Purification and characterization of a vitelline coat lysin from Ciona intestinalis spermatozoa.

In Ciona intestinalis a chymotrypsin-like activity is involved in sperm penetration of the egg vitelline coat. A chymotrypsin-like enzyme has been purified from spermatozoa by a protocol including ion exchange chromatography, gel filtration, and native polyacrylamide gel electrophoresis. The purified enzyme resulted homogeneous when analyzed by SDS-PAGE. The molecular weight of the chymotrypsin-like enzyme was estimated to be 35 kDa by gel filtration and 24 KDa by SDS-PAGE in nonreducing conditions. The pH optimum of the enzyme is 8.4 and its activity is enhanced by Ca2+. It shows the highest activity towards the synthetic substrate Suc-Ala-Ala-Pro-Phe-AMC. Furthermore, by electron microscopy, the purified enzyme affects the structure of egg vitelline coat, and thus it fulfills one of the criteria of a lysin.     

Participation of sperm proteasome in fertilization of the phlebobranch ascidian Ciona intestinalis

Sperm proteasomes are thought to be involved in sperm binding to and in sperm penetration through the vitelline coat of the eggs of the stolidobranch ascidian Halocynthia roretzi. However, it is not known whether they are involved in the fertilization of eggs of other ascidians. Therefore, we investigated whether sperm proteasomes are also involved in the fertilization of the eggs of the primitive phlebobranch ascidian Ciona intestinalis. Fertilization of the eggs of C. intestinalis was potently inhibited by the proteasome inhibitors MG115 and MG132 but not by the cysteine protease inhibitor E-64-d. On the other hand, neither fertilization of the vitelline coat-free eggs nor sperm binding to the vitelline coat was inhibited by the two proteasome inhibitors at a concentration sufficient to inhibit fertilization of intact eggs. These results indicate that the proteasome plays an essential role in sperm penetration through the vitelline coat rather than in sperm binding to the coat or in sperm-egg membrane fusion. The proteasome activity, which was detected in the sperm extract using Suc-Leu-Leu-Val-Tyr-MCA as a substrate, was strongly inhibited by both MG115 and MG132, and was weakly inhibited by chymostatin, whereas neither leupeptin nor E-64-d inhibited the activity. The molecular mass of the enzyme was estimated to be 600-kDa by Superose 12 gel filtration, and the activity in sperm extract was immunoprecipitated with an anti-proteasome antibody. These results indicate that the proteasome present in sperm of C. intestinalis is involved in fertilization, especially in the process of sperm penetration through the vitelline coat, probably functioning as a lysin. Mol. Reprod. Dev. 50:493–498, 1998. © 1998 Wiley-Liss, Inc.     

Sperm surface proteases in ascidian fertilization.

Ascidian eggs are surrounded by a noncellular layer and two cellular layers, which are penetrated by sperm. Three sperm surface proteases are essential for fertilization of eggs from the stolidobranch ascidian Halocynthia: spermosin, acrosin, and the proteasome. In the phlebobranch Ciona, a chymotrypsin-like protease and the proteasome are essential in fertilization. Sperm from the phlebobranch ascidians Phallusia mammillata, Ascidia (=Phallusia) nigra, and Ascidia columbiana, all express spermosin, acrosin, and the proteasomal chymotrypsin activities on their surfaces. Chymostatin blocks cleavage in phlebobranchs, but inhibitors of spermosin and acrosin only delay it by several minutes. Protease inhibitors have little effect upon sperm binding in Phallusia but strongly affect the rate of sperm passage through the vitelline coat. Peptide substrates and inhibitors to spermosin and acrosin cause a significant decline in the number of eggs undergoing pre-meiotic contractions at 3 min after fertilization. Thus while chymotrypsin activity is essential for penetration of the vitelline coat, spermosin and acrosin both function to increase the rate of fertilization. A crucial step in the divergence of the phlebobranchs and stolidobranchs may have been the conversion of spermosin and acrosin to essential proteases in the stolidobranchs, or, perhaps, their essential function was lost in the evolution of phlebobranchs. Aplousobranch ascidians are all colonial with very small zooids. Sperm from Aplidium californicum, Aplidium solidum (Polyclinidae), and Distaplia occidentalis (Holozoidae) have acrosin and chymotrypsin activities but lack spermosin activity. This enzyme is also missing from sperm of colonial phlebobranch and stolidobranch ascidians, suggesting that spermosin is not necessary for small zooids with internal fertilization.     

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.     

Effects of proteinase inhibitors on fertilization in sea lamprey (Petromyzon marinus)

A search for alternative sterilants in parasitic fish encouraged us to explore the usefulness of proteinase inhibitors for this purpose. Fertilization in sea lamprey species (Petromyzon marinus L.) was inhibited by chymotrypsin and trypsin inhibitors 4'-acetamidophenyl 4-guanidinobenzoate (AGB), chymostatin, tosyl-L-lysine chloromethyl ketone (TLCK), and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) when these substances were added into a fertilization medium at the time of fertilization. Preincubation of eggs before fertilization with 100 microM TPCK, but not TLCK, resulted in inhibition of fertilization. Conversely, preincubation of spermatozoa with TLCK, but not TPCK, produced inhibition of fertilization. These data suggest the involvement of the chymotrypsin-like activity of eggs and trypsin-like activity of spermatozoa in fertilization. However, enzymes present in sperm suspensions were able to hydrolyze a chymotrypsin substrate N-glutaryl-L-phenylalanine-p-nitroanilide (GPNA) but not trypsin substrate N-alpha-benzoyl-DL-arginine-p-nitroanilide (BAPNA). The nature of this activity can be characterized as serine protease and our results indicate the involvement of serine proteinases in the fertilization of sea lamprey.     

Sperm extract injection into ascidian eggs signals Ca(2+) release by the same pathway as fertilization

Injection of eggs of various species with an extract of sperm cytoplasm stimulates intracellular Ca(2+) release that is spatially and temporally like that occurring at fertilization, suggesting that Ca(2+) release at fertilization may be initiated by a soluble factor from the sperm. Here we investigate whether the signalling pathway that leads to Ca(2+) release in response to sperm extract injection requires the same signal transduction molecules as are required at fertilization. Eggs of the ascidian Ciona intestinalis were injected with the Src-homology 2 domains of phospholipase C gamma or of the Src family kinase Fyn (which act as specific dominant negative inhibitors of the activation of these enzymes), and the effects on Ca(2+) release at fertilization or in response to injection of a sperm extract were compared. Our findings indicate that both fertilization and sperm extract injection initiate Ca(2+) release by a pathway requiring phospholipase C gamma and a Src family kinase. These results support the hypothesis that, in ascidians, a soluble factor from the sperm cytoplasm initiates Ca(2+) release at fertilization, and indicate that the activating factor from the sperm may be a regulator, directly or indirectly, of a Src family kinase in the egg.     

Mechanism of the block to hybridization and selfing between the sympatric ascidians Ciona intestinalis and Ciona savignyi

The solitary ascidians Ciona intestinalis and Ciona savignyi co-occur in southern California harbors, but no hybrids have been recognized in nature. Numerous differences in their egg morphology were detected. Homologous (normal outcross) fertilization yielded 96-99% cleavage, where autologous (self) fertilization showed 3% and heterologous (hybrid) fertilization showed 0-1%. Acid treatment (pH 3.2) removed the block to selfing (P < 0.0001) but not hybridization for both species. Heterologous sperm bind to the vitelline coat (VC), but fail to penetrate. Enzymatic removal of the VC resulted in 91-97% cleavage with autologous and heterologous sperm (P < 0.0001). The vitelline coats of the two species differ in lectin binding to surface glycosides. Fertilization in both species is significantly inhibited by the lectins, fucose binding protein (P < 0.0001) and concanavalin A (P < 0.0001), and wheat germ agglutinin inhibits fertilization in C. intestinalis (P < 0.0001) but is without effect on C. savignyi fertilization. Self and hybrid blocks employ different mechanisms including glycoside composition and acid sensitivity.     

Molecular Reproduction & Development Volume 78, Issue 12 cover image

Signal transduction across plasma membrane is prerequisite for activation of sperm motility and sperm chemotaxis at fertilization. An oscillatory increase in intracellular calcium occurs upon activation of tunicate sperm (shown in false color in the foreground, clockwise displaying later time points; starting at 11:00). Zhu and Inaba (this issue) report that lipid rafts are essential for these calcium dynamics and for subsequent activation of adenylyl cyclase. Background image is of a single tunicate, Ciona intestinalis.     

Membrane hyperpolarization by sperm-activating and -attracting factor increases cAMP level and activates sperm motility in the ascidian Ciona intestinalis.

In the ascidian Ciona intestinalis (and C. savignyi), sperm-activating and -attracting factor (SAAF) is released from the egg at fertilization and stimulates both Ca(2+) influx and a transient increase in cAMP level of the sperm, leading to the activation of sperm motility (M. Yoshida et al., 1994, Dev. Growth Differ. 36, 589-595). In this paper we show in C. intestinalis that valinomycin, a potassium-selective ionophore, as well as SAAF, activated sperm motility, and this activation was suppressed by extracellular high K(+). Membrane potential measurements showed that both SAAF and valinomycin increase K(+) permeability of sperm and induce membrane hyperpolarization, the amplitude of which depends on the external K(+) concentration. The membrane potential and intracellular K(+) concentration of Ciona sperm without SAAF were estimated to be about -50 mV and 560 +/- 40 mM, respectively. After treatment with SAAF or valinomycin the membrane potential became almost equal to the equilibrium potential of K(+) (-100 mV), and the cAMP level increased in artificial seawater. A potent voltage-dependent K(+) channel blocker, MCD peptide, at the concentration of 10 microM blocked SAAF-induced hyperpolarization of the cells, increase in cAMP, and sperm motility. These results suggest that membrane hyperpolarization produced by the opening of K(+) channels elevates cAMP synthesis and leads to the activation of sperm motility in Ciona.     

Fertilization regulates apoptosis of Ciona intestinalis extra-embryonic cells through thyroxine (T4)-dependent NF-kappaB pathway activation during early embryonic development

In Ciona intestinalis, the elimination of extra-embryonic test cells during early stage of development is delayed by a fertilization signal. Test cells undergo a caspase-dependent apoptosis event repressed by thyroxine (T4)-activated NF-kappaB. When apoptosis was experimentally blocked, the hatching stage was delayed. The incubation of unfertilized eggs with a 1-h-fertilized egg extract or purified T4 restored apoptosis in test cells at a similar timing than found in fertilized eggs. Ciona expresses specific genes forming a functional IkappaB/NF-kappaB pathway. One, Ci-p65, was transiently induced upon fertilization via T4 and found to exert its anti-apoptotic role in test cells nuclei as well as in a reconstituted cell system. Blocking NF-kappaB activity by dexamethasone-induced overexpression of Ci-IkappaB abrogated the repression of apoptosis in test cells. Overall, the data are consistent for defining a central coupling role of both T4 and NF-kappaB during early embryo development.     

Self- and cross-fertilization in the solitary ascidian Ciona savignyi

Solitary ascidians are hermaphrodites that release sperm and eggs simultaneously. However, many species are self-sterile, owing to a self/non-self recognition system operating at the outer surface of the chorion during sperm-egg interaction. In Ciona intestinalis, self-incompatibility is thought to have a genetic basis. Here, we report a survey of the self-fertilization potential of a Santa Barbara, California, population of Ciona savignyi, a close relative of C. intestinalis. We found that, in contrast to reports on C. intestinalis, C. savignyi is highly self-fertile. However, using two nonlethal recessive mutant strains, aimless (aim) and immaculate (imc), and a stable transgenic strain that expresses green fluorescent protein (GFP) in the notochord to follow offspring genotype, we demonstrate that non-self sperm outcompete self-sperm in fertilization competition assays. When the chorion was removed, both self- and non-self sperm performed equally well in the competition assay. Thus the non-self/self gamete recognition in C. savignyi is not absolute but relative, and is mediated by one or more components in the chorion. We discuss the significance of this finding in the context of natural populations in the wild, where individuals of C. savignyi are typically found growing in large groups that spawn in unison and where self-fertilization would be expected to be very rare.     

Sperm Penetration of the Vitelline Envelope of Sicyonia ingentis Eggs is Mediated by a Trypsin-like Lysin of Acrosomal Vesicle Origin

The sperm of the decapod crustacean Sicyonia ingentis are nonmotile, unistellate cells. At spawning, mated females release both stored sperm and eggs. The sperm bind, via the tip of their anterior appendage, to the egg's vitelline envelope (VE), rapidly undergo acrosomal exocytosis, and penetrate the VE. In the present study we used protease inhibitors to show that sperm penetration of the VE is due to the activity of a sperm trypsin-like protease(s). Sperm extracts contained several proteases when examined using gelatin-substrate SDS-PAGE, with two major bands of relative molecular weight 46 kD and 30 kD. Using fluorescent peptidyl-MCA substrates, sperm extract showed trypsin-like and aminopeptidase-like activities, but no chymotrypsin-like activity. Sperm extracts were found to degrade isolated VEs. Using soybean trypsin inhibitor and anti-inhibitor antibodies, protease was localized at the light and electron microscope levels to the acrosome remnants of reacted sperm.     

Chymotrypsin-like protease activity associated with demembranated sperm of chum salmon.

Our previous study suggested that a chymotrypsin-like protease was involved in the motility of chum salmon sperm (Inaba K, Morisawa M, Biomed Res (1991) 12, 435-437). In this study, we examined the peptidase activity of demembranated sperm of chum salmon using ten synthetic peptides. When spermatozoa were treated with 0.04% Triton X-100 for extracting the plasma membrane and the suspension was separated into the Triton-soluble and insoluble fractions by centrifugation, only the hydrolytic activity towards succinyl (Suc)-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide (MCA), a typical substrate for chymotrypsin-like protease, was mostly retained in the insoluble fraction. The bulk of the activities toward other substrates was detected in the soluble fraction. Flagellar axonemes isolated from demembranated sperm showed considerable hydrolytic activity toward Suc-Leu-Leu-Val-Tyr-MCA and the activity was still retained in the axoneme even after further washing. The hydrolysis was activated by a low concentration of SDS, suggesting that the protease associated with the axonemes is a multicatalytic ATP-dependent proteinase (proteasome). Motility of demembranated sperm was inhibited by Suc-Leu-Leu-Val-Tyr-MCA in an ATP-concentration-dependent manner. These results suggest that proteasomes associated with flagellar axoneme regulate flagellar motility.     

Helper Function of Follicle Cells in Sperm-Egg Interactions of the Ascidian, Ciona intestinalis

Studies were made on the involvement in sperm-egg interactions of follicle cells of Ciona intestinalis , which are tall, vacuolated cells attached to the outer surface of the egg vitelline coat. The basal surface of the follicle cells is polygonal. The borders between cells could easily be observed by the binding of fluorescent SBA (soy bean agglutinin), a lectin recognizing N-acetylgalactosamine (GaINAc) residues. At fertilization many spermatozoa aggregate along these polygonal borders of cells on the vitelline coat, through which they entered the perivitelline space. The removal of follicle cells was sometimes associated with loss of SBA-binding sites, and in such cases the sperm did not show a hexagonal pattern of aggregation, but became dispersed all over the vitelline coat. Removal of the follicle sometimes delayed fertilization. Examination of sections of gametes stained with DAPI, a fluorescent dye staining DNA, showed that removal of the follicle reduced the number of spermatozoa bound to the vitelline coat and, more especially, the number of spermatozoa penetrating through the vitelline coat. The blockage of GalNAc residues on the vitelline coat with SBA did not appreciably affect the time course of fertilization or the number of sperm associated with eggs. These findings are discussed in relation to the role of follicle cells in facilitating sperm aggregation on the vitelline coat and their penetration through it.     

Sperm penetration through cumulus mass and zona pellucida

Mammalian fertilization requires sperm to penetrate the cumulus mass and egg zona pellucida prior to fusion with the egg. Although sperm penetration through these physical barriers is essential, the molecular mechanism has not yet been completely elucidated. In addition to sperm motility, hyaluronan-hydrolyzing and proteolytic enzymes of sperm have been suggested to participate in the penetration events. Here we focus on the functional roles of hyaluronidase and protease in sperm passage through the cumulus mass and zona pellucida.     

A fucosyl glycoprotein component with sperm receptor and sperm-activating activities from the vitelline coat of ciona intestinalis eggs

In this paper we describe a mild procedure which results in the extraction of a glycoprotein fraction from the vitelline coat (VC) of Ciona intestinalis while leaving behind the bulk of the VC components. When acting upon the spermatozoa this fraction inhibits sperm binding to the VC and fertilization and elicits sperm activation including the acrosome reaction. SDS-PAGE shows that it contains the same (fucosyl) glycoprotein components previously recognized in the total extracts of VC. It is suggested that this material contains the sperm receptors or those components of the receptors that are essential for their Chinese function.     

Self-Nonself Recognition Activity Extracted from Self-Sterile Eggs of the Ascidian, Ciona intestinalis

Eggs of the hermaphrodite, self-sterile ascidian, Ciona intestinalis , were washed with acid seawater (pH 3.2), and the washing solution was then adjusted to pH 8.2. This solution was found to inhibit only the binding of non-autologous sperm to the vitelline coat (VC) of eggs, indicating that it contained self-nonself recognition activity. This activity was heat-stable and insensitive to trypsin, but was destroyed by V-8 protease and α-glucosidase. Both the hydrophobic and hydrophilic components of a lyophilized powder of the extract showed allo-recognizing activity. On TLC, the hydrophobic components gave a major spot of glucose (Glc) and a peptide spot(s) containing mainly glutamic acid and/or glutamine (Glx). The glucosyl conjugate was purified by HPLC and shown to block sperm-egg binding to various extents. Individual peptide subfractions had no inhibitory activity, but in combination they showed inhibitory activity. These findings suggest that the acid extract of Ciona eggs contains a Glc-enriched nonspecific inhibitor of sperm-egg binding, which could be the primary effector of self-incompatibility, and Glx-enriched modulators, which serve as acceptors of allo-sperm. The cooperative interactions of these components may be responsible for the diversity of allo-recognition in Ciona gametes.     

Identification and localization of the sperm CRISP family protein CiUrabin involved in gamete interaction in the ascidian Ciona intestinalis

Ascidians are hermaphrodites, and most release sperm and eggs nearly simultaneously. Many species, including Halocynthia roretzi and Ciona intestinalis, are self-sterile. We previously reported that the interaction between a 12 EGF-like repeat-containing vitelline-coat (VC) protein, HrVC70, and a sperm GPI-anchored CRISP, HrUrabin, in lipid rafts plays a key role in self-/nonself-recognizable gamete interaction in H. roretzi. On the other hand, we recently identified two pairs of polymorphic genes responsible for self-incompatibility in C. intestinalis by positional cloning: The sperm polycystin 1-like receptors s-Themis-A/B and its fibrinogen-like ligand v-Themis-A/B on the VC. However, it is not known if the orthologs of HrVC70 and HrUrabin also participate in gamete interaction in C. intestinalis since they are from different orders. Here, we tested for a C. intestinalis ortholog (CiUrabin) of HrUrabin by searching the genome database and proteomes of sperm lipid rafts. The identified CiUrabin belongs to the CRISP family, with a PR domain and a GPI-anchor-attachment site. CiUrabin appears to be specifically expressed in the testis and localized at the surface of the sperm head, as revealed by Northern blotting and immunocytochemistry, respectively. The specific interaction between CiVC57, a C. intestinalis ortholog of HrVC70, and CiUrabin was confirmed by Far Western analysis, similarly to the interaction between HrVC70 and HrUrabin. The molecular interaction between CiVC57 and CiUrabin may be involved in the primary binding of sperm to the VC prior to the allorecognition process, mediated by v-Themis-A/B and s-Themis-A/B, during fertilization of C. intestinalis.     

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.     

Concanavalin A Modifies Allo-specific Sperm-Egg Interactions in the Ascidian, Ciona intestinalis

In the self sterile ascidian, Ciona intestinalis , the spermatozoa rarely bind to the vitelline coat of autologous eggs and never penetrate it. We report here that concanavalin A (ConA), a lectin recognizing mannose or glucose residues of carbohydrates, can modify these self- and nonself-specific sperm-egg interactions. When eggs were pretreated with 0.1–0.5 mg/ml of ConA, about two thousand spermatozoa became attached to the autologous vitelline coat within five minutes of insemination. The effect of ConA was not modified by the addition of D-mannose or pretreatment of spermatozoa with ConA, showing that ConA does not function merely as a ligand bridging the sperm and vitelline coat. In contrast to the marked enhancement of sperm-egg binding, ConA did not facilitate the penetration of spermatozoa through the autologous vitelline coat. Even in non-autologous insemination, it blocked the sperm penetration and, consequently, fertilization did not occur, as shown by Rosati et al. (1978). D-Mannose, when mixed with ConA in advance, completely abolished this inhibitory effect of ConA. Lotus agglutinin, a fucose-binding lectin, was less effective and wheat germ agglutinin and soy bean agglutinin had no effect on sperm entry in the perivitelline space. The results of this study are discussed in relation to the possible involvement of mannosyl and/or glucosyl glycoconjugates in allo-specific sperm-egg interactions.