Egg envelope conversion following fertilization in Bufo japonicus

The envelope of the Bufo japonicus egg becomes impenetrable to sperm following fertilization. Electrophoretic analysis of envelopes showed that two glycoprotein components with apparent molecular weights of 65,000 and 61,000 were hydrolyzed during fertilization to 62,000 and 58,000, respectively. These two envelope components were structurally related as shown by peptide mapping and deglycosylation studies. Hardening of the envelope following egg activation was also observed, as detected by an increase in the envelope melting temperature. The involvement of proteolytic activities in the envelope hydrolysis and hardening reactions was demonstrated using protease inhibitors, and was verified for the hydrolysis reaction by observing a loss of mass in deglycosylated envelope components obtained before and after fertilization. A low ionic strength medium (less than 50 mM) was required for both the hardening and hydrolysis reactions. Envelopes from eggs activated in a high ionic strength medium were resistant to lysin from sperm, indicating that neither hydrolysis nor hardening was necessary to block lysin activity on the envelope. Both envelope hydrolysis and hardening could be effected in the absence of sperm (i.e., when eggs were activated by electric shock) and after egg jelly had been removed, indicating that neither sperm nor jelly factors were required for the envelope modifications. In addition, when eggs were activated in the presence of NH4Cl to suppress cortical granule exocytosis, envelope hardening and hydrolysis were still observed, indicating that a cortical granule-derived factor may not be involved.     

A hatching enzyme substrate in the Xenopus laevis egg envelope is a high molecular weight ZPA homolog

The Xenopus laevis egg envelope is composed of six or more glycoproteins, three of which have been cloned and identified as the mammalian homologs ZPA (ZP2), ZPB (ZP1) and ZPC (ZP3). The remaining glycoproteins are a triplet of high molecular weight components that are selectively hydrolyzed by the hatching enzyme. We have isolated one of these proteins and cloned its cDNA. The mRNA for the protein was found to be expressed only in early stage oocytes, as are other envelope components. From the deduced amino acid sequence, it was indicated to be a secreted glycoprotein with a characteristic ZP domain in the C-terminal half of the molecule. The N-terminal half was unrelated to any known glycoprotein. Comparative sequence analysis of the ZP domain indicated that it was derived from an ancestor of ZPA and ZPB, with the greatest identity to ZPA. This envelope component has been designated ZPAX.     

Independent and hetero-oligomeric-dependent sperm binding to egg envelope glycoprotein ZPC in Xenopus laevis

Vitelline envelopes are composed of glycoproteins that participate in sperm-egg interactions during the initial stages of fertilization. In Xenopus laevis, the vitelline envelope is composed of at least 4 glycoproteins (ZPA, ZPB, ZPC, and ZPX). A sperm binding assay involving the covalent coupling of envelope glycoproteins to silanized glass slides was developed. In our assay, sperm bound to the egg envelopes derived from oviposited eggs but not activated eggs. The majority of the egg envelope ligand activity for sperm binding was derived from the complex N-linked oligosaccharides of ZPC. This sperm binding involved N-acetylglucosamine and fucose residues, as binding was abolished after treatment with cortical granule beta-N-acetylglucosaminidase and commercial beta-N-acetylglucosaminidases and was reduced by 44% after treatment with alpha-fucosidase. Although both the envelope glycoproteins ZPA and ZPC possessed independent ligand activity, ZPC was the major ligand for sperm binding (75%). Mixing of isolated ZPA, ZPB, and ZPC in a ratio of 1:4:4 (equal to that in the egg envelope) resulted in sperm binding that was greater than that of the sum of the separate components. The egg glycoproteins acted in synergy to increase sperm binding. Thus, ZPC possessed both independent and hetero-oligomeric-dependent ligand activities for sperm binding.     

The vitelline envelope to fertilization envelope conversion in eggs of Xenopus laevis

Fertilization of the Xenopus laevis egg causes the conversion of the vitelline envelope to the fertilization envelope, a change reflected in the loss of sperm penetrability of the egg and the appearance of an electron-dense layer on the outer aspect of the fertilization envelope. As seen by one-dimensional gel electrophoresis, two components with molecular weights of 69,000 and 64,000 in the vitelline envelope were converted to 66,000 and 61,000 in the fertilization envelope. By two-dimensional gel electrophoresis, the components in the 69,000 and 64,000 molecular weight regions of the vitelline envelope were seen to shift to more basic isoelectric points upon conversion to the fertilization envelope. Peptide mapping by limited proteolysis suggested that the 69,000 and 64,000 molecular weight components shared the same polypeptide chains but the smaller glycoprotein lacked a carbohydrate side chain found on the larger species. Similar sites on each glycoprotein were affected when the vitelline envelope was converted to the fertilization envelope. No N-terminal amino acids could be identified on the envelope components, indicating that these glycoproteins have blocked N-termini. Ionophore A23187-activation of jellied eggs (but not dejellied eggs) caused the molecular weight changes in the absence of sperm. Thus, factors from the jelly and the cortical granules but not from sperm apparently are involved in the processing of the 69,000 and 64,000 molecular weight components.     

Proteases released from Xenopus laevis eggs at activation and their role in envelope conversion

During fertilization of the Xenopus laevis egg, the egg envelope is converted so that further sperm contact with the egg is prevented. In this study two envelope conversion reactions were investigated, envelope hardening and limited hydrolysis of two structurally related envelope glycoproteins. Both of these reactions were shown to be sensitive to protease inhibitors. In an attempt to identify egg proteases involved in envelope conversion, the medium around activated dejellied eggs was collected and analyzed. The exudate was able to convert isolated envelopes and, when the exudate was analyzed using peptide substrates, two major activities were found, one with a preference for cleavage after argininyl peptide bonds and one with a preference for phenylalaninyl peptide bonds. Analysis of exudate using SDS-polyacrylamide gel electrophoresis with gelatin cast into the gel showed two bands of proteolytic activity, one at Mr 45,000 that was identified as the trypsin-like activity and one at Mr 30,000 that was identified as the chymotrypsin-like activity. When cortical granule exocytosis was suppressed using ammonium chloride, release of the two exudate proteases was also suppressed. Studies of the envelope conversion reactions using protease inhibitors indicated that the chymotrysin-like protease was involved in envelope conversion once it had been activated by the trypsin-like protease.     

Oviductin, the Xenopus laevis oviductal protease that processes egg envelope glycoprotein gp43, increases sperm binding to envelopes, and is translated as part of an unusual mosaic protein composed of two protease and several CUB domains

The glycoprotein envelope surrounding the Xenopus laevis egg is converted from an unfertilizable to a fertilizable form during transit through the pars recta portion of the oviduct. Envelope conversion involves the pars recta protease oviductin, which selectively hydrolyzes envelope glycoprotein gp43 to gp41. Oviductin cDNA was cloned, and sequence analysis revealed that the protease is translated as the N terminus of an unusual mosaic protein. In addition to the oviductin protease domain, a protease domain with low identity to oviductin was present, possessing an apparent nonfunctional catalytic site. Three CUB domains were also present, which are related to the mammalian spermadhesin molecules implicated in mediating sperm-envelope interactions. We propose that during post-translational proteolytic processing of the mosaic oviductin glycoprotein, the processed N-terminal protease domain is released coupled to two C-terminal CUB domains and constitutes the enzymatically active protease molecule. In functional studies, isolated coelomic egg envelopes treated with oviductin purified from the oviduct showed a dramatic increase in sperm binding. This observation established that oviductin alone was the oviductal factor responsible for converting the egg envelope to a sperm-penetrable form, via an increase in sperm binding. Trypsin mimicked oviductin's effect on envelope hydrolysis and sperm binding, demonstrating that gp43 processing is the only requirement for envelope conversion.     

An egg envelope component induces the acrosome reaction in sturgeon sperm

The acrosome reaction in Acipenser transmontanus sperm can be induced by a 66,000 dalton glycoprotein that is present in Layer 3 (L3) of the egg envelope and in egg water only following exposure of the eggs to fresh water. When egg water is fractionated on Sepharose CL 6B, the 66,000 dalton glycoprotein-containing fractions possess acrosome reaction inducing activity. Egg water may be species-specific in its ability to elicit the acrosome reaction, as demonstrated by the fact that it has no effect on the sperm of Acipenser fulvescens. Egg jelly possesses no acrosome reaction, inducing activity. The major carbohydrate-containing component of the egg envelope is L3, a layer that contains galactose residues. L3 possesses a 70,000 dalton glycoprotein prior to freshwater exposure and lacks the 66,000 dalton component. If isolated from polyacrylamide gels, the 70,000 dalton glycoprotein elicits acrosome reactions in what appears to be a species specific manner. After freshwater exposure, L3 contains both the 70,000 dalton glycoprotein and the 66,000 dalton glycoprotein that is also present in egg water. The appearance of the 66,000 dalton inducer can be blocked by the incubation of eggs in fresh water containing inhibitors of trypsin activity. Thus, the soluble inducer in egg water may be proteolytically derived from a higher molecular weight complex in the egg envelope.     

Identification and characterization of a unique Xenopus laevis egg envelope component,ZPD

We report the identification of a previously undetected Xenopus laevis egg envelope component discovered through cloning experiments. A cDNA sequence was found that represented a mature protein of 32 kDa. Peptide antibodies were generated to probe for the protein in egg envelope samples and reactivity was found to a glycoprotein of approximately 80 kDa. When deglycosylated egg envelope samples were probed, a 32 kDa protein was labeled, confirming the size of the translated cDNA sequence. A BLAST analysis showed that it is most closely related (34% amino acid identity) to the ZP domains of mammalian tectorin, uromodulin and ZPA. From a dendrogram of known egg envelope glycoproteins, the new glycoprotein was shown to be unique among egg envelope components and was designated ZPD. A similar glycoprotein was identified by immunocrossreactivity in Xenopus tropicalis and Xenopus borealis egg envelopes.     

Identification of the ZPC oligosaccharide ligand involved in sperm binding and the glycan structures of Xenopus laevis vitelline envelope glycoproteins

The Xenopus laevis egg vitelline envelope is composed of five glycoproteins (ZPA, ZPB, ZPC, ZPD, and ZPX). As shown previously, ZPC is the primary ligand for sperm binding to the egg envelope, and this binding involves the oligosaccharide moieties of the glycoprotein (Biol. Reprod., 62:766-774, 2000). To understand the molecular mechanism of sperm-egg envelope binding, we characterized the N-linked glycans of the vitelline envelope (VE) glycoproteins. The N-linked glycans of the VE were composed predominantly of a heterogeneous mixture of high-mannose (5-9) and neutral, complex oligosaccharides primarily derived from ZPC (the dominant glycoprotein). However, the ZPA N-linked glycans were composed of acidic-complex and high-mannose oligosaccharides, ZPX had only high-mannose oligosaccharides, and ZPB lacked N-linked oligosaccharides. The consensus sequence for N-linked glycosylation at the evolutionarily conserved residue N113 of the ZPC protein sequence was glycosylated solely with high-mannose oligosaccharides. This conserved glycosylation site may be of importance to the three-dimensional structure of the ZPC glycoproteins. One of the complex oligosaccharides of ZPC possessed terminal beta-N-acetyl-glucosamine residues. The same ZPC oligosaccharide species isolated from the activated egg envelopes lacked terminal beta-N-acetyl-glucosamine residues. We previously showed that the cortical granules contain beta-N-acetyl-glucosaminidase (J. Exp. Zool., 235:335-340, 1985). We propose that an alteration in the oligosaccharide structure of ZPC by glucosaminidase released from the cortical granule reaction is responsible for the loss of sperm binding ligand activity at fertilization.     

cDNA cloning and sequence analysis of the Xenopus laevis egg envelope glycoprotein gp43

The glycoproteins of the Xenopus laevis egg envelope function in fertilization and development. As the unfertilizable coelomic egg transits the pars recta region of the oviduct, it is converted to a fertilizable egg by limited proteolysis of the envelope glycoprotein gp43 to gp41. This conversion is caused by an oviductally secreted serine active site protease, oviductin. We cloned a cDNA for gp43 from an oocyte cDNA library. The cDNA encoded a 454 amino acid protein homologous to the ZPC family of glycoproteins previously shown to be present in mammalian and fish egg envelopes. Conserved ZPC domains and motifs present in the Xenopus sequence included a signal peptide sequence, an N-linked glycosylation site, and 12 aligned Cys residues. In mammalian and Xenopus sequences, a furin-like (convertase) site and a C-terminal transmembrane domain were present reflecting the biosynthesis of ZPC in these species via the secretory glycoprotein pathway. However, fish envelope glycoproteins lack these sequences since they are synthesized via a different route (in the liver, transported to the ovary, and assembled into the egg envelope surrounding the oocyte). Consensus amino acid residues were identified by sequence comparisons of seven ZPC family members; 19% of the amino acid residues were invariant and 48% of the residues were identical in at least four of the seven sequences. The consensus sequence was used to make structure-fertilization function predictions for this phylogenetically conserved family of glycoproteins.     

Factors inducing closure of the micropylar canal in the chum salmon egg

The micropylar canal of the chum salmon egg was almost completely closed following egg activation caused by incubation in a hypotonic salt solution (HSS) for I h. The closure occurred in both inseminated and parthenogenetically activated eggs. Incubation of isolated envelopes from non-activated eggs in HSS or perivitelline fluid (PVF) did not induce any modification in micropylar structure, indicating that normal organization of the egg is essential for inducing closure. To reduce the volume of the perivitelline fluid, the eggs were activated in PVF or HSS containing 8 mM Dextran, Although the envelope showed hardening, closure of the micropyle was not observed in these eggs. The wall of the micropylar canal, however, possessed a slightly rough surface. Following activation in a Ca-free hypotonic salt solution with 10 mM EDTA, hardening of the egg envelope was completely inhibited. Although such eggs possessed an apparent perivitelline space, neither closure of the micropylar canal nor roughening of the canal surface were detected. We conclude that the synergistic action of perivitelline turgor pressure and perivitelline material is responsible for the closure of the micropyle.     

Participation of a metalloprotease in the fertilization-associated conversion of the egg envelope (chorion) of the fish, Oryzias latipes

The unfertilized egg envelope of medaka ( Oryzias latipes ) consists of two major groups of subunits, ZI-1,2 (74–76 kDa) and ZI-3 (49kDa). During egg envelope hardening after egg activation, both subunit groups decreased in amount, new protein bands of 57–65, 110 and 125 kDa appeared and, finally, no bands were detectable on sodium dodecylsulfate-polyacrylamide gel electrophoresis. The 110 and 125 kDa bands are intermediates formed by polymerization of such subunit groups. In contrast, treatment with iodoacetamide, an inhibitor of polymerization, revealed that the 57–65 kDa intermediates originated from ZI-1,2 by limited hydrolysis. ZI-1,2 comprises at least three distinct proteins of quite similar structure with their N -termini undetectable by Edman degradation, while the 57–65 kDa intermediates also consist of at least three proteins with the same N -terminal amino acid sequence: DGKPSNPQQPQVPQYPSK-. This fact strongly suggests a participation of a protease in the conversion of ZI-1,2 into 57–65 kDa proteins. EDTA and 1,10-phenanthrolinium inhibited the conversion and both Ca²⁺ and Zn²⁺ recovered the inhibition. These results suggest that the assumed protease is a metalloprotease.     

Physicochemical characterization of progressive changes in the Xenopus laevis egg envelope following oviductal transport and fertilization

Previous studies have shown that the Xenopus laevis egg envelope exists in three forms with differing ultrastructural, macromolecular, and sperm penetrability properties. The coelomic envelope (CE) is derived from eggs released from the ovary into the body cavity of the female, the vitelline envelope (VE) from eggs which have passed through the oviduct, and the fertilization envelope (FE) from fertilized eggs. In the present study, the physicochemical characteristics of these three envelope types were differentiated. Investigation of envelope solubility, deformability, sulfhydryl reactivity, and hydrophobic dye and ferritin binding capacity demonstrated that profound physicochemical changes occur in envelope conversions CE----VE----FE. The physical strength of the envelopes, as evidenced by deformability studies, ranked FE greater than CE greater than VE. These differences were not accountable by differences in the number of disulfide bonds, although the CE sulfhydryl groups were significantly less accessible than those in the VE or FE. All three envelope forms were hydrophilic in nature, exhibiting little ability to bind 1-anilino-8-naphthalenesulfonic acid. The CE bound greater amounts of ferritin in comparison to the VE and FE, indicating the presence of a basic domain, presumably in the 43-kDa glycoprotein, which is lost upon proteolysis to 41 kDa during the CE----VE conversion. The envelope integrity of all three forms was maintained by both noncovalent and covalent (disulfide) bonds. Measurements of the effect of pH on envelope solubilization indicated the involvement of an ionizable group with pKa of 8.0 in maintaining envelope structure.     

The coelomic envelope to vitelline envelope conversion in eggs of Xenopus laevis

An amphibian egg recovered from the body cavity is enclosed by a coelomic egg envelope. Upon transport down the oviduct, the envelope is converted to the vitelline envelope. The coelomic and vitelline envelopes are distinct in terms of sperm penetrability, ultrastructural morphology, and radioiodination profiles. In this study, the macromolecular compositions of these two envelopes were determined. Isolated envelopes were compared by one- and two-dimensional gel electrophoresis, peptide mapping, and radiolabeling. A protein with a molecular weight of 57,000 (57K) was present in the vitelline envelope but was absent in the coelomic envelope. A glycoprotein with a molecular weight of 43K in the coelomic envelope was converted to a component with a molecular weight of 41K in the vitelline envelope. The 43K-molecular weight component of the coelomic envelopes could be radioiodinated by lactoperoxidase but no labeling of the 41K-molecular weight component occurred in the vitelline envelope. Peptide mapping using limited proteolysis established that the 43K-molecular weight component of the coelomic envelope was a precursor to the 41K-molecular weight component of the vitelline envelope. These molecular alterations may underlie the ultrastructural and physiological changes occurring in these envelopes.     

Characterization of the vitelline envelope of the sea urchin Strongylocentrotus purpuratus

The vitelline envelope (VE) is an extremely thin, acellular, proteinaceous coat that surrounds the extracellular surface of sea urchin eggs. Despite previous studies on VE composition, structure and function, our understanding of the envelope is still incomplete at the molecular level. We have isolated VE components from intact, unfertilized Strongylocentrotus purpuratus eggs by reduction with alkaline dithiothreitol-sea water solutions and have characterized the macromolecules by SDS-PAGE. There were eight major glycoprotein bands, including two high molecular weight components at 265 and 300 kDa, and several minor components. We have revealed, by lectin blot analysis, that most components contain mannose, while a subset of glycoproteins contain fucose and N -acetylglucosamine; galactose and sialic acid were also detected. The components in the VE preparations were compared with cell surface complex preparations by immunoblot analysis, using antisera against a VE preparation, a 305 kDa electrophoretically purified VE glycoprotein and an extracellular portion of the sea urchin egg recombinant 350 kDa sperm receptor. Serum against the recombinant sperm receptor reacted with a component of ∼350 kDa on blots, but did not react with the 300 kDa component found in VE preparations. Therefore, we suggest these two glycoproteins are not the same.     

Disulfide formation in bovine zona pellucida glycoproteins during fertilization: evidence for the involvement of cystine cross-linkages in hardening of the zona pellucida

The time for solubilization of the bovine zona pellucida in a hypotonic buffer containing 5% (v/v) beta-mercaptoethanol and 7 mol urea l-1 increased by 10% after fertilization. Coupling with a specific fluorescent thiol probe, monobromobimane (mBBr), was markedly greater in the zona pellucida of ovarian eggs compared with fertilized eggs, indicating that the cysteine residues in the zona pellucida of unfertilized eggs are oxidized to cystines during fertilization. After endo-beta-galactosidase digestion to remove N-acetyllactosamine repeats of the carbohydrate chains, three zona pellucida glycoproteins (ZPA, ZPB and ZPC) coupled with the fluorescent bimane groups were fractionated efficiently by reverse-phase HPLC. Estimation of bimane groups in the three components and SDS-PAGE revealed that intramolecular disulfide bonds in ZPA and intra- and intermolecular disulfide bonds in ZPB were formed during fertilization, but oxidation of cysteine residues in ZPC was low. Specific proteolysis of ZPA during fertilization was also observed. These results indicate that the formation of disulfide linkages together with specific proteolysis result in the construction of a rigid zona pellucida structure, which is responsible for hardening of the zona pellucida.     

Immunological evidence that a 305-kilodalton vitelline envelope polypeptide isolated from sea urchin eggs is a sperm receptor

Direct isolation of the sea urchin egg vitelline envelope with intact sperm receptors is difficult because the envelope is firmly attached to the egg plasma membrane. We now report a method for producing an inseminated egg preparation in Strongylocentrotus purpuratus (using soybean trypsin inhibitor [STI] and Ca²⁺, Mg²⁺-free seawater) that contains an elevated vitelline envelope (VE*-STI). The VE*-STI is devoid of cortical granule material, and supernumerary sperm do not detach postinsemination, suggesting that the VE*-STI contains active sperm receptors. VE*-STIs contain a 305-kD polypeptide and additional components that range from 225 to 31 kD, whereas the 305-kD polypeptide was considerably reduced in VE*s. Electrophoresis of sperm receptor hydrolase digests of VE*-STIs showed that the 305-kD polypeptide and several other envelope polypeptides are protease substrates. Univalent Fab fragments against VE*s, VE*-STIs, and 305 and 225-kD polypeptides blocked sperm binding and fertilization in an Fab concentration-dependent manner. The 305 and 225-kD polypeptides were localized in the VE*-STI using indirect immunofluorescence. Enzyme-linked immunosorbent assays showed that the 305 and 225-kD polypeptides share determinants, suggesting that the 225-kD polypeptide may be derived from the 305-kD polypeptide by the proteolysis that occurs at the cell surface during fertilization. Fab fragments against S purpuratus VE*-STI antigens neither bound to nor blocked homologous sperm binding and fertilization of Lytechinus variegatus eggs. Cross fertilizability occurred to the extent of 5% or less between L variegatus and S purpuratus, therefore, we conclude that the 305 kD-polypeptide isolated from S purpuratus is a species-specific vitelline envelope sperm receptor.     

The vitelline envelope of eggs from the giant keyhole limpet Megathura crenulata. I. Chemical composition and structural studies.

The egg vitelline envelope of the marine invertebrate Megathura crenulata is a glycoprotein composed of 37.3 mol % protein and 62.7 mol % carbohydrate. Of the total amino acid content, 61 mol % consists of a single amino acid, threonine. The carbohydrate content includes galactosamine, galactose, and fucose. The molar ratio of threonine to galactosamine is about 1:1. Most of the threonine residues are linked to galactosamine residues via O-glycosidic bonds. A single peptide that was purified following alkaline borohydride treatment of the vitelline envelope had the structure: Abu-Pro-Abu-(Abu6, Pro1, Thr1), where Abu is 2-aminobutyric acid. Several sugar residues have been isolated following the alkaline hydrolysis of the vitelline envelope that include an octasaccharide Gal4Fu4, an hexasaccharide Gal3Fu3, a trisaccharide Gal3, fucose, and galactose. It is proposed that the vitelline envelope of Megathura crenulata eggs is composed of polypeptide chains built to a large extent of closely spaced threonine residues. Almost every threonine residue is linked to a saccharide moiety.