Lectin binding sites in the vitelline envelope of Bufo arenarum oocytes: Role in fertilization

Incubation of dejellied spawned oocytes from Bufo arenarum with different lectins results in a decrease of oocyte fertility. Concanavalin A was the most effective lectin; phytohem-agglutinin P and wheat germ lectin were less effective. Agglutinin from soybean was scarcely active. These lectin effects could be ascribed to a hindering of specific sites for some proteases, since the same treatment renders the oocyte vitelline envelope insensitive to spermatolysin (an essential requisite for fertilization) and to trypsin. Also in this case concanavalin A was the most effective lectin. Univalent concanavalin A was also effective in blocking the fertility of dejellied oocytes. These results indicate that the residues of α-D-glucose and α-D-mannose present in the vitelline envelope are involved in gamete interactions in Bufo arenarum. This idea is also supported by the finding that dejellied oocytes (fertilizables) have a number of binding sites for concanavalin A that is three or four orders of magnitude higher than coelomic or fertilized oocytes (both not penetrable by spermatozoa).     

Sperm proteasomes are responsible for the acrosome reaction and sperm penetration of the vitelline envelope during fertilization of the sea urchin Pseudocentrotus depressus

The roles of sperm proteasomes in fertilization were investigated in the sea urchin Pseudocentrotus depressus. Two proteasome inhibitors, MG-132 and MG-115, inhibited fertilization at 100 microM, whereas chymostatin and leupeptin showed no inhibition. Among three proteasome substrates, Z-Leu-Leu-Glu-MCA showed the strongest inhibition toward fertilization. MG-132 inhibited the egg-jelly-induced, but not ionomycin-induced, acrosome reaction. In addition, MG-132, but not E-64-d, inhibited fertilization of dejellied eggs by acrosome-reacted sperm. MG-132 showed no significant inhibition toward the binding of reacted sperm to the vitelline layer. Proteasomes were detected by Western blotting in the acrosomal contents, which are partially released upon exocytosis. We also found that the inhibition pattern of the caspase-like activity of the proteasome in the acrosomal contents by chymostatin and proteasome inhibitors coincided well with their inhibitory abilities toward fertilization. Furthermore, the vitelline layer of unfertilized eggs appears to be ubiquitinated as revealed by immunocytochemistry and Western blotting. Extracellular ATP, required for the degradation of ubiquitinated proteins by the proteasome, was also necessary for fertilization. These results indicate that the sperm proteasome plays a key role not only in the acrosome reaction but also in sperm penetration through the vitelline envelope, most probably as a lysin, during sea urchin fertilization.     

Behaviour of the vitelline envelope in Bufo arenarum oocytes matured in vitro in blockade to polyspermy

During activation of amphibian eggs, cortical granule exocytosis causes elaborate ultrastructural changes in the vitelline envelope. These changes involve modifications in the structure of the vitelline envelope and formation of a fertilization envelope (FE) that can no longer be penetrated by sperm. In Bufo arenarum, as the egg traverses the oviduct, the vitelline envelope is altered by a trypsin-like protease secreted by the oviduct, which induces an increased susceptibility of the vitelline envelope to sperm lysins. Full-grown oocytes of B. arenarum, matured in vitro by progesterone, are polyspermic, although cortical granule exocytosis seems to occur within a normal chronological sequence. These oocytes can be fertilized with or without trypsin treatment, suggesting that the vitelline envelope is totally sperm-permeable. Vitelline envelopes without trypsin treatment cannot retain either gp90 or gp96. This suggests that these glycoproteins are involved in the block to polyspermy and that trypsin treatment of matured in vitro oocytes before insemination is necessary to enable vitelline envelopes to block polyspermy. The loss of the binding capacity in vitelline envelopes isolated from B. arenarum oocytes matured in vitro with trypsin treatment and activated by electric shock suggests that previous trypsin treatment is a necessary step for sperm block to occur. When in vitro matured oocytes were incubated with the product of cortical granules obtained from in vitro matured oocytes (vCGP), vitelline envelopes with trypsin treatment were able to block sperm entry. These oocytes exhibited the characteristic signs of activation. These results support the idea that B. arenarum oocytes can be activated by external stimuli and suggest the presence of unknown oocyte surface receptors linked to the activation machinery in response to fertilization. Electrophoretic profiles obtained by SDS-PAGE of solubilized vitelline envelopes from oocytes matured in vitro revealed the conversion of gp40 (in vitro matured oocytes, without trypsin treatment) to gp38 (ascribable to trypsin activity or cortical granule product activity, CGP) and the conversion of gp70 to gp68 (ascribable to trypsin activity plus CGP activity). Taking into account that only the vitelline envelopes of in vitro matured oocytes with trypsin treatment and activated can block sperm entry, we may suggest that the conversion of gp70 to gp68 is related to the changes associated with sperm binding.     

Binding of antibiotics to glycoproteins of the vitelline and fertilization envelopes of cherry salmon eggs

The binding of antibiotics (gentamicin, oleandomycin and chloramphenicol) to vitelline and fertilization envelopes and their extracts was investigated by immunohistochemical and immunocytochemical techniques and immunoblot analysis using mature and artificially activated eggs of the fish Oncorhynchus masou. Binding of antibiotics was detected in the vitelline and fertilization envelope outermost layers, the fertilization envelope inner surface and cortical alveolus exudates, with differences in immunoreactive intensity and deposition. The fertilization envelope outermost layer had the capacity to bind much greater amounts of the antibiotics than the vitelline envelope outermost layer. The greater capacity was caused by the deposition of cortical alveolus exudates, which were known to be responsible for functional roles of protection against bacteria, fungi and noxious materials. Treatment of the vitelline and fertilization envelopes with neuraminidase markedly reduced the binding of gentamicin and chloramphenicol but slightly increased that of oleandomycin; binding of the latter to the vitelline and fertilization envelope outermost layers was considerably reduced after treatment with alpha-fucosidase. Treatment of the two envelopes with alpha-mannosidase, beta-galactosidase or beta-SdD-glucosaminidase did not cause any alteration in immunoreactive intensity or number of immunoreactive deposits. Immunoblot analysis of the vitelline or fertilization envelope extracts indicated that many of the antibiotic-binding substances were glycoproteins, and several major bands were bound by all three antibiotics. These results suggest that the vitelline or fertilization envelopes may have the ability to protect the egg itself, or the embryo, respectively, by trapping antibiotics, and the trapping may be related to the presence of carbohydrate moieties, such as sialyl or fucosyl residues. This revised version was published online in November 2006 with corrections to the Cover Date.     

Binding of bacterial toxins to glycoproteins in the envelopes of rainbow trout eggs

Summary The ability of the vitelline and fertilization envelopes of rainbow trout eggs to trap toxins was investigated using cholera enterotoxin B and staphylococcal enterotoxin B in cytochemical or immunocytochemical experiments. Extracts from both envelopes were investigated by immunoblot analysis to identify toxin-binding proteins after SDS-PAGE. Binding studies of cholera enterotoxin B to vitelline envelopes and fertilization envelopes revealed a greater reactive intensity in the former. Treatment with neuraminidase enhanced the reactive intensity (or deposit) in the vitelline envelope and fertilization envelope outermost layers, with more conspicuous reactivity in the former. Cytochemical experiments showed that exogenous ganglioside GM₁ considerably enhanced cholera enterotoxin B binding to vitelline and fertilization envelopes. This enhancement was shown by an intense reactivity following the occurrence of new binding sites on the vitelline envelope inner surface and the inner wall of the zona radiata, a simultaneous extreme reduction in the reactivity of the vitelline envelope outermost layer, and a striking increase in reactive products in the fertilization envelope outermost layer. The surface region of the vitelline or fertilization envelope outermost layer was the binding site for staphylococcal enterotoxin B, and neuraminidase treatment caused a considerable reduction of reactive products in these areas. Immunoblot analysis of cholera enterotoxin Bor staphylococcal enterotoxin B-binding substances in extracts from the vitelline envelopes or fertilization envelopes demonstrated that the great majority of the binding substances are glycoproteins. The present results suggest that glycoproteins constituting the vitelline envelope or fertilization envelope may contribute to the protection of the egg itself or the embryo by trapping noxious toxins.     

Two major acrosomal proteins act on different parts of the oocyte vitelline coat in the abalone, Haliotis discus

Two proteins of molecular weights 20,000 (20K) and 15,500 (15.5K) are the major soluble substances released from the acrosomal vesicle of the abalone, Haliotis discus, spermatozoon. A crude preparation of them has been shown to possess lytic activity on the oocyte vitelline coat (VC). To elucidate the role(s) of each acrosomal protein (AP) in VC lysis, oocytes were examined after treatment with various AP preparations. The VC, which is about 1 micron thick, is composed of thin outer and inner electron-dense layers and a thick main layer of a fine filamentous feltwork. When oocytes were treated with a crude preparation containing both APs, the outer layer disappeared and the feltwork of the main layer loosened extensively. A preparation containing predominantly the 20K AP dissolved the outer layer completely and the main layer to some extent, whereas another preparation containing predominantly the 15.5K AP caused loosening of the main layer without alteration of the outer layer, suggesting that the 20K AP acts on the outer layer, whereas the 15.5K AP acts on the main layer. However, when purified, each AP by itself failed to dissolve the VC, although lysis occurred in a 1:1 mixture of these preparations. Moreover, when the oocytes were pretreated with the 20K AP and thoroughly washed, the 15.5K AP alone could induce lysis. These results suggest that the lysis of the outer layer requires both APs but not simultaneously. The 15.5K AP, which is located posteriorly in the acrosomal vesicle, must be released to act on the VC following the action of the 20K AP.     

Involvement of sperm proteases in the binding of sperm to the vitelline envelope in Xenopus laevis

Sperm binding to the vitelline envelope in dejellied Xenopus laevis eggs was effectively inhibited by inhibitors for trypsin (soybean trypsin inhibitor and p-toluenesulfonyl-L-lysine chloroethyl ketone) and aminopeptidase B (o-phenanthroline, bestatin, and arphamenine B). Likewise, synthetic 4-methylcoumaryl-7-amide (MCA) substrates (t-butoxycarbonyl-GlyArgArg-MCA, benzyloxycarbonyl-ArgArg-MCA, and Arg-MCA) inhibited binding. Consistently, when jellied eggs were inseminated in the presence of these substrates or inhibitors for proteases, fertilization was effectively blocked. The medium in which live sperm or the sperm membrane fraction were suspended exhibited hydrolyzing activities against the synthetic substrates mentioned above, and these activities were effectively inhibited by the protease inhibitors. Ultracentrifugal fractionation of the sperm suspension following induction of the acrosome reaction by a calcium ionophore, A23187, indicated that a considerable amount of the total tryptic and aminopeptidase B activity was released into the medium. On this occasion, part of the tryptic and aminopeptidase B activity was definitely estimated to be discharged in association with a vesiculated membrane, supporting the notion that the proteases involved in binding to the vitelline envelope are present on the sperm plasma membrane.     

An ultrastructural analysis of the gametes and early fertilization in two bivalve molluscs,Chama macerophylla and Spisula solidissima with special reference to gamete binding

Summary An ultrastructural investigation of the gametes and their interaction during the early events of fertilization in molluscs has been performed. A gamete binding event involving large numbers of sperm has been identified and examined in detail. The surface of the oocyte is projected into numerous microvilli which extend through the vitelline envelope. Tufts of fibrillar material radiate from the tips of these microvilli, forming a layer external to the vitelline envelope. The acrosomal vesicle of the mature spermatozoon contains two major components, which function differently during fertilization. The vesicle is indented at its adnuclear surface, constituting a preformed acrosomal tubule. This tubule does not elongate during the acrosome reaction. Completion of the reaction results in the formation of an extracellular coat, derived from one component of the acrosomal vesicle, on the anterior surface of the sperm. Sperm-egg binding is accomplished by an association of the extracellular coat on the reacted sperm and the fibrous tufts on the tips of the microvilli of the oocyte. Evidence that gamete membrane fusion occurs by fusion of the acrosomal tubule and a microvillus is presented. These observations provide a generalized pattern of molluscan fertilization.The assistance of Mr. B. Calloway in identifying and obtaining the organisms is gratefully acknowledged. This investigation was supported by NSF grants PCM 76-13459 and PCM 76-09654 and performed at the Bermuda Biological Station with instruments made available through the courtesy of Philips, Inc., DuPont-Sorvall, and L.K.B. Inc. Bermuda Biological Station Contribution No. 709     

Sperm binding and fertilization envelope formation in a cell surface complex isolated from sea urchin eggs

An isolated surface complex consisting of the vitelline layer, plasma membrane, and attached secretory vesicles has been examined for its ability to bind sperm and to form the fertilization envelope. Isolated surface complexes (or intact eggs) fixed in glutaraldehyde and then washed in artificial sea water are capable of binding sperm in a species-specific manner. Sperm which bind to the isolated surface complex exhibit the acrosomal process only when they are associated with the exterior surface (vitelline layer) of the complex. Upon resuspension of the unfixed surface complex in artificial sea water, a limiting envelope is formed which, based on examination of thin sections and negatively stained surface preparations, is structurally similar to the fertilization envelope formed by the fertilized egg. These results suggest that the isolated egg surface complex retains the sperm receptor, as well as integrated functions for the secretion of components involved in assembly of the fertilization envelope.     

Ascidian sperm lysin system

Fertilization is a precisely controlled process involving many gamete molecules in sperm binding to and penetration through the extracellular matrix of the egg. After sperm bind to the extracellular matrix (vitelline coat), they undergo the acrosome reaction which exposes and partially releases a lytic agent called "lysin" to digest the vitelline coat for the sperm penetration. The vitelline coat sperm lysin is generally a protease in deuterostomes. The molecular mechanism of the actual degradation of the vitelline coat, however, remains poorly understood. In order to understand the lysin system, we have been studying the fertilization mechanism in ascidians (Urochordata) because we can obtain large quantities of gametes which are readily fertilized in the laboratory. Whereas ascidians are hermaphrodites, which release sperm and eggs simultaneously, many ascidians, including Halocynthia roretzi, are strictly self-sterile. Therefore, after sperm recognize the vitelline coat as nonself, the sperm lysin system is thought to be activated. We revealed that two sperm trypsin-like proteases, acrosin and spermosin, the latter of which is a novel sperm protease with thrombin-like substrate specificity, are essential for fertilization in H. roretzi. These molecules contain motifs involved in binding to the vitelline coat. We found that the proteasome rather than trypsin-like proteases has a direct lytic activity toward the vitelline coat. The target for the ascidian lysin was found to be a 70-kDa vitelline coat component called HrVC70, which is made up of 12 EGF-like repeats. In addition to the proteasome system, the ubiquitination system toward the HrVC70 was found to be necessary for ascidian fertilization. In this review, I describe recent progress on the structures and roles in fertilization of the two trypsin-like proteases, acrosin and spermosin, and also on the novel extracellular ubiquitin-proteasome system, which plays an essential role in the degradation of the ascidian vitelline coat.     

Proteasomal interference prevents zona pellucida penetration and fertilization in mammals

The ubiquitin-proteasome pathway has been implicated in the penetration of ascidian vitelline envelope by the fertilizing spermatozoon (Sawada et al., Proc Natl Acad Sci U S A 2002; 99:1223-1228). The present study provides experimental evidence demonstrating proteasome involvement in the penetration of mammalian zona pellucida (ZP). Using porcine in vitro fertilization as a model, penetration of ZP was completely inhibited by specific proteasomal inhibitors MG-132 and lactacystin. Three commercial rabbit sera recognizing 20S proteasomal core subunits beta-1i, beta-2i, alpha-6, and beta-5 completely blocked fertilization at a very low concentration (i.e., diluted 1/2000 to 1/8000 in fertilization medium). Neither proteasome inhibitors nor antibodies had any effects on sperm-ZP binding and acrosome exocytosis in zona-enclosed oocytes or on fertilization rates in zona-free oocytes, which were highly polyspermic. Consistent with a possible role of ubiquitin-proteasome pathway in ZP penetration, ubiquitin and various alpha and beta type proteasomal subunits were detected in boar sperm acrosome by specific antibodies, immunoprecipitated and microsequenced by MALDI-TOF from boar sperm extracts. Antiubiquitin-immunoreactive substrates were detected on the outer face of ZP by epifluorescence microscopy. This study therefore provides strong evidence implicating the ubiquitin-proteasome pathway in mammalian fertilization and zona penetration. This finding opens a new line of acrosome/ZP research because further studies of the sperm acrosomal proteasome can provide new tools for the management of polyspermia during in vitro fertilization and identify new targets for contraceptive development.     

Morphological Modifications of Oocyte Vitelline Envelope from Bufo arenarum during Different Functional States

The submicroscopic morphology of the vitelline envelope of Bufo arenarum's oocyte change significantly during the maturation and fertilization processes. The morphological changes are related to physiological activity in vivo and can be triggered in vitro by experimental procedures. It is our scope to present the ultrastructure differences of fascicular components of the vitelline envelope in coelomic, "pars recta" conditioned, oviductal, oviposited and fertilized oocytes. Our experimental results indicate that artificial "pars recta" treatment of coelomic oocytes arrange the fascicular components as those of oviposited oocyte, although differences still remain indicating that additional maturation processes take place while the egg pass througth the oviduct. Fertilized or activated oocytes which are refractary to sperm penetration, change the vitelline envelope fascicular components orientation giving a submicroscopical image quite different to those of none fertilized oocytes. These ultrastructural changes define in a clear cut manner the functional states of Bufo arenarum's oocyte.     

Purification and immunocytochemical localization of the vitelline coat lysin of abalone spermatozoa

Spermatozoa of the abalone Haliotis discus were treated with high-calcium seawater to induce the acrosome reaction. The soluble components released from the sperm acrosomal vesicles showed potent lytic activity on the egg vitelline coat. A vitelline coat lysin was purified by salting-in, preparative polyacrylamide gel electrophoresis, and high-performance liquid chromatography. Its molecular weight was 15,500 and its isoelectric point 9.6. These properties were similar to those of other molluskan vitelline coat lysins. The lysin was immunocytochemically localized using a protein A-gold technique, in the posterior half of the acrosomal vesicle.     

Egg jelly of the newt, Cynops pyrrhogaster contains a factor essential for sperm binding to the vitelline envelope

The acrosome reaction of newt sperm is induced at the surface of egg jelly and the acrosome-reacted sperm acquire the ability to bind to the vitelline envelope. However, because the substance that induces the acrosome reaction has not been identified, the mechanism by which the acrosome-reacted sperm bind to the vitelline envelope remains unclear. We found here that a Dolichos biforus agglutinin (DBA) specifically mimicked the acrosome reaction immediately upon its addition in the presence of milimolar level Ca(2+). Fluorescein isothiocyanate-labeled DBA bound specifically to the acrosomal cap of the intact sperm in the presence of a Ca(2+)-chelating agent, EDTA, suggesting that binding of DBA to the native receptor for the egg jelly substance on the acrosomal region took the place of the egg jelly substance-induced acrosome reaction. In contrast, the sperm that had been acrosome reacted by DBA treatment did not bind to the vitelline envelope of the egg whose jelly layers were removed. Subsequent addition of jelly extract caused the sperm binding to vitelline envelope, indicating that the egg jelly of the newt contains substances that are involved in not only inducing the acrosome reaction but also binding to the vitelline envelope. This is the first demonstration of the involvement of egg jelly substance in the binding of acrosome-reacted sperm to the vitelline envelope.     

Induction of germinal vesicle breakdown in Xenopus laevis oocytes: response of denuded oocytes to progesterone and insulin

Denuded oocytes freed of their vitelline envelope have been prepared by two methods, enzymatically with pronase and manually by microdissection. The response of denuded oocytes to progesterone, in terms of germinal vesicle breakdown (GVBD), was similar to that obtained with defolliculated oocytes (separated with collagenase from follicle cells, but still keeping their vitelline membrane). The same conclusion was drawn with respect to morphological features of the oocyte surface observed by transmission and scanning electron microscopy, before and after progesterone-induced GVBD. The synergistic effect of insulin and progesterone in denuded oocytes was comparable to that observed in defolliculated oocytes. Multiplication stimulating activity (MSA) had the same effect as insulin. These observations indicate that hormones act directly upon oocytes, without interference of the surrounding vitelline envelope and follicle cells.     

Crystal structure and subunit dynamics of the abalone sperm lysin dimer: egg envelopes dissociate dimers, the monomer is the active species

Lysin is a 16-kD acrosomal protein used by abalone spermatozoa to create a hole in the egg vitelline envelope (VE) by a nonenzymatic mechanism. The crystal structure of the lysin monomer is known at 1.9 A resolution. The surface of the molecule reveals two tracks of basic residues running the length of one surface of the molecule and a patch of solvent-exposed hydrophobic residues on the opposite surface. Here we report that lysin dimerizes via interaction of the hydrophobic patches of monomers. Triton X-100 dissociates the dimer. The crystal structure of the dimer is described at 2.75 A resolution. Fluorescence energy transfer experiments show that the dimer has an approximate KD of 1 microM and that monomers exchange rapidly between dimers. Addition of isolated egg VE dissociates dimers, implicating monomers as the active species in the dissolution reaction. This work represents the first step in the elucidation of the mechanism by which lysin enables abalone spermatozoa to create a hole in the egg envelope during fertilization.     

Cross fertilization between sea urchin eggs and oyster spermatozoa

Interphylum crossing was examined between sea urchin eggs (Temnopleurus hardwicki) and oyster sperm (Crassostrea gigas). The eggs could receive the spermatozoa with or without cortical change. The fertilized eggs that elevated the fertilization envelope began their embryogenesis. Electron microscopy revealed that oyster spermatozoa underwent acrosome reaction on the sea urchin vitelline coat, and their acrosomal membrane fused with the egg plasma membrane after the appearance of an intricate membranous structure in the boundary between the acrosomal process and the egg cytoplasm. Oyster spermatozoa penetrated sometimes into sea urchin eggs without stimulating cortical granule discharge and consequently without fertilization envelope formation. The organelles derived from oyster spermatozoa seemed to be functionally inactive in the eggs whose cortex remained unchanged.     

EFFECT OF TRYPSIN INHIBITORS AND CONCANAVALIN A ON BUFO ARENARUM FERTILIZATION

The presence of some trypsin inhibitors (soybean trypsin inhibitor and lima bean trypsin inhibitor) in the inseminating media, is effective in blocking Bufo arenarum fertilization. Ovomucoid does not significantly affect fertilization, even at concentrations as high as 10 mg/ml. Similarly the presence of Concanavalin A in the inseminating media results in fertility impairment. The possible mechanisms of action of these substances are discussed.     

鲍配子识别蛋白的研究

配子相互作用的生化机制对于进一步阐明生殖过程具有重要作用,它是深入了解细胞内识别的理想体系。精卵细胞相互作用包括一系列的步骤,开始于精子与卵细胞外被的接触,终止于两性细胞的融合及精子核进入卵细胞质中,而精卵细胞的识别具有建立于各自性细胞表面成分基础上的种的特异性,鲍则是研究精卵识别的好材料。鲍精子在发生顶体反应时释放出两种蛋白质——细胞溶素(1ysin)和18ku糖蛋白(spl8),其中的细胞溶素与其卵黄膜上的受体紧密结合,并利用非酶反应在卵黄膜上穿一个小孔,整个精子则从此孔穿过卵黄膜与卵细胞融合;spl8释放后则覆盖到精子细胞膜表面,起到溶解卵细胞脂质体的作用,即spl8介导精、卵细胞膜的融合。鲍卵细胞膜上存在细胞溶素受体,它是大的不分支的糖蛋白分子,占据了卵黄膜30％的组分,可以专一性地与细胞溶素相结合。这些配子识别蛋白共同进化且速度很快,其中细胞溶素和18ku糖蛋白通过正向选择进化,而细胞溶素受体进行协同进化。