Studies on fertilization in the ascidians : Fucosyl sites on vitelline coat of Ciona intestinalis

The vitelline coat (originally called chorion) of the ascidian egg is the site where species-specific recognition and binding of spermatozoa occurs. Recent findings from this laboratory have suggested that fucosyl residues are present on the vitelline coat of Ciona intestinalis eggs and play an important role in the process of fertilization. The results reported in this paper confirm and extend those findings. With Fucose Binding Protein (FBP) and Fucosyl-Ferritin as markers, fucosyl sites have been localized on the fibrillar tufts emerging from the outer surface of the vitelline coat, both on glycerol-treated eggs and on living eggs deprived of follicle cells at pH 5. Observations on isolated vitelline coats have shown that the inner surface does not contain fucosyl sites. Studies performed with ¹²⁵I-FBP on glycerol-treated eggs have indicated that two distinct classes of fucosyl sites are present on the vitelline coat. The association constants for FBP of the high affinity and of the low affinity class are 2.3×10⁶ and 4.1×10⁵ respectively. The gel electrophoresis of the proteins extracted in sodium dodecyl sulfate (SDS) from sonicated vitelline coats has shown three fucosyl-containing polypeptide bands.     

Bacillus subtilis spore coats: complexity and purification of a unique polypeptide component.

Extensively washed, dormant spores of Bacillus subtilis were disrupted with glass beads in buffer at pH 7 in the presence of protease inhibitors. Approximately 31% of the total spore protein was soluble, and another 14% was removed from the insoluble fraction by hydrolysis with lysozyme and washing with 1 M KCl and 0.1% sodium dodecyl sulfate. The residual spore integuments comprised 55% of the total spore proteins and consisted of coats and residual membrane components. Treatment of integuments with sodium dodecyl sulfate and reducing agents at pH 10 solubilized 40% of the total spore protein. Seven low-molecular-weight polypeptide components of this solubilized fraction comprised 27% of the total spore protein. They are not normal membrane components and reassociated to form fibrillar structures resembling spore coat fragments. The residual insoluble material (15% of the total spore protein) was rich in cysteine and was probably also derived from the spore coats. A solubilized coat polypeptide of molecular weight 12,200 has been purified in good yield (4 to 5% of the total spore protein). Five amino acids account for 92% of its total amino acid residues: glycine, 19%; tyrosine, 31%; proline, 23%; arginine, 13%; and phenylalanine, 6%.     

Influence of buffer ions and divalent cations on coated vesicle disassembly and reassembly

Disruption of the coat of coated vesicles is accompanied by the release of clathrin and other proteins in soluble form. The ability of solubilized coated vesicle proteins to reassemble into empty coats is influenced by Mg²⁺, Tris ion concentration, pH, and ionic strength. The proteins solubilized by 2 M urea spontaneously reassemble into empty coats following dialysis into isolation buffer (0.1 M MES–1 mM EGTA–1 mM MgCl₂–0.02% NaN₃, pH 6.8). Such reassembled coats have sedimentation properties similar to untreated coated vesicles. Clathrin is the predominant protein of reassembled coats; most of the other proteins present in native coated vesicles are absent. We have found that Mg²⁺ is important in the coat assembly reaction. At pH 8 in 0.01 M or 0.1 M Tris, coats dissociate; however, 10 mM MgCl₂ prevents dissociation. If the coats are first dissociated at pH 8 and then the MgCl₂ raised to 10 mM, reassembly occurs. These results suggest that Mg²⁺ stabilizes the coat lattice and promotes reassembly. This hypothesis is supported by our observations that increasing Mg²⁺ (10 μM–10 mM) increases reassembly whereas chelation of Mg²⁺ by (EGTA) inhibits reassembly. Coats reassembled in low-Tris (0.01 M, pH 8) supernatants containing 10 mM MgCl₂ do not sediment, but upon dialysis into isolation buffer (pH 6.8), these coats become sedimentable. Nonsedimentable coats are noted also either when partially purified clathrin (peak I from Sepharose CL4B columns) is dialyzed into low-ionic-strength buffer or when peaks I and II are dialyzed into isolation buffer. Such nonsedimentable coats may represent intermediates in the assembly reaction which have normal morphology but lack some of the physical properties of native coats. We present a model suggesting that tightly intertwined antiparallel clathrin dimers form the edges of the coat lattice.     

Egg coats of the rock shrimp Rhynchocinetes typus: ultrastructural characterization and their function during fertilization

The aim of the present work was to characterize structurally and ultrastructurally the egg coats of the rock shrimp, Rhynchocinetes typus, and to describe their functional roles during fertilization. Oocytes fixed directly from the ovary, have a total diameter of 549 microm and are covered by a 10- microm-thick transparent envelope. Electron microscope sections (dehydrated) of the egg envelope revealed an electron-dense external coat of 0.4 microm covered by filamentous processes, and a granular inner coat of 4- microm thickness. Oocytes placed for 5 min in seawater had a significantly larger diameter (573 microm), because of the increase in the thickness of the egg coats (32 microm) and the formation of a 16- microm perivitelline space. The diameter of the egg proper was reduced by the same extent as the size of the perivitelline space. All these changes were associated to the loss of the egg fertilizability. SDS-PAGE of isolated and solubilized egg coats with 20% beta-mercaptoethanol or 25 mM dithiothreitol (DTT) showed bands between 58 and 105 kDa and between 44 and 103 kDa, respectively. During normal fertilization, the sperm undergoes a drastic change in shape after first contact with the egg. We observed a similar change when solubilized egg coats were placed with vas deferens sperms. When the solubilized egg coat proteins were ultrafiltrated with a membrane of 10,000 MWCO (pore size) and then assayed for their effect on fertilization, an inhibitory effect of 30%, 41%, and 59% was found when oocytes were incubated with spermatozoa pre-treated with 30, 60, and 120 microg/ml of proteins solubilized with beta-mercaptoethanol. A similar inhibitory effect was found when egg coat proteins solubilized with 25 mM DTT were used. Our results suggest that, in the shrimp, the egg coats play an active role during the morphological changes of the sperm during their passage through them.     

Pre-fusion events of sperm-oocyte interaction in the marine shrimp,Sicyonia ingentis

The non-motile sperm of Sicyonia ingentis, mixed with eggs by a spawning female, undergo a primary binding to the vitelline envelope (VE) of the oocyte. Once bound to the VE, sperm undergo exocytosis of the acrosomal vesicle, penetrate the VE, and secondarily bind to a surface coat that is closely associated with the oolemma. Unreacted sperm preincubated with solubilized VE components exhibit diminished binding to VEs in a concentration dependent manner. The ligand responsible for this binding is a carbohydrate moiety in the VE. The ligand preferentially binds to the anterior tip of unreacted sperm, as demonstrated with anti-VE polyclonal antibodies. Acrosome intact sperm will not bind to surface coats; however, acrosome reacted sperm do bind to surface coats via an externalized acrosomal granule.     

On the contents of the cortical granules from Xenopus laevis eggs

The extruded contents of the cortical granules in eggs of Xenopus laevis were solubilized by exposure to divalent metal ion chelators. Chelator extraction of cortical granule (CG) material from intact fertilized or artificially activated eggs was quantitated by fluorescence spectroscopy. The isolated fertilization envelope, formed upon interaction between CG material and the preexisting vitelline envelope, was also subject to extraction. An ultrastructural analysis revealed that chelator exposure resulted in the disruption of the structural integrity of the CG-derived F-component of the fertilization envelope. CG material was isolated from Xenopus ova by three procedures: (1) extrusion from artificially activated, dejellied eggs; (2) extraction of intact, fertilized eggs; and (3) extraction of isolated fertilization envelopes. Only 4–5% of the CG protein recovered by extrusion or by extraction of the intact fertilized egg could be associated with the isolated fertilization envelopes. One predominant polypeptide fraction with an identical relative mobility was demonstrated in all CG preparations upon polyacrylamide gel electrophoresis in SDS. Polymeric forms of CG protein were detected in chelator extracted preparations. The presence of an intact jelly coat during CG breakdown was a prerequisite to the transformation of the vitelline envelope to a fertilization envelope with altered physicochemical characteristics. Further, the CG-derived F-component of the fertilization envelope did not appear to play a critical role in determining the physicochemical properties of the fertilization envelope.     

ELECTRON MICROSCOPIC DEMONSTRATION OF A DITHIOTHREITOL-LABILE VITELLINE COAT SURROUNDING THE UNFERTILIZED EGG OF COMANTHUS JAPONICA (ECHINODERMATA: CRINOIDEA)*

In Comanthus, the unfertilized egg is surrounded by a vitelline coat, which is separated from the underlying plasma membrane by a space several hundred Ångstroms wide. By electron microscopy, the vitelline coat is a distinct layer 100 to 150 Å thick, which consists of finely granular material of moderate electron density. Treatment for 3 min in 0.01 M dithiothreitol in sea water buffered to pH 9.2 almost completely removes the vitelline coat and causes the irregularly shaped egg to become spherical. After such DTT-treated eggs have been washed for 2 min in sea water, they cannot be fertilized, but they can undergo a cortical reaction when treated with ionophore A23187. This cortical reaction consists of the exocytosis of cortical granule material directly into the surrounding sea water. By several hours after DTT treatment, most of the eggs, whether exposed to ionophore or not, fragment into spheres of diverse sizes.     

THE ISOLATION OF A MAJOR STRUCTURAL ELEMENT OF THE SEA URCHIN FERTILIZATION MEMBRANE

Procedures for isolating the contents of the cortical granules from the ova of the sea urchin, Strongylocentrotus purpuratus, are reported. Dithiothreitol is used to remove the vitelline coat; the "demembranated" eggs are then subsequently activated with butyric acid. By means of these procedures, the hyaline protein and crystalline or paracrystalline material have been isolated from the cortical granules. The crystalline material consists of sheets of cylinders or tubules 150–200 A in diameter. This material is believed to be a major structural element of the fertilization membrane which, in the absence of the vitelline coat, does not form.     

Phorbol Ester Induces Elevation of the Vitelline Coat of Eggs of the Ascidian Halocynthia roretzi

Elevation of the vitelline coat of eggs of the ascidian, Halocynthia roretzi , was induced by 12-O-tetradecanoylphorbol-13-acetate or phorbol-12, 13-didecanoate, but not by their 4-epimers. After elevation of the vitelline coat in this way, eggs were unable to undergo cleavage on addition of sperm. This elevation of the vitelline coat by phorbol esters was inhibited by specific inhibitors of trypsin-like enzyme, calmodulin, phospholipase A₂, and protein kinase C. In association with elevation of the vitelline coat, a trypsin-like enzyme was released from the eggs. The properties of this enzyme were similar to those of the enzyme released by calcium ionophore, which also induced elevation of the vitelline coat, suggesting that similar exocytosis of intracellular granules occurred in both cases. Calmodulin, possibly involved in the elevation process, was isolated from eggs and characterized.     

Vitelline coat of Unio elongatulus: II. Biochemical properties of the 220- and 180-kD components

In a previous study we found that two glycoproteins with apparent molecular weights of 220 kD and 180 kD account for 80–90% of the material dissolved from the vitelline coat of the egg of the bivalve mollusk, Unio elongatulus (Focarelli and Rosati, 1993: Mol Reprod Dev 35:44–51). In this study we isolated and purified these glycoproteins by electroelution. The two proteins differ in many respects: the 180-kD molecule is acidic in nature and highly heterogeneous, whereas the 220-kD protein is neutral and less heterogeneous. Both seem to have O- and N-linked oligosaccharide chains. The 180-kD protein contains 13–16% carbohydrate, whereas the 220-kD molecular contains only 7–8%. Amino acid analysis and peptide mapping also show that each protein represents a unique polypeptide chain. © 1995 Wiley-Liss, Inc.     

Oviducal pars recta-induced degradation of vitelline coat proteins in relation to acquisition of fertilizability of toad eggs

In the toad Bufo bufo japonicus the vitelline coat (VC) of the uterine egg (UEVC) is more readily lysed by the sperm lysin than the VC of coelomic egg (CEVC). Fluorometric determinations of released proteins after incubation of the VC with the sperm lysin in vitro revealed that the CEVC is not completely refractory to the lysin but increases in susceptibility after treatment with a pars recta extract (PRE). Experiments employing isolated pars recta granules showed that both this increase of VC susceptibility and the acquisition of egg fertilizability are ascribable to the contents of the granules. SDS-PAGE analyses of VC proteins revealed that in comparison with CEVC, UEVC lacks 40–52K proteins concomitant with the increased stainability of 39K protein and the appearance of 36K protein. These changes in SDS-PAGE profiles were observed either in oviducal eggs after passage through the pars recta or in coelomic eggs treated with PRE but were inhibited when coelomic eggs were treated with PRE containing soybean trypsin inhibitor (SBTI) and leupeptin. Likewise, the acquisition of fertilizability by treatment of coelomic egg with PRE was inhibited by SBTI. Dejellied uterine eggs were successfully fertilized when pretreated with trypsin inhibitors before insemination but were not fertilized when pre-treated with concanavalin A. We propose that the hydrolytic degradation of certain VC proteins due to the tryptic activity of pars recta granules renders the VC susceptible to the sperm lysin, so that the eggs are made receptive to a fertilizing sperm.     

The Properties of the Oviducal Pars Recta Protease which Mediates Gamete Interaction by Affecting the Vitelline Coat of a Toad Egg

SDS-PAGE analyses of the vitelline coats (VCs) of coelomic eggs (CEVC) and uterine eggs (UEVC) of Bufo japonicus revealed that the UEVC lacks the 40K–52K molecular weight components present in the CEVC; this is concomitant with the increased stainability of a 39K component and the appearance of a 36K component. These macromolecular alterations, accompanied by the acquisition of egg fertilizability, were induced when coelomic eggs were treated with the contents of secretory granules obtained from the oviducal pars recta (PRG). Gel-filtration of PRG in combination with hydrolytic assays employing either fluorescamine-labeled CEVC or a variety of synthetic substrates showed that the CEVC to UEVC alterations are ascribable to the action of a protease hydrolyzing specifically peptidyl-Arg-MCAs in a highly Ca²⁺-dependent way. This enzyme, which has an optimal pH of 8.0–8.2, is inhibited by soybean trypsin inhibitor and leupeptin, as well as by such serine protease inhibitors as DFP and p-APMSF. On the basis of a SDS-PAGE analysis, its molecular weight is estimated to be 66K. Treatment of coelomic eggs with the partially purified PR protease did not render the eggs fertilizable, although CEVC to UEVC macromolecular alterations were effected. We conclude that the action of this oviducal protease in partially hydrolyzing the VC is a prerequisite but insufficient in itself to render the coelomic eggs fully accessible to a fertilizing sperm.     

Synthesis and function of the fibrous layers covering the eggs of Siphlonurus lacustris (Ephemeroptera, Siphlonuridae)

Ultrastructural analysis (transmission and electron scanning microscopy) of the eggs of the mayfly Siphlonurus lacustris (Eaton) showed that they are wrapped in a thick coat composed of a network of tightly entwined filaments. Groups of twisted filaments form slightly uplifted buttons that are scattered on the coat surface. After experimentally induced egg deposition, egg–water interaction promotes marked cohesion of the eggs and their firm adhesion to the substrate. Egg masses include numerous gametes; the covering of those located close to the substrate greatly extends to anchor the whole mass. Eggs removed from the coat reveal a slightly punctuated smooth chorion and tagenoform micropyles (three to five). The coat increases egg size by about 20%. The lack of female reproductive accessory glands in Ephemeroptera transfers the synthesis of the adhesive coats to the follicle cells, which are typically competent for insect egg shell deposition (vitelline envelope and chorionic layers). This covering results from electron‐dense granules that give rise to filaments progressively organized to form superimposed layers variously orientated around the egg. In addition to egg adhesion to the substrate, a trophic function and protection from shear stress are postulated for this covering.     

Origin of 33 kDa protein of vitelline membrane of quail egg: Immunological studies

The inner layer of vitelline membrane is an investment of avian ovum at the time of ovulation, but its formation is poorly understood. In order to elucidate the origin of the inner layer of vitelline membrane, a 33 kDa protein, one of the components of the inner layer, was purified from quail eggs and polyclonal antibody was raised against this protein. The tissue distribution of protein interacted with the antibody was studied by Western blotting technique. No immunoreactive component could be observed in extracts of liver, kidney, heart, lung, small intestine, brain, infundibulum, albumen-secreting region of oviduct, uterus, and wall of small white follicles. The intensive band was detected in the granulosa layer, which was isolated from the large preovulatory follicles as a monolayer of granulosa cells sandwiched between the inner layer of vitelline membrane and the basal lamina. The granulosa cells isolated from the granulosa layer also reacted with this antibody. Theca layer had no immunoreactive components. The position of the band of the 33 kDa protein on SDS-PAGE was sifted to higher molecular weight in follicular tissues as compared with that in the laid eggs, indicating that the structural change of the protein occurs after ovulation. These studies indicate that the material reactive to the antibody raised against a 33 kDa protein of quail vitelline membrane is synthesized by the granulosa cells.     

Coat and enterotoxin-related proteins in Clostridium perfringens spores

Coat proteins from mature spores of two enterotoxin-positive (Ent+) and two enterotoxin-negative (Ent-) strains of Clostridium perfringens were solubilized using 50 mM-dithiothreitol and 1% sodium dodecyl sulphate at pH 9.7, and alkylated using 110 mM-iodoacetamide to prevent aggregation. The coat proteins and C. perfringens type A enterotoxin (CPE) were separated by SDS-PAGE and analysed by Western blotting using anti-CPE antibody. As previously reported, CPE aggregated in the presence of SDS, but no aggregation occurred at concentrations below 15 micrograms CPE ml-1. Two CPE-related proteins (34 and 48 kDa) were found in the solubilized spore coat protein of Ent+ strains while only the 48 kDa CPE-related protein was found in the spore coat fraction of Ent- strains. CPE-related proteins comprised 2.7% and 0.8% of the total solubilized coat protein of Ent+ and Ent- strains respectively. CPE-related proteins could be extracted from the spores with 1% SDS alone. They could also be released by disruption of whole spores, indicating that the CPE-related proteins may be in the spore core or trapped between the core and coat layers. The results suggest that CPE is not a major structural component of the coat fraction of C. perfringens spores.     

Proteins of the sea urchin egg vitelline layer

The vitelline layers (VL) of unfertilized sea urchin eggs were isolated, and the diversity of their polypeptide constitutents estimated by two-dimensional polyacrylamide gel electrophoresis. At least 25 components are reproducibly observed. While VL polypeptides are almost certainly synthesized in the growing oocyte, they are not among the more prevalent newly synthesized proteins detected in oocytes that were isolated and labeled in vitro for 4 hr. A set of monoclonal antibodies was raised against VL components and partially characterized. The 31 monoclonals analyzed fell into 11 classes with respect to their avidity for VL proteins solubilized under mild and under strongly denaturing conditions, and to their reactions with surface components of the VLs of living eggs. Fluorescence microscopy showed diverse patterns of surface reactivity when different monoclonal antibodies were compared. Two of the monoclonal antibodies reacted with specific sets of three proteins each on VL protein blots. It is concluded that the VL is a complex structure containing a large number of different polypeptide components, the genes for several of which should now be experimentally accessible.     

Isolation, characterization, and localization of a sperm-bound N-acetylglucosaminidase that is indispensable for fertilization in the ascidian, Phallusia mammillata

N-Acetylglucosaminidase (GlcNAc'ase), which possesses by far the highest activity of all Phallusia mammillata sperm glycosidases, was isolated and purified using DEAE-cellulose, phenyl-Sepharose, and concanavalin A affinity chromatography. The molecular size of the native enzyme estimated by G-200 gel permeation was 158 kDa. On SDS-PAGE, the denatured enzyme migrated as a single band with a Mr of 78 kDa. This indicates that under nondenaturing conditions the GlcNAc'ase prevails as a dimer. The molecular activity of the enzyme was determined to be 3.7 x 10(5) U/mumole, the Km for p-NP-GlcNAc was 0.65 mM, and the Ki for GlcNAc was 5.5 mM. It has been suggested that gamete binding in ascidians might be mediated by an enzyme-substrate complex established between a sperm glycosidase and corresponding glycosides on the vitelline coat. Thus, the GlcNAc'ase should be present as an exoenzyme at the proper place on the sperm surface membrane, i.e., on the sperm tip and possibly over the mitochondrial region. We localized the enzyme with fluorescence and electron microscopy using the neoglycoprotein BSA-p-aminophenyl-N-acetyl-beta-D-glucosaminide (BSA-GlcNAc) or concanavalin A coupled either to fluorochromes or gold particles. Labeling of unreacted and activated sperm revealed three distinct binding sites, namely at the sperm tip, over the mitochondrion, and at the head-tail junction. In reacted sperm strong labeling was observed over the translocated mitochondrion as well as at the sperm tip. An intensive binding was observed along the rim which borders the cap-like structure at the sperm tip. The distribution of the enzyme reflected by these binding patterns accounts well for the suggested function. Using N-acetylglucosaminono-1,5-lactone oxime, a novel, highly specific inhibitor of GlcNAc'ase, we were able to show that this enzyme is indispensable for fertilization of intact eggs, but not of eggs deprived of their vitelline coat. These observations are discussed in terms of functional relationships which may exist between this enzyme, sperm binding, gamete recognition, and penetration of the vitelline coat.     

Properties of the Bacillus subtilis spore coat.

About 70% of the protein in isolated Bacillus subtilis spore coats was solubilized by treatment with a combination of reducing and denaturing agents at alkaline pH. The residue, consisting primarily of protein, was insoluble in a variety of reagents. The soluble proteins were resolved into at least seven bands by sodium dodecyl sulfate gel electrophoresis. About one-half of the total was four proteins of 8,000 to 12,000 daltons. These were relatively tyrosine rich, and one was a glycoprotein. There was also a cluster of proteins of about 40,000 daltons and two or three in the 20,000- to 25,000-dalton range. The insoluble fraction had an amino acid composition and N-terminal pattern of amino acids very similar to those of the soluble coat proteins. A major difference was the presence of considerable dityrosine in performic acid-oxidized preparations of insoluble coats. Coat antigen including a 60,000-dalton protein not present in extracts of mature spores was detected in extracts of sporulating cells by immunoprecipitation. This large antigen turned over in a pulse-chase experiment. Antibodies to either the array of 8,000- to 12,000-dalton coat polypeptides or to the larger coat proteins reacted with this 60,000-dalton species, suggesting a common precursor for many of the mature coat polypeptides. Spore coats seem to be assembled by processing of proteins and by secondary modifications including perhaps dityrosine formation for cross-linking.     

Immunoelectron Microscopic Demonstration of Cortical Granule Lectins in Coelomic, Unfertilized and Fertilized Eggs of Xenopus laevis

Immunoelectron microscopic studies demonstrated cortical granule lectins (CGLs) in coelomic, unfertilized and fertilized eggs of Xenopus laevis . An antiserum raised against purified cortical granule lectin 1 specifically reacted with the CGLs in immunoblotting and agar diffusion tests. When ultrathin sections were treated with the antiserum and protein A-gold solution, gold particles, indicating antigenic sites, were seen over cortical granules of coelomic and unfertilized eggs, and over the perivitelline space, the vitelline coat and the condensed region of the fertilization layer of fertilized eggs. The pre-fertilization layer immediately adjacent to the outer margin of the vitelline coat in unfertilized eggs was free from gold particles. These observations suggest that released CGLs permeate through the vitelline coat of fertilized eggs and interact with the pre-fertilization layer mainly at the outer margin of the vitelline coat, resulting in formation of the fertilization layer which acts as a block to polyspermy.     

Specific inhibition of sperm β-N-acetylglucosaminidase by the synthetic inhibitor N-acetylglucosaminono-1,5-lactone O-(phenylcarbamoyl)oxime inhibits fertilization in the ascidian, Phallusia mammillata

Increasing evidence has evolved from studies in ascidians and mammals that sperm β- N -acetylglucosaminidase (GlcNAc'ase) plays a crucial role in fertilization. In the ascidian Phallusia mammillata , GlcNAc'ase is the predominant sperm-bound glycosidase and N-acetylglucosamine (GlcNAc) is the prevailing glycoside residue on the vitelline coat. We report here that the GlcNAc'ase inhibitor O -(2-acetamido-2-deoxy-D-glucopyrano-sylidene)-amino- N -phenylcarbamate (PUGNAC) is a potent competitive inhibitor of sperm-bound GlcNAc'ase in P. mammillata . The inhibitor constant K_i for the isolated enzyme is 47 nmol/L. Fertilization of eggs is inhibited by PUGNAC in a dose dependent competitive manner with 50% inhibition at an inhibitor concentration of 85 μmol/L. Further experiments, in which intact eggs possessing an egg coat were mixed with eggs from which the coat had been removed, showed that only fertilization of intact eggs was inhibited by PUGNAC. This finding suggests that PUGNAC prevents the binding of the sperm-associated GlcNAc'ase to terminal GlcNAc residues on the vitelline coat, thus inhibiting sperm binding and subsequently fertilization. Furthermore and most importantly, it shows that treatment with PUGNAC does not affect the viability of sperm and that the process of sperm-egg fusion is not affected.