Labeling of cell surface sperm proteins was carried out with the cell-impermeable NHS-LC biotin, extracted with Triton X-114, and mixing with avidin-agarose beads. Nonpermeabilized sperm were incubated with an anti-proteasome monoclonal antibody and evaluated by indirect immunofluorescence. The results indicate that sperm extracts as well as intact sperm had proteasome activity; the sperm proteasome was involved in IVF, specifically during sperm-zona pellucida binding and the acrosome reaction; soluble sperm membrane proteins exhibited proteasome activity; biotin experiments indicated the presence of proteasomes on the sperm surface, which was corroborated by indirect immunofluorescence experiments. All these observations indicate that the mouse sperm proteasome participates in the binding to the zona pellucida and the acrosome reaction and that there is a pool of proteasomes located on the sperm head.     

Sperm surface galactosyltransferase activities during in vitro capacitation

Studies using genetic and biochemical probes have suggested that mouse sperm surface galactosyltransferases may participate during fertilization by binding N- acetylglucosamine (GlcNAc) residues in the egg zona pellucida. In light of these results, we examined sperm surface galactosyltransferase activity during in vitro capacitation to determine whether changes in enzymatic activity correlated with fertilizing ability. Results show that surface galactosyltransferases on uncapacitated sperm was preferentially loaded with poly N-acetyllactosamine substrates. As a consequence of capacitation in Ca(++)-containing medium, these polylactosaminyl substrates are spontaneously released from the sperm surface, thereby exposing the sperm galactosyltransferase for binding to the zona pellucida. Sperm capacitation can be mimicked, in the absence of Ca(++), either by washing sperm in Ca(++)-free medium, or by pretreating sperm with antiserum that reacts with the galactosyltransferase substrate. In both instances, sperm galgactosylation of endogenous polylactosaminyl substrates is reduced, coincident with increased galactosylation of exogenous GlcNAc, and increased binding to the zona pellucida. Binding of capacitated sperm to the egg can be inhibited by pronase-digested high molecular weight polyactosaminyl glycoside extracted from epidymal fluids or from undifferentiated F9 embryonal carninoma cells. Thus, these glycosides function as “decapacitation factors” when added back to in vitro fertilization assays. These glycoside “decapacitation factors” inhibit sperm-egg binding by competeing for the sperm surface galactosyltransferase, since (a) they are galactosylated by sperm in the presence of UDP[(3)H]galactose, and (b) enzymatic removal of terminal GlcNAc residues reduces “decapacitation factio” competition. On the other hand “conventional” low molecular weight glycosides, isolated from either epididymal fluid or differentiated F9 cells, fail to inhibit capacitated sperm binding to the zona pellucida. These results define a molecular mechanism for one aspect of sperm capacitation, and help explain why removal of “decapacitation factos” is a necessary prerequisite for sperm binding to the zona pellucida.