Immunocytochemical localization of the 35-kDa sea urchin egg trypsin-like protease and its effects upon the egg surface

Trypsin-like protease in sea urchin eggs is thought to reside in cortical granules since it is secreted at fertilization and has been isolated with cortical granule fractions from unfertilized eggs. A 35-kDa serine protease has been purified from Strongylocentrotus purpuratus eggs by soybean trypsin inhibitor-affinity chromatography. For this report the protease was localized by immunocytochemistry before and after fertilization, and its potential biological activity was examined by application of the isolated enzyme to the unfertilized egg surface. The protease was localized on sections by immunofluorescence and immunoelectron microscopy, and was found to reside in the spiral lamellae of S. purpuratus cortical granules and in the electron-dense stellate core of Arbacia punctulata granules. At fertilization the enzyme is secreted into the perivitelline space and accumulates only very briefly between the hyaline layer and the nascent fertilization envelope. Shortly thereafter the enzyme is lost from the perivitelline space and immunological reactivity is no longer associated with the egg surface. The 35-kDa cortical granule protease has vitelline delaminase activity but does not appear to destroy vitelline envelope sperm receptors as judged by the fertility of protease-treated eggs.     

A novel procedure for obtaining denuded sea urchin eggs and observations on the role of the vitelline layer in sperm reception and egg activation

A procedure is described for the complete removal of the vitelline layer of the eggs of the sea urchin, Strongylocentrotus purpuratus. The method involves treatment of unfertilized eggs with an S. purpuratus cortical granule protease preparation followed by incubation in an alkaline dithiothreitol seawater solution. Eggs denuded of their vitelline layers react metabolically to parthenogenetic agents and sperm like unfertilized eggs, whereas the fertilizability of denuded eggs and receptivity to sperm is much less than controls. The present method is superior to previous methods using mercaptans in that all of the vitelline layer is removed and to procedures using other proteolytic enzymes in that no ¹²⁵I-labelled plasma membrane proteins are extensively modified. Thus the cortical granule protease dithiothreitol procedure is ideal for studies of the plasma membrane of the unfertilized egg and for studies on the role of the vitelline layer in normal fertilization and development.     

Mastoparan induces Ca2+-independent cortical granule exocytosis in sea urchin eggs

In most species, cortical granule exocytosis is characteristic of egg activation by sperm. It is a Ca(2+)-mediated event which results in elevation of the vitelline coat to block permanently the polyspermy at fertilization. We examined the effect of mastoparan, an activator of G-proteins, on the sea urchin egg activation. Mastoparan was able to induce, in a concentration-dependent manner, the egg cortical granule exocytosis; mastoparan-17, an inactive analogue of mastoparan, had no effect. Mastoparan, but not sperm, induced cortical granule exocytosis in eggs preloaded with BAPTA, a Ca(2+) chelator. In isolated egg cortical lawns, which are vitelline layers and membrane fragments with endogenously docked cortical granules, mastoparan induced cortical granule fusion in a Ca(2+)-independent manner. By contrast, mastoparan-17 did not trigger fusion. We conclude that in sea urchin eggs mastoparan stimulates exocytosis at a Ca(2+)-independent late site of the signaling pathway that culminates in cortical granule discharge.     

Partial purification and characterization of sperm receptor hydrolase,a cortical granule proteoesterase,from eggs of the sea urchin Strongylocentrotus purpuratus

Sperm receptor hydrolase, one of two classes of cortical granule proteoesterases (E.C.3.4.4.4) was purified approximately 30-fold with 80% yield from Strongylocentrotus purpuratus cortical granule exudate. Sperm receptor hydrolase preparations were free of vitelline delaminase activity (the other class of cortical granule proteoesterase) and had less than 1% of the starting levels of cortical granule peroxidase and β-1,3-glucanohydro-lase activities. Native polyacrylamide gel electrophoresis coupled with a protease activity stain showed that three proteases were present in the most highly purified preparations of sperm receptor hydrolase. Each of the three proteases has the same molecular weight of 60,000, but different isoelectric points of 2.4, 3.8, and 5.5. The K_m value of the mixture of proteases for α-N-benzoyl-L-arginine ethyl ester as substrate was 263 μM at pH 8.4 and 30°C; the pH dependence of V_m showed a single prototrophic group with a pK of 6.7 and an enthalpy of ionization of 8.6 kcal-mol^?1. The values of these kinetic parameters are consistent with an enzyme-active site containing histidine. Phenylmethyl sulfonyl fluoride, tosyl lysine chloromethyl ketone, several proteinaceous trypsin inhibitors, and p-aminobenzamidine inhibited the esterase activity of the proteases. These data suggest that sperm receptor hydrolases are serine proteases.     

Polyspermic fertilization of sea urchin eggs treated with protease inhibitors: Localization of sperm receptor sites at the egg surface

The normal elevation of the fertilization membrane and the establishment of the block to polyspermy are retarded in Arbacia punctulata eggs by specific protease inhibitors, soybean trypsin inhibitor (SBTI), leupeptin, and antipain. Ultrastructural observations show that the vitelline layer remains attached to the plasma membrane of fertilized SBTI treated eggs at numerous sites (cortical projections). Quantitive morphometric analysis indicates that the vitelline layer elevates from about 65% of the surface of SBTI treated eggs during the first 3 min post insemination. However, the vulnerability of SBTI treated eggs to refertilization (polyspermy) only declined during the subsequent gradual detachment of the vitelline layer from the cortical projections over the next 15 min. Antipain and leupeptin (10^?5 to 10^?3M) also promoted polyspermy in Arbacia eggs by a process of refertilization extending for a 10- to 15-min period after the initial monospermic insemination. Normal cleavage and development was obtained when eggs were placed in leupeptin and antipain (10^?3M) after the fertilization membrane had elevated. The data indicate that the normal secretory function (or functions) of the cortical granule protease in establishing the block to polyspermy is retarded by these protease inhibitors, and that the vitelline layer is transformed into a mechanical barrier to prevent penetration by supernumerary sperm during its detachment from the plasma membrane of the egg. Furthermore, the vitelline layer in unfertilized eggs appears to be a mosaic structure, with sperm receptor sites localized in regions of the egg's surface, which give rise to cortical projections in the presence of SBTI.     

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.     

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.     

An ultrastructural study of cross-fertilization (Arbacia female x Mytilus male)

Insemination of sea urchin (Arbacia) ova with mussel (Mytilus) sperm has been accomplished by treating eggs with trypsin and suspending the gametes in seawater made alkaline with NaOH. Not all inseminated eggs undergo a cortical granule reaction. Some eggs either elevate what remains of their vitelline layer or demonstrate no cortical modification whatsoever. After its incorporation into the egg, the nucleus of Mytilus sperm undergoes changes which eventually give rise to the formation of a male pronucleus. Concomitant with these transformations, a sperm aster may develop in association with the centrioles brought into the egg with the spermatozoon. Both the male pronucleus and the sperm aster may then migrate centrad to the female pronucleus. Evidence is presented which suggests that fusion of the male pronuclei from Mytilus sperm with female pronuclei from Arbacia eggs may occur, although this was not directly observed. These results demonstrate that Mytilus sperm nuclei are able to react to conditions within Arbacia eggs and differentiate into male pronuclei.     

Sea urchin egg-cortical granule protease has arginyl proteolytic specificity

Sea urchin eggs secrete esteroproteolytic activity at fertilization. This enzyme has been shown to be proteolytic toward embryo protein and casein, but a systematic study of its substrate specificity has not been done. In this communication we present data that demonstrates for the first time that the cortical granule protease from Strongylocentrotus purpuratus eggs cleaves arginyl residues in a protein substrate, lysozyme. We have developed a sensitive reverse-phase high-performance liquid chromatography (RP-HPLC) assay that detects femtomole levels of trypsin and chymotrypsin protease activity [Green, 1986: Anal Biochem 152:83–88]. In the sea urchin system, we have detected protease activity from as few as 50 eggs. Correlating the RP-HPLC analysis with a spectrophotometric Nα-benzoyl-L-arginine ethyl ester assay, we have found that each egg secretes approximately 40 attomoles of trypsin-like activity. This general method should be quite useful in investigations into the natural substrate of the egg protease.     

Purification and characterization of trypsin-like enzyme from sea urchin eggs: substrate specificity and physiological role

A trypsin-like enzyme has been purified to homogeneity from eggs of the sea urchin, Strongylocentrotus intermedius. The purified enzyme efficiently hydrolyzed Z-Phe-Arg-4- methylcoumaryl -7-amide (MCA) and Pro-Phe-Arg-MCA among 12 peptidyl-Arg (or Lys)- MCAs . The substrate specificity of the enzyme was closely similar to that of the enzyme activity in the egg cortical granule exudate. Among various peptidyl-argininal (Arg-H) derivatives, Z-Phe-Arg-H and Z-Phe-Leu-Arg-H showed the strongest inhibition against both the activity of the purified enzyme and the elevation of vitelline coat. Thus, the trypsin-like enzyme of sea urchin possesses a narrow substrate specificity and participates at least in the elevation of vitelline coat during fertilization.     

Identification of a new sea urchin vitelline envelope sperm binding glycoprotein

The sea urchin egg vitelline envelope (VE) is composed of eight major glycopolypeptides that are heavily mannosylated and contain fucose and N-acetylglucosamine moieties based on lectin staining. In the present study, the macromolecular composition of the VE and the potential role of a purified VE glycoprotein in initial gamete binding was investigated. The VE components were solubilized from the surface of intact, dejellied eggs with dithiothreitol in divalent cation-free seawater, and analyzed using native, reduced electrophoresis and immunoblotting. Three major VE glycoproteins, VE-A, VE-B and VE-C, and one minor component, VE-D, were identified with antisera against whole VE preparations and against glutaraldehyde-fixed, unfertilized eggs. The electrophoretically purified glycoproteins resolved into a common subunit doublet and one unique subunit each of decreasing size on blots of sodium dodecylsulfate polyacrylamide gels. Lectin affinity chromatography was used for analysis and purification of reduced VE components; a glycoprotein eluted from Con A columns with methyl-mannoside comigrated with VE-B when analyzed by immunoblotting. Whole VE preparations and VE-B obtained from Con A columns were found to inhibit fertilization when preincubated with sperm, thus directly establishing a role for VE-B in gamete binding.     

Cortical Granules of Sea Urchin Eggs do not Undergo Exocytosis at the Site of Sperm-egg Fusion*

Microscopic observations of sea urchin egg fertilization (phase contrast, Nomarski and transmission electron microscope) reveal that the cortical granules in the area of sperm egg-fusion do not undergo exocytosis. These intact granules remain associated with the sperm, moving into the egg cytoplasm with the entering sperm. This sperm-cortical granule association occurs before the sperm centriole affects microtubule organization and the sperm-cortical granule association is not affected by cytochalasin D or griseofulvin. We discuss the possibility that a reorganization of the egg cytoplasm ensues from the sperm-egg interaction at the site of sperm-egg fusion. Other possibilities are that the retention of cortical granules is not related to egg reorganization, but is necessary for successful sperm incorporation or reflects an unrelated component of the activation process.     

Effects of protease inhibitors on sperm-related events in sea urchin fertilization

Evidence for sperm-borne proteolytic enzymes exposed during the acrosome reaction in sea urchin sperm has been accumulating. To investigate the possible role(s) such enzymes have in fertilization, we studied the effects of several protease inhibitors on sperm-related events. Soybean trypsin inhibitor, Nα-p-tosyl-l-lysine, chloromethyl ketone, phenylmethylsulfonyl fluoride, and chymostatin neither reduced the number of acrosome reactions nor interfered with gamete binding. p-Nitrophenyl-p′-guanidinobenzoate caused sperm to fuse into irregular clumps, rendering them unable to fertilize eggs. However, l-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK), an inhibitor of chymotrypsin, prevented the acrosome reaction in Strongylocentrotus purpuratus, S. droebachiensis, and Lytechinus pictus. The effects of TPCK on sperm in subsequent steps of fertilization were also investigated. First, gamete binding assays were performed on fixed eggs. This precluded any effects TPCK might have had on egg-derived secretions (e.g., proteases). Binding of prereacted sperm occurred with both fixed and living eggs. However, fertilization of living eggs in the presence of TPCK was greatly reduced, even though sperm had been prereacted with egg jelly. Vitelline coats were then removed from eggs by trypsin treatment. Eggs in TPCK fertilized and developed normally after the above treatment. These observations are consistent with the hypothesis of a sperm protease participating in the acrosome reaction and the penetration of the egg vitelline coat in the sea urchin.     

Changes in the topography of the sea urchin egg after fertilization

Changes in the topography of the sea urchin egg after fertilization were studied by scanning and transmission electron microscopy. Strongylocentrotus purpuratus eggs were treated with dithiothreitol to modify the vitelline layer and to prevent formation of a fertilization membrane. Dithiothreitol treatment caused the microvilli to become more irregular in shape, length, and diameter than those of untreated eggs. The microvilli were similarly modified by trypsin treatment. This effect did not appear to be due to disruption of cytoskeletal elements beneath the plasma membrane, for neither colchicine nor cytochalasin B altered microvillar morphology. Thus, it appears that the vitelline layer may act in the maintenance of surface form of unfertilized eggs. Since dithiothreitol-treated eggs did not elevate a fertilization membrane, scanning electron microscopy could be used to directly observe modifications in the egg plasma membrane after fertilization. The wave of cortical granule exocytosis initiated at the point of attachment of the fertilizing sperm was characterized by the appearance of pits that subsequently opened, releasing the cortical granule contents and leaving depressions upon the egg surface. The perigranular membranes inserted during exocytosis were seen as smooth patches between the microvillous patches remaining from the original egg surface. This produced a mosaic surface with more than double the amount of membrane of unfertilized eggs. The mosaic surface subsequently reorganized to accommodate the inserted membrane material by elongation of microvilli. Blebs and membranous whorls present before reorganization suggested the existence of an unstable intermediate state of plasma membrane reorganization. Exocytosis and mosaic membrane formation were not blocked by colchicine or cytochalasin B, but microvillar elongation was blocked by cytochalasin B treatment.     

Novel procedures for collection of sea urchin egg cortical granule exudate: Partial characterization and evidence for postsecretion processing

We have developed two procedures to collect total cortical granule exudate in a soluble form from eggs of the sea urchin Strongylocentrotus purpuratus. Egg suspensions were either treated with dithiothreitol to disrupt the vitelline envelope or divalent cations were removed postinsemination to prevent the normal vitelline-to-fertilization envelope transition. Rapid acidification of the insemination mixture (dithiothreitol-treated eggs) to pH 6.0 prevented precipitation of the paracrystalline protein fraction described by Bryan [1970a]. Exudate was partitioned into three fractions. The pH 8.0-insoluble fraction appeared to be identical to the paracrystalline protein fraction. The pH 8.0-soluble fraction was separated into pH 4.0-soluble and-insoluble fractions. Analysis for peroxidase and protease activities showed that peroxidase activity was localized in all three fractions whereas protease activity was restricted to the pH 4.0 insoluble fraction as reported [Carroll and Epel, 1975]. A minimum of six major proteins were detected on native polyacrylamide gels of total exudate. Under reducing and denaturing conditions, 12 polypeptides ranging from 19,000 to 165,000 in molecular weight were detected in total exudate; six polypeptides were recovered in the pH 8.0-insoluble fraction. To test the hypothesis that protease and peroxidase activities process cortical granule proteins after secretion, we inseminated eggs in solutions containing peroxidase and protease inhibitors. The paracrystalline protein fraction crystallized slowly from insemination mixtures containing both inhibitors compared to controls and there were dramatic differences in exudate electrophoretic patterns. We suggest that cortical granule protease and peroxidase activities process the exudate so that the paracrystalline protein fraction rapidly crystallizes during normal fertilization.     

Monoclonal antibodies to the sea urchin egg vitelline layer inhibit fertilization by blocking sperm adhesion

Thirty-one mouse hybridomas were produced against the vitelline layer (VL) of the egg of the sea urchin S. purpuratus. Ascites fluids of eight of the 31 bound to the VL surface in the high ionic strength conditions of sea water. Binding was specific to the VL, since immunofluorescence showed that the antibodies elevated from the egg surface with the fertilization envelope after activation with ionophore A23187. Antibody binding was strictly species-specific, the eggs of L. pictus showing no reaction. An immunoperoxidase surface-binding assay showed a wide range in the amount of each monoclonal antibody binding to the VL surface at saturation. All eight monoclonals inhibit fertilization by inhibiting the binding of sperm to the VL. None of the eight ascites fluids reacted with egg jelly. The inhibition of fertilization correlates positively with amount of antibody binding the egg surface. In contrast to the effects of polyclonal rabbit antisera raised against whole eggs or egg cortices, these eight monoclonal antibodies to the VL do not induce the wrinkling of the egg, the cortical granule reaction, the centering of pronuclei, or any other visual indication of metabolic activation.     

The Program of and Mechanisms of Fertilization in the Echinoderm Egg

Current research on the mechanisms of sperm-egg fusion, theblock to polyspermy, and metabolic activation are described.A cinemicrographic analysis of fertilization reveals that fusionof sperm and egg occurs between non-motile gametes, indicatingthat the flagellar motion of sperm is not required. The blockto polyspermy is reviewed, emphasizing recent work on the roleof cortical granule protease in altering sperm receptors ofthe vitelline layer. Metabolic activation or derepression at fertilization is highlyregulated and occurs in a definite sequence. The primary eventappears to be release of intracellular Ca²⁺. The timing of metabolicderepression is different in starfish oocytes. Here, a partof the derepression occurs during maturation and another partat fertilization.     

Ovostatin,an endogenous trypsin inhibitor of sea urchin eggs: Purification and characterization of ovostatin from eggs of the sea urchin,Strongylocentrotus intermedius

A trypsin inhibitor, termed ovostatin, has been purified approximately 265-fold with 82% yield, from unfertilized eggs of the sea urchin Strongylocentrotus intermedius, using trypsin coupled Sepharose 4B as an affinity column for chromatography. The isolated ovostatin is homogeneous in sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the estimated molecular weight being 20K–21.5K. Ovostatin inhibits preferentially trypsin-like endogenous protease purified from the eggs of the same species and bovine pancreatic trypsin and also bovine pancreatic chymotrypsin. Values of IC₅₀ (amount causing 50% inhibition of enzymes) for trypsin-like protease purified from eggs of the same species, bovine pancreatic trypsin, and bovine pancreatic chymotrypsin, are 0.91 ± 0.13 μg/ml (4.55 ± 0.65 × 10^?8 M), 3.0 ± 0.28 μg/ml (1.5 ± 0.14 × 10^?7 M), and 4.8 ± 0.2 μg/ml (2.4 ± 0.1 × 10^?7 M), respectively, in the experimental condition used. Kinetic studies indicate that ovostatin is a noncompetitive inhibitor of trypsin. The inhibitor is relatively heat labile. NaCl (0.025–0.01 M) enhances the inhibitor activity, whereas KCl is inhibitory. Ovostatin requires a low concentration of Ca²⁺ for activity. The activity is higher in unfertilized eggs than in fertilized eggs; total activity and specific activity in unfertilized eggs is about 1.67-fold and 1.85-fold higher than those in fertilized eggs, respectively. We believe that ovostatin may regulate the function of the cortical granule protease and other trypsin-like proteases that are activated in sea urchin eggs during fertilization.     

Evidence for the existence of two assembly domains within the sea urchin fertilization envelope.

The sea urchin fertilization envelope (FE) is a complex, macromolecular aggregate assembled by the addition of cortical granule secretions to the vitelline layer. The completed, trilaminar structure has a dense layer sandwiched between surface coats of paracrystalline material. Two cortical granule enzymes, ovoperoxidase and protease, and a cell surface transglutaminase are required for the assembly process. We have examined, by quick-freeze, deep-etch, rotary-shadow electron microscopy, the effects of inhibiting each of these enzymes upon FE assembly. These experiments reveal two domains within the FE, distinguishable by their enzymatic requirements for proper maturation. The first domain consists of the microvillar casts which require both protease and transglutaminase activities to obtain a normal paracrystalline coat. The second domain comprises the regions between casts and appears to mature by ovoperoxidase-mediated cross-linking of paracrystalline material to the envelope.     

Bioelectric responses of sea urchin eggs inseminated with oyster spermatozoa: a sperm evoked potential without egg activation

Multiple oyster spermatozoa can enter sea urchin eggs with or often without fertilization membrane formation (Osanai and Kyozuka, 1982). In the present work, electrical responses of sea urchin (Temnopleurus hardwicki) eggs inseminated with oyster (Crassostrea gigas) sperm were examined and correlated to the failure of monospermy and egg activation. With diluted sperm, a transient depolarization of the membrane with a constant pattern appeared repeatedly and discretely, and the depolarizations (sperm evoked potentials, SEPs) were not associated with fertilization membrane elevation. With dense sperm, the SEPs occurred consecutively, and sometimes an assembled consecutive depolarization was followed by an activation potential associated with cortical granule discharge. When the membrane potential was artificially held at positive levels, the frequency of SEPs was strongly suppressed but not completely blocked. The present results indicate that an individual heterologous spermatozoon neither produces a depolarization sufficient to block additional sperm entry, nor stimulates egg activation, and that simultaneous entries of multiple heterologous spermatozoa, as possibly reflected by the assembled consecutive depolarizations, induce cortical granule discharge and egg activation.