Oxidative stress and the role of novel thiol compounds at fertilization

A new class of thiols, the 1-methyl-4-mercaptohistidines, has been found in high concentrations in invertebrate eggs. This family, called the ovothiols, has unusual redox properties, including the ability to confer a CN- -resistant NAD(P)H oxidase activity on ovoperoxidase, the enzyme that catalyzes the physiological crosslinking of the fertilization envelope with dityrosine residues. Ovothiol has a redox potential of 44 mV positive to glutathione and thus is maintained in the reduced state in eggs by reduced glutathione, without the need for an ovothiol reductase. We propose that high concentrations of reduced ovothiol are present in eggs to protect them from the oxidative stress caused by the respiratory burst of fertilization.     

The relationship between a novel NAD(P)H oxidase activity of ovoperoxidase and the CN- -resistant respiratory burst that follows fertilization of sea urchin eggs

The extracellular protein coat of the sea urchin egg is cross-linked after fertilization via dityrosyl linkages made by an exocytosed ovoperoxidase. The source of oxidant for this reaction is unknown, but eggs produce H2O2 in amounts equivalent to the cyanide-insensitive O2 uptake "respiratory burst" that follows fertilization. Several possible H2O2-forming oxidase activities, including glucose, xanthine, fatty acyl, and fatty-acyl CoA oxidases, were absent from the egg cortex. However, an NAD(P)H-O2 oxidoreductase activity was found in the egg cortex and was completely accounted for by ovoperoxidase. Homogeneous ovoperoxidase exhibits two types of NAD(P)H oxidase activity. One of these activities is similar to that of horseradish peroxidase and lactoperoxidase; it is dependent on Mn2+ ions and catalytic amounts of phenols, such as 2,4-dichlorophenol and N-acetyltyrosinamide, and is greater than 95% inhibited by 0.1 mM cyanide. A second, novel oxidase activity utilizes Ca2+ and an unidentified, heat-stable, Mr less than 1000 factor that can be extracted by ethanol from egg homogenates. This NADH oxidase activity is only 40% inhibited by 0.1 mM cyanide and is maximally stimulated by 10 mM Ca2+. It has an apparent Km for NADH of 50 microM. The stoichiometry of NADH:O2 consumption is 1.6:1, but approaches 2:1 in the presence of 20 micrograms/ml superoxide dismutase or 200 micrograms/ml catalase. This indicates that complete reduction of O2 to water occurs and that the reaction does not produce H2O2 stoichiometrically. However, nearly complete inhibition of the reaction by higher catalase concentrations suggests that H2O2 is an intermediate. The properties of this novel oxidase activity suggest that it may play such a role in vivo.     

Superoxide peroxidase activity of ovoperoxidase, the cross-linking enzyme of fertilization

Ovoperoxidase, an enzyme secreted by the eggs of the sea urchin Stronglycocentrotus purpuratus upon activation, catalyzes the formation of dityrosine residues in the fertilization envelope. This cross-linking reaction requires extracellular H2O2, which is produced by the egg during the cyanide-insensitive "respiratory burst" of fertilization. While investigating the possibility that the sea urchin oxidase might generate O2- as a precursor to H2O2, we discovered that ovoperoxidase possessed O2- degrading activity. Ovoperoxidase catalyzed the breakdown of O2- in a reaction that was sensitive to inhibition by catalase, indicating a requirement for H2O2. High concentrations of either O2- or H2O2 inhibited the O2- degrading activity of ovoperoxidase, as did the peroxidase inhibitors aminotriazole, azide, and phenylhydrazine. When ovoperoxidase was heated at 56 degrees C, it lost O2- degrading activity in parallel with peroxidase activity. In contrast, the copper-chelating agent diethyldithiocarbamate, which completely inactivated CuZn superoxide dismutase, failed to affect ovoperoxidase. The requirement for H2O2 and the inhibition by aminotriazole, azide, and phenylhydrazine support the hypothesis that ovoperoxidase catalyzes the breakdown of O2- by a peroxidative mechanism. Ovoperoxidase may play a role in protecting the developing embryo from oxidants derived from O2-.     

Ovothiols as free-radical scavengers and the mechanism of ovothiol-promoted NAD(P)H-O2 oxidoreductase activity

Racemic ovothiol A [(+/-)-1a] and the ovothiol model compound 1,5-dimethyl-4-mercaptoimidazole (DMI, 2) were found to scavange the free radicals Fremy's salt (4) and Banfield' radical (5) much more rapidly than did the thiol antioxidant glutathione. Ovothiol A also scavenges the tyrosyl radical, with efficiency comparable to that of ascorbic acid and the tocopherol analogue trolox (3). The ovothiol model compound DMI was found to scavenge superoxide with a rate constant comparable to that of the reaction between superoxide and glutathione. These results suggest both a free-radical scavenging role for the ovothiols and a mechanism by which the ovothiols confer NAD(P)H-O2 oxidoreductase activity upon the enzyme ovoperoxidase. Investigation of this mechanism implicates the ovothiol thiyl radical and the NAD radical as key intermediates. The ovothiyl radical appears to be unreactive toward oxygen but highly reactive toward NADH. An estimate of the one-electron oxidation potential of the ovothiol anion is presented. The physical basis for the stability of the ovothiol free radical is discussed.     

Regulation of extracellular matrix assembly: in vitro reconstitution of a partial fertilization envelope from isolated components

At fertilization, the glycocalyx (vitelline layer) of the sea urchin egg is transformed into an elevated fertilization envelope by the association of secreted peptides and the formation of intermolecular dityrosine bonds. Dityrosine cross-links are formed by a secreted ovoperoxidase that exists in a Ca2+-stabilized complex with proteoliaisin in the fertilization envelope. By using purified proteins, we now show that proteoliaisin is necessary and sufficient to link ovoperoxidase to the egg glycocalyx. Specifically, we have found that ovoperoxidase can associate with the vitelline layer only when complexed with proteoliaisin; proteoliaisin binds to the vitelline layer independently of its association with ovoperoxidase; proteolytic modification of the vitelline layer is not required for this interaction to occur; the binding of proteoliaisin to the vitelline layer is mediated by the synergistic action of the two major seawater divalent cations, Ca2+ and Mg2+; the number of proteoliaisin-binding sites on the vitelline layer of unfertilized eggs is equivalent to the amount of proteoliaisin secreted at fertilization; and the binding of ovoperoxidase to the vitelline layer, via proteoliaisin, permits the in vitro cross-linking of these two in vivo substrates. The association of purified ovoperoxidase and proteoliaisin with the vitelline layer of unfertilized eggs reconstitutes part of the morphogenesis of the fertilization envelope.     

Chorion peroxidase-mediated NADH/O(2) oxidoreduction cooperated by chorion malate dehydrogenase-catalyzed NADH production: a feasible pathway leading to H(2)O(2) formation during chorion hardening in Aedes aegypti mosquitoes

A specific chorion peroxidase is present in Aedes aegypti and this enzyme is responsible for catalyzing chorion protein cross-linking through dityrosine formation during chorion hardening. Peroxidase-mediated dityrosine cross-linking requires H(2)O(2), and this study discusses the possible involvement of the chorion peroxidase in H(2)O(2) formation by mediating NADH/O(2) oxidoreduction during chorion hardening in A. aegypti eggs. Our data show that mosquito chorion peroxidase is able to catalyze pH-dependent NADH oxidation, which is enhanced in the presence of Mn(2+). Molecular oxygen is the electron acceptor during peroxidase-catalyzed NADH oxidation, and reduction of O(2) leads to the production of H(2)O(2), demonstrated by the formation of dityrosine in a NADH/peroxidase reaction mixture following addition of tyrosine. An oxidoreductase capable of catalyzing malate/NAD(+) oxidoreduction is also present in the egg chorion of A. aegypti. The cooperative roles of chorion malate/NAD(+)oxidoreductase and chorion peroxidase on generating H(2)O(2) with NAD(+) and malate as initial substrates were demonstrated by the production of dityrosine after addition of tyrosine to a reaction mixture containing NAD(+) and malate in the presence of both malate dehydrogenase fractions and purified chorion peroxidase. Data suggest that chorion peroxidase-mediated NADH/O(2) oxidoreduction may contribute to the formation of the H(2)O(2) required for chorion protein cross-linking mediated by the same peroxidase, and that the chorion associated malate dehydrogenase may be responsible for the supply of NADH for the H(2)O(2) production.     

The heme environment of ovoperoxidase as determined by optical spectroscopy

Native ovoperoxidase exhibited an optical absorption spectrum with certain similarities to lactoperoxidase, but not horseradish peroxidase, over the pH range 4.5-11.5. Ovoperoxidase had three distinct spectral forms dependent on pH, with transitions at apparent pKa values of 6.6 and 3.0. Complexes of ovoperoxidase with CN-, N3-, F-, or when reduced and ligated to carbon monoxide, CN-, or pyridine, were distinct from other peroxidases. Ovoperoxidase formed two specific and different spectral derivatives at pH 6.0 and 8.0, either in the native state, or when combined with CN-, when reduced, or when reduced and ligated to CN-. The position of the Soret band when mixed with near-stoichiometric amounts of H2O2. This cycling was inhibited by phenylhydrazine, 3-amino-1,2,4-triazole, or low pH (less than or equal to 6). Compound II was formed when ovoperoxidase was mixed with ethyl hydrogen peroxide in a 1:3 ratio, but not with H2O2. With a great excess of H2O2, Compound III was formed at pH 8.0; at pH 6.0 or below, the Soret band shifted slightly with excess of H2O2, but Compound III was never formed. Even when ovoperoxidase was bound to proteoliaisin (Weidman, P. J., and Shapiro, B. M. (1987) J. Cell Biol. 105, 561-567), ovoperoxidase exhibited spectral characteristics of the free enzyme.     

Hierarchies of protein cross-linking in the extracellular matrix: involvement of an egg surface transglutaminase in early stages of fertilization envelope assembly

The involvement of transglutaminase activity in fertilization envelope (FE) formation was investigated using eggs from the sea urchin, Strongylocentrotus purpuratus. Eggs fertilized in the presence of the transglutaminase inhibitors, putrescine and cadaverine, had disorganized and expanded FEs with inhibition of the characteristic I-T transition. The permeability of the FE was increased by these agents, as revealed by the loss of proteins from the perivitelline space and the appearance of ovoperoxidase activity in supernates from putrescine- treated eggs. [3H]putrescine was incorporated into the FE during fertilization in a reaction catalyzed by an egg surface transglutaminase that could also use dimethylcasein as a substrate in vitelline layer-denuded eggs. Egg secretory products alone had no transglutaminase activity. The cell surface transglutaminase activity was transient and maximal within 4 min of activation. The enzyme was Ca2+ dependent and was inhibited by Zn2+. We conclude that sea urchin egg surface transglutaminase catalyzes an early step in a hierarchy of cross-linking events during FE assembly, one that occurs before ovoperoxidase-mediated dityrosine formation (Foerder, C. A., and B. M. Shapiro. 1977. Proc. Natl. Acad. Sci. USA. 74:4214-4218). Thus it provides a graphic example of the physiological function of a cell surface transglutaminase.     

Generation of O2- and tyrosine cation-mediated chemiluminescence during the fertilization of sea urchin eggs

Sea urchin eggs emit light in the visible region during their fertilization. Judging from the chemiluminescence spectra, one of the excited species generated is considered to have originated from a tyrosine cation radical-mediated reaction. Chemiluminescence probes such as luminol or a cypridina luciferin analog (See text) are useful for detecting the ovoperoxidase +H2O2 reaction associated with membrane hardening and O2- generation, respectively, during fertilization of sea urchin eggs.     

Ovothiols, a family of redox-active mercaptohistidine compounds from marine invertebrate eggs

We have previously reported a novel thiol compound, 1-methyl-N alpha,N alpha-dimethyl-4-mercaptohistidine, or ovothiol, present at high concentration in the eggs of the sea urchin Strongylocentrotus purpuratus [Turner, E., Klevit, R., Hopkins, P. B., & Shapiro, B. M. (1986) J. Biol. Chem. 261, 13056-13063]. Here we report two related compounds, 1-methyl-N alpha-methyl-4-mercaptohistidine, or ovothiol B, from the scallop Chlamys hastata, and 1-methyl-4-mercaptohistidine, or ovothiol A, from the starfish Evasterias troschelii. These two compounds, as well as the S. purpuratus compound now designated ovothiol C, were isolated from eggs or ovarian tissue by S-carboxymethylation with [3H]iodoacetic acid, ion-exchange chromatography and ion-pairing high-pressure liquid chromatography. The structures of S-(carboxymethyl)ovothiols A and B were determined by 1H NMR, and that of ovothiol A was confirmed by comparison with authentic methylhistidine samples after desulfuration with Raney nickel. In the ovary of each species, the predominant methylation form of ovothiol accounts for at least 80% of the total 4-mercaptohistidine. The ovothiol concentration of the ovary far exceeds that of the testis or somatic tissues. The ovothiol C content of unfertilized S. purpuratus eggs is 1.14 mumol/10(6) eggs, equivalent to approximately 4.3 mM average concentration; the glutathione (GSH + GSSG) content is 0.9 mumol/10(6) eggs. In this species, high ovothiol levels persisted for the first 2 weeks of embryonic development. Ovothiol and glutathione account for virtually all of the trichloroacetic acid soluble-SH groups in the egg; these results are compared to several previous studies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational control of ovoperoxidase catalysis in the sea urchin fertilization membrane

The ovoperoxidase-catalyzed oxidation of iodide has been investigated as a function of pH for the homogeneous enzyme and for ovoperoxidase incorporated into several forms of the egg fertilization membrane. The pH dependent hysteresis previously observed in purified ovoperoxidase (Deits, T. L., Shapiro, B. M. (1985) J. Biol. Chem. 260, 7882-7888) is entirely absent in ovoperoxidase incorporated into the mature fertilization membrane, where the enzyme is bound noncovalently in vivo. The pH activity profile of ovoperoxidase incorporated into the mature fertilization membrane closely resembles the profile observed only transiently in purified ovoperoxidase subjected to a rapid downward pH shift. These observations can be accounted for by our previously presented mechanism for ovoperoxidase hysteresis (ibid.). We hypothesize that ovoperoxidase, upon incorporation into the fertilization membrane, is restricted to a limited subset of the conformational states available to the purified enzyme. This matrix-dependent conformational restriction is a novel control mechanism that serves to enhance the catalytic activity of ovoperoxidase upon its assembly into the fertilization membrane and thereby modulates ovoperoxidase catalysis in the vicinity of the developing egg.     

Localization and developmental fate of ovoperoxidase and proteoliaisin, two proteins involved in fertilization envelope assembly

Fertilization of the sea urchin egg leads to the assembly of an extracellular matrix, the fertilization envelope. Ovoperoxidase, the enzyme implicated in hardening the fertilization envelope, is inserted into the assembling structure via a Ca2+-dependent interaction with the protein proteoliasin (P. Weidman and B. M. Shapiro, 1987, J. Cell Biol. 105, 561-567). In the present report, polyclonal antisera were raised to ovoperoxidase and proteoliasin (purified from eggs of Strongylocentrotus purpuratus) and characterized by Western blot analysis and an enzyme-linked immunoabsorbent assay (ELISA). By indirect immunofluorescence microscopy all cortical granules of unfertilized eggs, as well as the fertilization envelope, contained both proteoliasin and ovoperoxidase. At the ultrastructural level both proteins are localized to the electron-dense spiral lamellae of the cortical granules. Western blot analysis revealed that ovoperoxidase and proteoliasin persist in early embryos until hatching, but are absent from later developmental stages. Homogenates of eggs of several other echinoderm species (Strongylocentrotus droebachiensis, Strongylocentrotus franciscanus, Pisaster ochraceus, Dendraster excentricus, and Lytechinus pictus) also contain proteins antigenically similar to ovoperoxidase and proteoliaisin, indicating that many echinoderms utilize a similar strategy for assembly of the fertilization envelope. The results underline the need for postsecretory controls in the extracellular matrix modifications that accompany the cortical reaction.     

Adult body weight is programmed by a redox-regulated and energy-dependent process during the pronuclear stage in mouse

In mammals fertilization triggers a series of Ca(2+) oscillations that not only are essential for events of egg activation but also stimulate oxidative phosphorylation. Little is known, however, about the relationship between quantitative changes in egg metabolism and specific long-term effects in offspring. This study assessed whether post-natal growth is modulated by early transient changes in NAD(P)H and FAD(2+) in zygotes. We report that experimentally manipulating the redox potential of fertilized eggs during the pronuclear (PN) stage affects post-natal body weight. Exogenous pyruvate induces NAD(P)H oxidation and stimulates mitochondrial activity with resulting offspring that are persistently and significantly smaller than controls. Exogenous lactate stimulates NAD(+) reduction and impairs mitochondrial activity, and produces offspring that are smaller than controls at weaning but catch up after weaning. Cytosolic alkalization increases NAD(P)(+) reduction and offspring of normal birth-weight become significantly and persistently larger than controls. These results constitute the first report that post-natal growth rate is ultimately linked to modulation of NAD(P)H and FAD(2+) concentration as early as the PN stage.     

Regulated proteolysis by cortical granule serine protease 1 at fertilization

Cortical granules are specialized organelles whose contents interact with the extracellular matrix of the fertilized egg to form the block to polyspermy. In sea urchins, the granule contents form a fertilization envelope (FE), and this construction is critically dependent upon protease activity. An autocatalytic serine protease, cortical granule serine protease 1 (CGSP1), has been identified in the cortical granules of Strongylocentrotus purpuratus eggs, and here we examined the regulation of the protease activity and tested potential target substrates of CGSP1. We found that CGSP1 is stored in its full-length, enzymatically quiescent form in the granule, and is inactive at pH 6.5 or below. We determined the pH of the cortical granule by fluorescent indicators and micro-pH probe measurements and found the granules to be pH 5.5, a condition inhibitory to CGSP1 activity. Exposure of the protease to the pH of seawater (pH 8.0) at exocytosis immediately activates the protease. Activation of eggs at pH 6.5 or lower blocks activation of the protease and the resultant FE phenotypes are indistinguishable from a protease-null phenotype. We find that native cortical granule targets of the protease are beta-1,3 glucanase, ovoperoxidase, and the protease itself, but the structural proteins of the granule are not proteolyzed by CGSP1. Whole mount immunolocalization experiments demonstrate that inhibition of CGSP1 activity affects the localization of ovoperoxidase but does not alter targeting of structural proteins to the FE. The mistargeting of ovoperoxidase may lead to spurious peroxidative cross-linking activity and contribute to the lethality observed in protease-null cells. Thus, CGSP1 is proteolytically active only when secreted, due to the low pH of the cortical granules, and it has a small population of targets for cleavage within the cortical granules.     

pH-induced hysteretic transitions of ovoperoxidase

Ovoperoxidase, the enzyme that catalyzes the dityrosine cross-linking of fertilization membranes of eggs from the sea urchin Stronglyocentrotus purpuratus, exhibits slow changes in catalytic activity upon alterations of pH, with attendant changes in spectral properties. For ovoperoxidase pre-equilibratated at pH 8, abrupt decreases in pH are accompanied by a slow loss in activity that is temporally associated with a change in absorbance at the Soret band. With enzyme pre-equilibrated at pH 4.5 and then shifted to higher pH, there was a slow increase in catalytic activity following a rapid change in the Soret band absorbance. These changes were reversible and led to the same equilibrium state, regardless of the direction of pH shift. The rate of approach to the equilibrium state of ovoperoxidase was independent of enzyme concentration, the presence of substrates, or temperature (from 6.5 to 39.7 degrees C). The pH-induced interconversions of catalytic and spectral properties indicate that ovoperoxidase undergoes hysteretic transitions, in which alterations in the heme environment accompany, but are not sufficient for, the expression of catalytic activity. We present a kinetic mechanism for the hysteretic relaxations and suggest how these transitions may have relevance to the assembly of the fertilization membrane in vivo.     

Identification of a magnesium-dependent NAD(P)(H)-binding domain in the nicotinoprotein methanol dehydrogenase from Bacillus methanolicus

The Bacillus methanolicus methanol dehydrogenase (MDH) is a decameric nicotinoprotein alcohol dehydrogenase (family III) with one Zn(2+) ion, one or two Mg(2+) ions, and a tightly bound cofactor NAD(H) per subunit. The Mg(2+) ions are essential for binding of cofactor NAD(H) in MDH. A B. methanolicus activator protein strongly stimulates the relatively low coenzyme NAD(+)-dependent MDH activity, involving hydrolytic removal of the NMN(H) moiety of cofactor NAD(H) (Kloosterman, H., Vrijbloed, J. W., and Dijkhuizen, L. (2002) J. Biol. Chem. 277, 34785-34792). Members of family III of NAD(P)-dependent alcohol dehydrogenases contain three unique, conserved sequence motifs (domains A, B, and C). Domain C is thought to be involved in metal binding, whereas the functions of domains A and B are still unknown. This paper provides evidence that domain A constitutes (part of) a new magnesium-dependent NAD(P)(H)-binding domain. Site-directed mutants D100N and K103R lacked (most of the) bound cofactor NAD(H) and had lost all coenzyme NAD(+)-dependent MDH activity. Also mutants G95A and S97G were both impaired in cofactor NAD(H) binding but retained coenzyme NAD(+)-dependent MDH activity. Mutant G95A displayed a rather low MDH activity, whereas mutant S97G was insensitive to activator protein but displayed "fully activated" MDH reaction rates. The various roles of these amino acid residues in coenzyme and/or cofactor NAD(H) binding in MDH are discussed.     

Assembly of the sea urchin fertilization membrane: isolation of proteoliaisin, a calcium-dependent ovoperoxidase binding protein

Fertilization of the sea urchin egg is accompanied by the assembly of an extracellular glycoprotein coat, the fertilization membrane. Assembly of the fertilization membrane involves exocytosis of egg cortical granules, divalent cation-mediated association of exudate proteins with the egg glycocalyx (the vitelline layer), and cross- linking of the assembled structure by ovoperoxidase, a fertilization membrane component derived from the cortical granules. We have identified and isolated a new protein, which we call proteoliaisin, that appears to be responsible for inserting ovoperoxidase into the fertilization membrane. Proteoliaisin is a 250,000-Mr protein that binds ovoperoxidase in a Ca2+-dependent manner, with half-maximal binding at 50 microM Ca2+. Other divalent cations are less effective (Ba2+, Mn2+, and Sr2+) or ineffective (Mg2+ and Cd2+) in mediating the binding interaction. Binding is optimal over the physiological pH range of fertilization membrane assembly (pH 5.5-7.5). Both proteoliaisin and ovoperoxidase are found in isolated, uncross-linked fertilization membranes. We have identified several macromolecular aggregates that are released from uncross-linked fertilization membranes after dilution into divalent cation-free buffer. One of these is an ovoperoxidase- proteoliaisin complex that is further disrupted only upon the addition of EGTA. These results suggest that a Ca2+-stabilized complex of ovoperoxidase and proteoliaisin forms one structural subunit of the fertilization membrane.     

The [4B-3H] NADH-H2O exchange reaction of the mitochondrial NADH dehydrogenase

The purified mitochondrial NADH dehydrogenase enzyme has been shown to catalyze a rapid [4B-3H] NADH-H2O exchange reaction. When the enzyme is subjected to a single freeze-thaw cycle there is a complete loss of NADH dehydrogenation without a measurable decrease in the [4B-3H] NADH-H2O exchange. Complete loss of the [4B-3H] NADH-H2O exchange follows brief exposure to ultraviolet photoirradiation. The differential sensitivity of the water exchange reaction and the dehydrogenase activity suggests a direct involvement of the enzymes flavin cofactor in the catalysis of the [4B-3H] NADH-H2O exchange. Arylazido-beta-alanyl NAD+ (A3'-0-[3-[N-4-azido-2-nitrophenyl)amino] propionyl]NAD+) is shown to be a potent photodependent inhibitor of the [4B-3H] NADH-H2O exchange activity following photoirradiation with visible light. This is consistent with the observed photodependent inhibition of the dehydrogenase activity by this photoprobe (Chen, S. and Guillory, R.J. (1981) J. Biol. Chem. 256, 8318-8323).     

The alphaBbetaC integrin is expressed on the surface of the sea urchin egg and removed at fertilization

Integrins are expressed on the surface of some vertebrate eggs where they are thought to have a role in fertilization. The objective of this study is to determine if integrins are expressed on sea urchin eggs. The alphaB and betaC subunits were cloned using the homology polymerase chain reaction. Monoclonal and polyclonal antibodies were developed against bacterially expressed fragments of the extracellular domains of the betaC subunit and the alphaB subunit. As well, a monoclonal antibody was developed against a synthesized peptide corresponding to part of the cytoplasmic domain of betaC. Analysis of biotinylated egg cortex extracts immunoprecipitated with either anti-betaC or anti-alphaB yields bands of 130 and 225 kDa. Immunoblots confirm that betaC is part of the complex immunoprecipitated with anti-alphaB. Confocal immunofluorescence and immunogold electron microscopy show that betaC is present on the surface of the unfertilized egg at the tips of microvilli and in cortical granules. During the cortical reaction, immunoreactivity with antibodies to the extracellular domains of betaC and alphaB disappears from the egg surface, and microvillar casts on the fertilization envelope become immunoreactive. With antibodies to the cytoplasmic domain of betaC, immunoreactivity is lost from the surface of the egg, but the fertilization envelope does not immediately become immunoreactive. In immunoblots of egg cortex there are immunoreactive bands of the predicted sizes for alphaB and betaC. However, in fertilization envelopes, a second band that is slightly lower in molecular weight is also present. Eggs fertilized in the presence of soybean trypsin inhibitor have elongated microvilli that remain bound to the elevating fertilization envelope and immunoreactive to anti-betaC antibodies. Eggs fertilized in the presence of an ovoperoxidase inhibitor, 3-amino-1,2,4-triazole, have a patchy distribution of betaC immunoreactivity in fertilization envelopes. Together, these data suggest that alphaBbetaC integrins are expressed on the surface of unfertilized eggs and, during the cortical reaction, the extracellular domains are cleaved by proteases and cross-linked into the fertilization envelope by ovoperoxidase. The alphaBbetaC integrin receptors may have several potential functions prior to their removal at fertilization, including attachment of the vitelline envelope to the egg surface and anchoring the cortical cytoskeleton.     

Reconstitution of Src-dependent phospholipase Cgamma phosphorylation and transient calcium release by using membrane rafts and cell-free extracts from Xenopus eggs

We reported previously that egg membrane rafts serve as a subcellular microdomain for sperm-dependent tyrosine kinase signaling in Xenopus fertilization. Moreover, we demonstrated that raft-associated Src tyrosine kinase was activated by sperm in vitro. Here we show that egg rafts incubated with sperm or hydrogen peroxide (H2O2) can promote Src-dependent phosphorylation of phospholipase Cgamma (PLCgamma) and transient calcium release in the extracts of unfertilized Xenopus eggs. In vivo egg activation by sperm or H2O2 also promotes tyrosine phosphorylation and raft-translocalization of PLCgamma. Immunodepletion of PLCgamma from the egg extracts inhibits the raft-dependent calcium release. Rafts prepared from H2O2-activated eggs also promote Src-dependent dephosphorylation of p42 mitogen-activated protein kinase and cell cycle transition from metaphase II to interphase in egg extracts. PLCgamma phosphorylation and calcium release in egg extracts can be promoted by rafts prepared from COS-7 cells expressing the Xenopus Src gene. These results demonstrate that the signaling events elicited by fertilization in Xenopus eggs can be reconstituted in vitro. The development of such experimental platforms will allow us to dissect the molecular mechanism of sperm-dependent activation of raft-associated Src and subsequent up-regulation of PLCgamma and egg activation machinery in Xenopus eggs.