Evidence for the involvement of metalloendoproteases in the acrosome reaction in sea urchin sperm

An essential initial step in fertilization in the sea urchin Strongylocentrotus purpuratus is an intracellular membrane fusion event in the sperm known as the acrosome reaction. This Ca2+-dependent, exocytotic process involves fusion of the membrane of the acrosomal vesicle and the plasma membrane. Recently, metalloendoproteases requiring divalent metals have been implicated in several Ca2+-dependent membrane fusion events in other biological systems. In view of the suggested involvement of Zn2+ in the sea urchin sperm acrosome reaction (Clapper, D.L., Davis, J.A., Lamothe, P.J., Patton, C., and Epel, D. (1985) J. Cell Biol. 100, 1817-1824) and the fact that Zn2+ is a metal cofactor for metalloendoproteases, we investigated the potential role of this protease in the acrosome reaction. A soluble metalloendoprotease was demonstrated and characterized in sperm homogenates using the fluorogenic protease substrate succinyl-alanine-alanine-phenylalanine-4-aminomethylcoumarin. The protease was inhibited by the metal chelators EDTA and 1,10-phenanthroline, and activity of the inactive apoenzyme could be reconstituted with Zn2+. The metalloendoprotease substrate and inhibitors blocked the acrosome reaction induced either by egg jelly coat or by ionophore, but had no effect on the influx of Ca2+. These observations suggest that inhibition occurs at a step independent of Ca2+ entry. Overall, the results of this study provide strong indirect evidence that the acrosome reaction requires the action of metalloendoprotease.     

Evidence that metalloendoproteases are involved in gamete fusion of Ciona intestinalis, ascidia.

The use of specific inhibitors and substrates of metalloendoproteases provides evidence that in many systems these enzymes are involved in membrane fusion events. In this study, we investigated whether metalloendoproteases are involved in Ciona sperm-egg fusion. In vitro fertilization assays with the metal chelator 1,10-phenanthroline, specific metalloendoprotease substrates, and the vital stain Hoechst 33342 suggested that a Zn(2+)-dependent metalloendoprotease(s) takes part in Ciona sperm-egg fusion. Furthermore, electrophysiological recordings showed that insemination carried out in the presence of either 1,10-phenanthroline or the substrate CBZ-Gly-Phe-NH2 fails to induce fertilization potential or any other change in membrane potential. These results support the hypothesis that in Ciona intestinalis, a metalloendoprotease(s) is functional in gamete fusion.     

Inhibitors of metalloendoproteases block spiculogenesis in sea urchin primary mesenchyme cells

Metalloendoproteases have been implicated in a variety of fusion processes including plasma membrane fusion and exocytosis. As a prerequisite to skeleton formation in the sea urchin embryo, primary mesenchyme cells undergo fusion via filopodia to form syncytia. The spicule is formed within the syncytial cable by matrix and mineral deposition. To investigate the potential involvement of a metalloendoprotease in spiculogenesis, the effect of inhibitors of this enzyme on skeleton formation was studied. Experiments with primary mesenchyme cells in vitro and in normal embryos revealed that skeleton formation was blocked by these inhibitors. These findings implicate a metalloendoprotease in spiculogenesis; such an enzyme has been demonstrated in homogenates of primary mesenchyme cells. The most likely site of action of the metalloendoprotease is at the cell membrane fusion stage and/or at subsequent events requiring membrane fusion.     

Evidence for the involvement of muscle tropomyosin in the contractile elements of the coelom-esophagus complex in sea urchin embryos

The sea urchin morphogenesis, especially formation of the coelom-esophagus complex, was observed correlating the distribution of tropomyosin-specific immunofluorescence. Coelomic cells arranged at both sides of the esophagus extended their pseudopods toward the esophagus to form the contractile bands, which surrounded the esophagus and brought about the contraction of the esophagus. The earliest stage at which the tropomyosin-specific immunofluorescence was recognized coincided with the appearance of the coelomic pseudopods. The tropomyosin-specific immunofluorescence located at the contractile bands and the cell bodies from which they derived, when the ectoderm-disrupted embryos were used to investigate the detailed distribution of tropomyosin. The tropomyosin-specific immunofluorescence remained in the same regions when the embryos were stained with the antiserum absorbed with egg tropomyosin, which detected only muscle tropomyosin. From these observations, the coelomic pseudopod-forming cells were conclusively shown to be muscle cells.     

Role of a vitelline layer-associated 350 kDa glycoprotein in controlling species-specific gamete interaction in the sea urchin

The process of sperm-egg binding is one of the barriers to cross-fertilization between related sea urchin species. A 350 kDa glycoprotein in the egg vitelline layer of Strongylocentrotus purpuratus has been shown to be a sperm-binding protein (SBP). Sulfated O-linked oligosaccharide chains on the 350 kDa glycoprotein, as well as domains of the polypeptide chain, serve as ligands for this binding process. The hypothesis that species-specific sperm-egg binding is attributed to the interaction between the sperm and the 350 kDa glycoprotein was tested using S. purpuratus and S. franciscanus. It was found that both species had a 350 kDa glycoprotein on the egg surface that cross-reacted immunologically using antibodies prepared against a recombinant form of the SBP. Because earlier studies had implicated the carbohydrate chains of the 350 kDa glycoprotein of S purpuratus in sperm binding, differences in carbohydrate chains on the 350 kDa glycoproteins of these species were examined. It was found that among the lectins tested only wheat germ agglutinin and Sambucus nigra agglutinin showed a significant difference in reactivity to the 350 kDa glycoproteins between species. Finally, using a bead-binding assay, it was shown that the isolated 350 kDa glycoproteins exhibited species-specific sperm-binding activity.     

Evidence for a secretory pathway Ca2+-ATPase in sea urchin spermatozoa

Plasma membrane, sarco-endoplasmic reticulum and secretory pathway Ca2+-ATPases (designated PMCA, SERCA and SPCA) regulate intracellular Ca2+ in animal cells. The presence of PMCA, and the absence of SERCA, in sea urchin sperm is known. By using inhibitors of Ca2+-ATPases, we now show the presence of SPCA and Ca2+ store in sea urchin sperm, which refills by SPCA-type pumps. Immunofluorescence shows SPCA localizes to the mitochondrion. Ca2+ measurements reveal that approximately 75% of Ca2+ extrusion is by Ca2+ ATPases and 25% by Na+ dependent Ca2+ exchanger/s. Bisphenol, a Ca2+ ATPase inhibitor, completely blocks the acrosome reaction, indicating the importance of Ca2+-ATPases in fertilization.     

Correct cell-type-specific expression of a fusion gene injected into sea urchin eggs

A fusion gene construct containing the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of CyIIIa actin gene regulatory sequences was injected into unfertilized eggs of the urchin Strongylocentrotus purpuratus, and early pluteus stage embryos that developed from these eggs were fixed and sectioned for analysis by in situ hybridization. A [3H]RNA antisense probe for CAT mRNA was hybridized to 5-micron embryo sections. Autoradiographic signal denoting the presence of CAT mRNA was detected only over aboral ectoderm cells, in which the CyIIIa gene is normally expressed, and not over any recognizable regions of gut or oral ectoderm included in the same sections.     

Analysis of microtubule polymerization inhibitors in sea urchin egg extracts: Evidence for a protease

Inhibitors of microtubule polymerization have been found in extracts of unfertilized sea urchin eggs using neural tubulin polymerization assays without glycerol. The inhibitory activity is partially destroyed by boiling or by reduction and carboxymethylation and is nondialyzable. When chromatographed on DEAE-cellulose, the inhibitory activity is eluted over a broad NaCl gradient and is in association with several peaks. This partially purified inhibitor is not destroyed by incubation with RNase A. When the partially purified inhibitor is incubated with brain microtubule protein under conditions which support microtubule polymerization, both high molecular weight-microtubule associated proteins and tubulin appear to be digested when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteolytic digestion as well as inhibition of microtubule polymerization depend upon similar concentrations of partially purified inhibitor present in the polymerization reaction. It appears as though at least part of the microtubule polymerization inhibitory activity present in unfertilized sea urchin eggs is due to this protease.     

Evidence of an acrosin-like enzyme in sea urchin sperm.

An enzyme which hydrolyzes benzoyl arginine ethylester has been demonstrated in sperm of the sea urchin Strongylocentrotus purpuratus using a sensitive assay employing tritiated substrate. Eighty percent of the enzyme is exposed but not solubilized by treatment with either 30 mM CaCl₂ or solubilized egg jelly coat. The enzymatic activity is masked in control sperm which have not been treated with these agents. The exposed enzyme is inhibited by diisopropyl phosphofluoridate (DFP), soybean trypsin inhibitor, or phenylmethane sulfonyl fluoride, suggesting that it is a serine protease and the invertebrate counterpart of vertebrate acrosin. Inhibition of the exposed enzyme with DFP prevents subsequent fertilization.     

Evidence of phospholipase A2 in the sea urchin egg: Its possible involvement in the cortical granule reaction

Fertilization of the sea urchin egg involves an exocytotic event known as the cortical granule reaction (CGR). In many cell systems, phospholipase A₂ is implicated in regulation of the secretory event. Indirect evidence suggests that phospholipase A₂ mediates the CGR; however, there has been no direct demonstration of phospholipase A₂ activity in the sea urchin egg. We report here evidence of phospholipase A₂ activity in egg homogenate of the sea urchin Lytechinus pictus. The enzyme was calcium-dependent and had a pH optimum near the intracellular pH of the unfertilized egg. Neither exogenous calmodulin nor trifluoperazine had any apparent effect on enzyme activity. Quinacrine, a phospholipase A₂ inhibitor, blocked the enzyme activity in the egg homogenate. In intact eggs, quinacrine blocked the CGR in a dose-dependent, egg-concentration-dependent manner. The inhibitory effect of quinacrine on the CGR could not be overcome by the phospholipase A₂ activator melittin or by the calcium ionophore A23187. Quinacrine did not inhibit sperm-egg binding or sperm incorporation. These results lend further support to the hypothesis that phospholipase A₂ is involved in the CGR.     

Natural hybridization in the sea urchin genus Pseudoboletia between species without apparent barriers to gamete recognition

Marine species with high dispersal potential often have huge ranges and minimal population structure. Combined with the paucity of geographic barriers in the oceans, this pattern raises the question as to how speciation occurs in the sea. Over the past 20 years, evidence has accumulated that marine speciation is often linked to the evolution of gamete recognition proteins. Rapid evolution of gamete recognition proteins in gastropods, bivalves, and sea urchins is correlated with gamete incompatibility and contributes to the maintenance of species boundaries between sympatric congeners. Here, we present a counterexample to this general pattern. The sea urchins Pseudoboletia indiana and P. maculata have broad ranges that overlap in the Indian and Pacific oceans. Cytochrome oxidase I sequences indicated that these species are distinct, and their 7.3% divergence suggests that they diverged at least 2 mya. Despite this, we suspected hybridization between them based on the presence of morphologically intermediate individuals in sympatric populations at Sydney, Australia. We assessed the opportunity for hybridization between the two species and found that (1) individuals of the two species occur within a meter of each other in nature, (2) they have overlapping annual reproductive cycles, and (3) their gametes cross-fertilize readily in the laboratory and in the field. We genotyped individuals with intermediate morphology and confirmed that many were hybrids. Hybrids were fertile, and some female hybrids had egg sizes intermediate between the two parental species. Consistent with their high level of gamete compatibility, there is minimal divergence between P. indiana and P. maculata in the gamete recognition protein bindin, with a single fixed amino acid difference between the two species. Pseudoboletia thus provides a well-characterized exception to the idea that broadcast spawning marine species living in sympatry develop and maintain species boundaries through the divergence of gamete recognition proteins and the associated evolution of gamete incompatibility.     

In vitro fusion and separation of sea urchin primary mesenchyme cells

Time-lapse videomicroscopy of cultured primary mesenchyme cells from mesenchyme blastulae of the sea urchin Lytechinus pictus demonstrates the dramatic ability of these cells to undergo cell fusion and cell separation. Although this plasticity of cell associations is presumed to play a role in the formation of the syncytial cables that secrete the larval skeleton, the surfaces of these cells must be specialized for fusion and cell separation.     

The involvement of O-linked oligosaccharide chains of the sea urchin egg receptor for sperm in fertilization

Recent investigations on the sea urchin egg receptor for spermhave led to its sequencing and the demonstration that it isa 350 kDa glycoprotein. In the current study, the N- and O-linkedoligosaccharide chains were cleaved from the protein fractionatedon concanavalin A-agarose. The putative O-linked oligosaccharidechains that did not bind to the lectin were further fractionatedby anion-exchange chromatography. Using a competition bioassaythat measured the ability of these oligosaccharide chains toinhibit fertilization, it was found that the N-linked chainswere devoid of inhibitory activity. Rather, the inhibitory activitywas localized to the O-linked chains, with the most highly charged,sulphated chains showing the highest inhibitory activity. Thebioactive oligosaccharides were labelled by reduction and assayedfor binding to sperm. The results of the binding assay, coupledwith the fertilization bioassay, indicate that the oligosaccharidesinhibit fertilization by binding to acrosome-reacted sperm.The bioactive oligosaccharide lacked species specificity infertilization bioassays, unlike the intact receptor and a recombinantaglyco protein containing only the extracellular domain of thereceptor. Since previous work showed that the recombinant proteininhibits fertilization species specifically and binds to acrosome-reactedsperm, a two-step model of sperm-egg interaction is proposed.The first step is postulated to be a low-affinity ionic interactionof the sulphated O-linked oligosaccharide chains of the receptorwith sperm that is not species specific. This is followed bya high affinity, species-specific interaction of the sperm withone or more binding sits on the polypeptide chain of the receptor. fertilization oligosaccharide receptor sea urchin egg sea urchin sperm     

Evidence for a cannabinoid receptor in sea urchin sperm and its role in blockade of the acrosome reaction

Delta-9-tetrahydrocannabinol ((?)δ⁹ THC), the primary psychoactive cannabinoid in marihuana, reduces the fertilizing capacity of sea urchin sperm by blocking the acrosome reaction that normally is stimulated by a specific ligand in the egg's jelly coat. The bicyclic synthetic cannabinoid [ H]CP-55,940 has been used as a ligand to demonstrate the presence of a cannabinoid receptor in mammalian brain. We now report that [ H]CP-55,940 binds to live sea urchin (Strongylocentrotus purpuratus) sperm in a concentration, sperm density, and time-dependent manner. Specific binding of [ H]CP-55,940 to sperm, defined as total binding displaced by (?)δ⁹ THC, was saturable: K_D 5.16 ± 1.02 nM; Hill coefficient 0.98 ± 0.004. This suggests a single class of receptor sites and the absence of significant cooperative interactions. Sea urchin sperm contain 712 ± 122 cannabinoid receptors per cell. Binding of [ H]CP-55,940 to sperm was reduced in a dose-dependent manner by increasing concentrations of CP-55,940, (?)δ⁹ THC, and (+)δ⁹ THC. The rank order of potency to inhibit binding of [ H]CP-55,940 to sperm and to block the egg jelly stimulated acrosome reaction was: CP-55,940 > (?)δ⁹THC > (+)δ⁹THC. These findings show that sea urchin sperm contain a stereospecific cannabinoid receptor that may play a role in inhibition of the acrosome reaction. The radioligand binding data obtained with live sea urchin sperm are remarkably similar to those previously published by other investigators using [ H]CP-55,940 on mammalian brain and nonneural tissues. The cannabinoid binding properties of this receptor appear to have been highly conserved during evolution. We postulate that the cannabinoid receptor may modulate cellular responses to stimulation. © 1993 Wiley-Liss, Inc.     

Multiple GTP-binding proteins in sea urchin sperm: Evidence for Gs and small G-proteins

Sea urchin sperm plasma membranes isolated from heads and flagella were used to examine the presence of Gs (stimulatory guanine nucleotide-binding regulatory protein) and small G-proteins. Flagellar plasma membranes incubated with [³²P]NAD and cholera toxin (CTX) displayed radiolabeling in a protein of 48 kDa, which was reactive by immunoblotting with a specific antibody against mammalian Gs. CTX-catalyzed [³²P]ADP-ribosylation in conjunction with immunoprecipitation with anti-Gs, followed by electrophoresis and autoradiography, revealed one band of 48 kDa. Head plasma membranes, in contrast, did not show substrates for ADP-ribosylation by CTX. In flagellar and head plasma membranes pertussis toxin (PTX) ADP-ribosylated the same protein described previously in membranes from whole sperm; the extent of ADP-ribosylation by PTX was higher in flagellar than in head membranes. Small G-proteins were investigated by [³²P]GTP-blotting. Both head and flagellar plasma membranes showed three radiolabeled bands of 28, 25 and 24 kDa. Unlabeled GTP and GDP, but not other nucleotides, interfered with the [α-³²P]GTP-binding in a concentration-dependent manner. A monoclonal antibody against human Ras p21 recognized a single protein of 21 kDa only in flagellar membranes. Thus, sea urchin sperm contain a membrane protein that shares characteristics with mammalian Gs and four small G-proteins, including Ras . Gs, Gi and Ras are enriched in flagellar membranes while the other small G-proteins do not display a preferential distribution along the sea urchin sperm plasma membrane. The role of these G-proteins in sea urchin sperm is presently under investigation.