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.     

Cross-fertilization and structural comparison of egg extracellular matrix glycoproteins from Xenopus laevis and Xenopus tropicalis

While the anuran amphibian Xenopus laevis is a widely used vertebrate model system, it is not optimal for genetic manipulations due to its tetraploid genome and long generation time. A current alternative amphibian model system, Xenopus tropicalis, has the advantages of a diploid genome and a much shorter generation time. We undertook a comparative investigation of X. tropicalis egg extracellular matrix glycoproteins in relation to those already characterized in X. laevis. Fertilization methods and isolation of egg extracellular molecules were directly transferable from X. laevis to X. tropicalis. Cross-fertilizations were successful in both directions, indicating similar molecules involved in sperm-egg interactions. Egg envelopes analyzed by SDS-PAGE were found to have almost identical gel patterns, whereas jelly component profiles were similar only for the larger macromolecules (>90 kDa). The cDNA sequences for egg envelope glycoproteins ZPA, ZPB, ZPC, ZPD and ZPAX, and also egg cortical granule lectin involved in the block to polyspermy, were cloned for X. tropicalis and showed a consistent approximately 85% amino acid identity to the X. laevis sequences. Thus, homologous egg extracellular matrix molecules perform the same functions, and the molecular and cellular mechanisms of fertilization in these two species are probably equivalent.     

The Primary Egg Envelope of the Anuran Lepidobatrachus laevis: Physicochemical and Macromolecular Alterations During Development

The coelomic egg envelope (CE) of the frog Lepidobatrachus laevis has a network of fibrillar bundles which disperse after transit through the oviduct. Following oviposition, the egg vitelline envelope (VE) has an additional amorphous zone on the exterior surface. The fertilization envelope (FE) formed after fertilization, appears to be very similar to the VE. The CEs, VEs, FEs and hatched envelopes (FE_h) were manually isolated. The CE, VE and FE were solubilized at 100° using denaturing conditions, but were only partially solubilized in phosphate buffer, pH 7.0. All envelopes and several purified polypeptides from the VE and FE were analyzed using gel electrophoresis and one-dimensional peptide mapping. Each of the envelopes contained 9 major polypeptides ranging from 118.5 to 22 kD and 8–12 minor polypeptides. Several envelope components were added/removed in the conversions based on the results of experiments in which preparations were incubated with activated egg exudate and crude hatching enzyme; some of these transformations were mimicked by tryptic and chymotryptic digestions. Therefore, serine proteases may be involved in envelope processing in vivo. Lepidobatrachus CE polypeptides and several major components from the VE, FE and FE_h were crossreactive with antibodies against Xenopus VE^*.     

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.     

Proteolysis of Xenopus laevis egg envelope ZPA triggers envelope hardening

The egg envelope of most animal eggs is modified following fertilization, resulting in the prevention of polyspermy and hardening of the egg envelope. In frogs and mammals a prominent feature of envelope modification is N-terminal proteolysis of the envelope glycoprotein ZPA. We have purified the ZPA protease from Xenopus laevis eggs and characterized it as a zinc metalloprotease. Proteolysis of isolated egg envelopes by the isolated protease resulted in envelope hardening. The N-terminal peptide fragment of ZPA remained disulfide bond linked to the ZPA glycoprotein moiety following proteolysis. We propose a mechanism for egg envelope hardening involving ZPA proteolysis by an egg metalloprotease as a triggering event followed by induction of global conformational changes in egg envelope glycoproteins.     

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.     

Membrane-associated lamins in Xenopus egg extracts: identification of two vesicle populations

Nuclear lamin isoforms of vertebrates can be divided into two major classes. The B-type lamins are membrane associated throughout the cell cycle, whereas A-type lamins are recovered from mitotic cell homogenates in membrane-free fractions. A feature of oogenesis in birds and mammals is the nearly exclusive presence of B-type lamins in oocyte nuclear envelopes. In contrast, oocytes and early cleavage embryos of the amphibian Xenopus laevis are believed to contain a single lamin isoform, lamin LIII, which after nuclear envelope breakdown during meiotic maturation is reported to be completely soluble. Consequently, we have reexamined the lamin complement of Xenopus oocyte nuclear envelopes, egg extracts, and early embryos. An mAb (X223) specific for the homologous B-type lamins B2 of mouse and LII of Xenopus somatic cells (Hoger, T., K. Zatloukal, I. Waizenegger, and G. Krohne. 1990. Chromosoma. 99:379-390) recognized a Xenopus oocyte nuclear envelope protein biochemically distinct from lamin LIII and very similar or identical to somatic cell lamin LII. Oocyte lamin LII was detectable in nuclear envelopes of early cleavage embryos. Immunoblotting of fractionated egg extracts revealed that approximately 20-23% of lamin LII and 5-7% of lamin LIII were membrane associated. EM immunolocalization demonstrated that membrane-bound lamins LII and LIII are associated with separate vesicle populations. These findings are relevant to the interpretation of nuclear reconstitution experiments using Xenopus egg extracts.     

Identification of Xenopus laevis sperm and egg envelope binding components on nitrocellulose membranes

Interacting egg envelope and sperm surface components were identified for Xenopus laevis using blotting methods. Sperm were extracted with sodium dodecyl sulfate (SDS), the extracted proteins separated by gel electrophoresis and blotted, and the blots treated with 125I-labeled heat solubilized envelopes. The converse experiment was also performed where envelope components were separated by gel electrophoresis, blotted, and the blots treated with 125I-labeled sperm components. Blotted sperm components with apparent molecular weights of 14K, 19K, 25K, and 35K selectively bound the solubilized envelopes. All of the envelope binding components were found to be localized on the sperm surface by radioiodinating intact sperm using Iodo-Gen. The blotted egg envelope component with an apparent molecular weight of 37K selectively bound to solubilized sperm components, and this binding was due to the protein moiety of the glycoprotein. 125I-labeled heat solubilized envelopes from unfertilized and fertilized eggs showed the same pattern of binding to blotted sperm components. Selected sulfated carbohydrates (fucoidan, dextran sulfate, and heparin, but not chondroitin sulfate) inhibited fertilization and binding of 125I-labeled heat solubilized envelopes to blotted sperm extract. Thus, the binding of heat solubilized envelopes to electrophoretically separated and blotted sperm proteins may reflect cellular interactions.     

Fish hatching strategies: a review

The paper presents differences in the distribution of hatching gland cells, and the location of egg envelope digestion, the significance of hatching movements, and the ways larvae escape from egg envelopes. A review of the literature on the hatching orientation of 34 fish species is compared. No correlation was seen between hatching orientation and egg diameter or newly hatched larva length, nor newly hatched larvae length ratio to egg diameter. Photographs of twelve freshwater species present the moment of hatching either head first or tail first. Some differences were shown in swelling between eggs incubated in commercial hatchery and developed in natural conditions, as well as possible effect of these differences on hatching.     

A homologue of cysteine-rich secretory proteins induces premature degradation of vitelline envelopes and hatching of Xenopus laevis embryos

We cloned Xenopus laevis CRISP, XCRISP, a homologue of the mammalian family of cysteine-rich secretory proteins (CRISPs), which has been previously identified as a Wnt3a/noggin responsive gene in an expression screen [Mech. Dev. 87 (1999) 21]. We detected XCRISP expression exclusively in the hatching gland. XCRISP enters the secretory pathway and accumulates on the surface of presumptive hatching gland cells. Overexpression studies of XCRISP and XCRISP-mutants show that XCRISP induces premature hatching of embryos preceded by degradation of the vitelline envelope. A deletion mutant that lacks a 35 amino acid domain even accelerates hatching, while further deletion of the carboxy-terminus reverses these effects. From our studies, we conclude that XCRISP is sufficient to induce degradation of vitelline envelopes and that this activity maps to the most C-terminal amino acids, while the adjacent domain regulates XCRISP activity.     

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.     

The hatching enzyme from xenopus laevis: Limited proteolysis of the fertilization envelope

Hatching in the amphibian Xenopus laevis involves release of an embryo-secreted hatching enzyme, a protease, which weakens the envelope surrounding the embryo. The envelope is not totally solubilized, which infers that only selected envelope components are hydrolyzed by the enzyme. The susceptibility of the glycoprotein components composing the envelope to hydrolysis by the hatching enzyme was investigated. Isolated envelopes in various physical states, ie, particulate and solubilized, were treated with the hatching enzyme, and the resulting envelope hydrolysis products were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The susceptibility of the envelope components to proteolysis was not a function of the state of the envelope. The envelope components most susceptible to proteolysis were the 125K and 11 8K components followed by the 60K and 71 – 77K components. These components are minor constituents of the envelope. The major constituents, 33K and 40K, were relatively resistant to hydrolysis by the hatching enzyme. From these observations, we infer that the envelope components hydrolyzed are components that link or bind together the major structural elements of the envelope, eg, the 33K and 40K components. Selective destruction of the components required for maintaining the structural integrity of the envelope, eg, the “nuts and bolts” of the structure, permits a weakening of the envelope that allows the embryo to hatch without having to destroy totally (hydrolyze) the envelope.     

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.     

Radioiodination studies of the envelopes from Xenopus laevis eggs

To investigate the molecular basis of the observed morphological and biological characteristics of coelomic egg envelopes (CE), vitelline envelopes (VE), and fertilization envelopes (FE) of Xenopus laevis eggs, envelopes were radioiodinated under a variety of conditions: in situ, isolated and intact, or solubilized. The distribution of 125I in envelope components was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Each envelope type displayed unique profiles when iodinated in the intact state. A major constituent of VE, the 41,500 molecular weight component, was not labeled in the intact state, although the corresponding component of CE was heavily labeled. After dissociation of the envelope by guanidine-HCl or sodium dodecyl sulfate, all of the components could be radioiodinated. However, when the envelopes (VE and FE) were dissolved by heating and subsequently radioiodinated by lactoperoxidase, the resulting radioactivity profile was similar to that of the intact envelopes, suggesting that in the heat-dissolved envelope, the individual components retain similar structural relations as in the intact envelope. Quantitative but not qualitative differences were found between the inner and outer aspects of VE and FE. The significance of these findings is discussed in relation to what is known about the morphological, biological, and molecular properties of the envelopes.     

Molecular identification and characterization of Xenopus egg uroplakin III, an egg raft-associated transmembrane protein that is tyrosine-phosphorylated upon fertilization

Here we describe mass spectrometric identification, molecular cloning, and biochemical characterization of a lipid/membrane raft-associated protein that is tyrosine-phosphorylated upon Xenopus egg fertilization. This protein is homologous to mammalian uroplakin III, a member of the uroplakin family proteins (UPs) that constitute asymmetric unit membranes in the mammalian urothelial tissues, thus termed Xenopus uroplakin III (xUPIII). xUPIII contains N-linked sugars and is highly expressed in Xenopus eggs, ovary, urinary tract, and kidney. In unfertilized eggs, xUPIII is predominantly localized to the lipid/membrane rafts and exposed on the cell surface, as judged by surface biotinylation experiments and indirect immunofluorescent studies. After fertilization or hydrogen peroxide-induced egg activation, xUPIII becomes rapidly phosphorylated on tyrosine residue-249, which locates in the carboxyl-terminal cytoplasmic tail of the molecule. Raft localization and tyrosine phosphorylation of xUPIII can be reconstituted in HEK293 cells by coexpression of xUPIII, and Xenopus c-Src, a tyrosine kinase whose fertilization-induced activation in egg rafts is required for initiation of development. In mammals, UPIII is forming a complex with a tetraspanin molecule uroplakin Ib. As another tetraspanin, CD9, is known to be a critical component for sperm-egg fusion in the mouse, we have assumed that xUPIII is involved in sperm-egg interaction. An antibody against the extracellular domain of xUPIII blocks sperm-egg interaction, as judged by the occurrence of egg activation and first cell cleavage. Thus, xUPIII represents an egg raft-associated protein that is likely involved in sperm-egg interaction as well as subsequent Src-dependent intracellular events of egg activation in Xenopus.     

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.     

Signal transduction pathways leading to Ca2+ release in a vertebrate model system: lessons from Xenopus eggs

At fertilization, eggs unite with sperm to initiate developmental programs that give rise to development of the embryo. Defining the molecular mechanism of this fundamental process at the beginning of life has been a key question in cell and developmental biology. In this review, we examine sperm-induced signal transduction events that lead to release of intracellular Ca(2+), a pivotal trigger of developmental activation, during fertilization in Xenopus laevis. Recent data demonstrate that metabolism of inositol 1,4,5-trisphosphate (IP(3)), a second messenger for Ca(2+) release, is carefully regulated and involves phospholipase C (PLC) and the tyrosine kinase Src. Roles of other potential regulators in this pathway, such as phosphatidylinositol 3-kinase, heterotrimeric GTP-binding protein, phospholipase D (PLD) and phosphatidic acid (PA) are also discussed. Finally, we address roles of egg lipid/membrane microdomains or 'rafts' as a platform for the sperm-egg membrane interaction and subsequent signaling events of egg activation.     

Gamete Interactions in Xenopus laevis: Identification of Sperm Binding Glycoproteins in the Egg Vitelline Envelope

A quantitative assay was developed to study the interaction of Xenopus laevis sperm and eggs. Using this assay it was found that sperm bound in approximately equal numbers to the surface of both hemispheres of the unfertilized egg, but not to the surface of the fertilized egg. To understand the molecular basis of sperm binding to the egg vitelline envelope (VE), a competition assay was used and it was found that solubilized total VE proteins inhibited sperm-egg binding in a concentration-dependent manner. Individual VE proteins were then isolated and tested for their ability to inhibit sperm binding. Of the seven proteins in the VE, two related glycoproteins, gp69 and gp64, inhibited sperm-egg binding. Polyclonal antibody was prepared that specifically recognized gp69 and gp64. This gp69/64 specific antibody bound to the VE surface and blocked sperm binding, as well as fertilization. Moreover, agarose beads coated with gp69/64 showed high sperm binding activity, while beads coated with other VE proteins bound few sperm. Treatment of unfertilized eggs with crude collagenase resulted in proteolytic modification of only the gp69/64 components of the VE, and this modification abolished sperm-egg binding. Small glycopeptides generated by Pronase digestion of gp69/64 also inhibited sperm-egg binding and this inhibition was abolished by treatment of the glycopeptides with periodate. Based on these observations, we conclude that the gp69/64 glycoproteins in the egg vitelline envelope mediate sperm-egg binding, an initial step in Xenopus fertilization, and that the oligosaccharide chains of these glycoproteins may play a critical role in this process.