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 glycosylphosphatidylinositol-anchored CRISP-like protein as a binding partner for an allorecognizable sperm receptor on the vitelline coat

Although ascidians are hermaphroditic, many species including Halocynthia roretzi are self-sterile. We previously reported that a vitelline coat polymorphic protein HrVC70, consisting of 12 EGF (epidermal growth factor)-like repeats, is a candidate allorecognition protein in H. roretzi, because the isolated HrVC70 shows higher affinity to nonself-sperm than to self-sperm. Here, we show that a sperm 35-kDa glycosylphosphatidylinositol-anchored CRISP (cysteine-rich secretory protein)-like protein HrUrabin in a low density detergent-insoluble membrane fraction is a physiological binding partner for HrVC70. We found that HrVC70 specifically interacts with HrUrabin, which had been separated by SDS-PAGE and transferred onto a nitrocellulose membrane. HrUrabin has an N-linked sugar chain, essential for binding to HrVC70. HrUrabin mRNA is expressed in the testis but not in the ovary, and the protein appears to be localized on the surface of sperm head and tail. Anti-HrUrabin antibody, which neutralizes the interaction between HrUrabin and HrVC70, potently inhibited fertilization and allorecognizable sperm-binding to HrVC70-agarose. However, no significant difference in the binding ability of HrUrabin to HrVC70 was observed in autologous and allogeneic combinations by Far Western analyses. These results indicate that sperm-egg binding in H. roretzi is mediated by the molecular interaction between HrUrabin on the sperm surface and HrVC70 on the vitelline coat, but that HrUrabin per se is unlikely to be a direct allorecognition protein.     

Highly polymorphic vitelline-coat protein HaVC80 from the ascidian, Halocynthia aurantium: structural analysis and involvement in self/nonself recognition during fertilization

Ascidians release sperm and eggs simultaneously, but self-fertilization is effectively blocked by unknown mechanisms. We previously reported that a 70-kDa sperm receptor HrVC70 on the egg vitelline coat (VC) consisting of 12 EGF-like repeats is a candidate self/nonself recognition molecule during fertilization of the ascidian, Halocynthia roretzi. Here, we report that Halocynthia aurantium also utilizes a homolog (HaVC80) of HrVC70 as an allorecognizable sperm receptor. HaVC80 is attached to the VC during the acquisition of self-sterility and is detached from the VC by acid treatment, allowing self-fertilization. A cDNA clone of the HaVC80 precursor, HaVC130, consists of 3726 nucleotides and encodes an open reading frame of 1208 amino acids. The structure of HaVC130 is very similar to the HrVC70 precursor HrVC120, but the number of EGF-like repeats of HaVC130/VC80 is one repeat larger than that of HrVC120/VC70. There are several amino acid substitutions between different individuals, and two alleles of the HaVC80 sequence were detected in each individual. Genomic DNA sequence analysis reveals that each EGF-like domain corresponds to a specific exon, and HaVC130 may have been evolutionarily generated from HrVC120 by duplication of the 8th EGF-like repeat. The data support the hypothesis that HaVC80 is a highly polymorphic protein responsible for self-sterility in H. aurantium.     

Extracellular ubiquitin system implicated in fertilization of the ascidian, Halocynthia roretzi: isolation and characterization

The ubiquitin-proteasome system is essential for intracellular protein degradation, but there are few studies of this system in the extracellular milieu. Recently, we reported that a 70-kDa sperm receptor, HrVC70, on the vitelline coat is ubiquitinated and then degraded by the sperm proteasome during fertilization of the ascidian, Halocynthia roretzi. Here, we investigated the mechanism of extracellular ubiquitination. The HrVC70-ubiquitinating enzyme activity was found to be released from the activated sperm during the fertilization process. This enzyme was purified from an activated sperm exudate, by chromatography on DEAE-cellulose and ubiquitin-agarose columns, and by glycerol density gradient centrifugation. The molecular mass of the enzyme was estimated to be 700 kDa. The purified enzyme requires CaCl2 and MgATP for activity, and is active in seawater. The purified enzyme preparation, but not the crude enzyme preparation, showed narrow substrate specificity to HrVC70. Moreover, ATP and ubiquitin are released from the activated sperm to the surrounding seawater during fertilization. These results indicate that ascidian sperm release a novel extracellular ubiquitinating enzyme system together with ATP and ubiquitin during penetration of the vitelline coat of the egg, which catalyzes the ubiquitination of the HrVC70, an essential component of ascidian fertilization.     

Proteins interacting with the ascidian vitelline-coat sperm receptor HrVC70 as revealed by yeast two-hybrid screening

Ascidians are hermaphrodites releasing sperm and eggs nearly simultaneously, but many species are self sterile. We have previously reported that HrVC70 consisting of 12 EGF-like repeats is a major component of the vitelline coat, functioning as a self/nonself-recognizable sperm receptor during fertilization of the ascidian Halocynthia roretzi. Here, in order to identify the binding partner of HrVC70, we explored HrVC70-interacting proteins by yeast two-hybrid screening. HrVC70 is capable of interacting with HrVC70 precursor HrVC120 itself and also with three additional extracellular and/or transmembrane proteins, HrVLP-1, -2, and HrTTSP-1. Specific interaction of HrVC120, HrVLP-1, -2, and HrTTSP-1 with HrVC70 was confirmed by exchanging prey and bait, and also by a pulldown assay using the GST-fusion proteins. HrVLP-1 and -2 are proteins structurally related to HrVC120; both are expressed in the oocytes and may be novel components of the ascidian vitelline coat. HrTTSP-1 appears to be a member of the serine protease family with type II transmembrane topology. HrTTSP-1 is expressed in the testis and its gene product contains multiple conserved motifs known to be involved in protein-protein or protein-carbohydrate interactions. Close inspection revealed that the protease domain of HrTTSP-1 is considerably divergent, in particular around the region of the catalytic center Ser residue. Possible roles of these proteins in ascidian fertilization are also discussed.     

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.     

Localization and roles in fertilization of sperm proteasomes in the ascidian Halocynthia roretzi

We previously reported that sperm proteasome is responsible for degradation of the ubiquitinated vitelline-coat during fertilization in the ascidian Halocynthia roretzi. Here, we report the roles in fertilization and localization in the sperm cell surface of H. roretzi sperm proteasome. An anti-proteasome antibody, as well as the proteasome inhibitors MG115 and MG132, inhibited the fertilization, indicating that the sperm proteasome functions extracellularly in ascidian fertilization. In order to further assess this issue, the sperm surface proteasome activity was labeled with a cell-impermeable labeling reagent, NHS-LC-biotin, extracted with 0.1% CHAPS, and was subjected to a pull-down assay with avidin-agarose beads. It was found that a substantial amount of sperm proteasome is exposed to the cell surface. Partition analysis with Triton X-114 also revealed that a considerable amount of the sperm proteasome activity is partitioned into a lipid layer. Localization of the proteasome activity was investigated by fluorescence microscopy with succinyl-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide as a substrate. The sperm proteasome activity was specifically detected in the sperm head region, and it was markedly activated upon sperm activation. The membrane-associated proteasome was purified from the membrane fraction of H. roretzi sperm by affinity chromatography using an anti-20S proteasome antibody-immobilized Sepharose column. SDS-PAGE of the purified preparation showed a similar pattern of subunit composition to that of the 26S proteasome of mammalian origin. Taken together, these results indicate that H. roretzi sperm has the membrane-associated proteasome on its head, which is activated upon sperm activation, and that sperm proteasome plays an essential role in H. roretzi fertilization.     

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.     

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.     

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.     

Gamete self-discrimination in ascidians: a role for the follicle cells

Gamete self-incompatibility in the hermaphrodite tunicate Ciona intestinalis is a useful system with which to study self-nonself recognition. We have used in vitro fertilization of oocytes isolated from the gonad of Ciona intestinalis to identify the cellular source of self-sterility elements present on the egg envelopes. Here we show for the first time that self-discrimination, which occurs on the egg vitelline coat, is established there in late oogenesis and is contributed or controlled by products of the overlying follicle cells. The acquisition of self-sterility by the oocyte is prevented by the ionophore monensin, which suggests that the follicle cell self-sterility controlling factor is a glycoprotein.     

Re-examination of sibling cross-sterility in the ascidian,Ciona intestinalis: genetic background of the self-sterility

Self-sterility of solitary ascidians is a typical example of the allogeneic recognition, though its molecular mechanism remains an open question. In this paper we analyze the fertility between siblings from selfed and crossed eggs to understand the genetic basis of self-sterility in the ascidian, Ciona intestinalis. First, we show that the self-sterility is strict and stable, and the individuality expressed in gametes is highly diversified in the wild population that we used. Secondly, we show one-way cross-sterility and reciprocal cross-sterility within the siblings that are self-sterile but fertile with non-siblings. Thirdly, we show self-sterility and cross-sterility share some natures and both are closely related to the sperm capacity not to bind to the vitelline coat of the autologous eggs or the eggs sterile to the sperm concerned. In all, this paper shows that the self-sterility is genetically governed by a multiple-locus system, and that most probably individual-specific determinants are haploid expression in sperm and diploid expression in eggs, given they recognize self but not non-self.     

Proteomic analysis of bovine sperm YWHA binding partners identify proteins involved in signaling and metabolism

Posttranslational modification of proteins by phosphorylation is involved in regulation of sperm function. Protein phosphatase 1 gamma isoform 2 (PPP1CC_v2) and protein YWHA (also known as 14-3-3) are likely to be key molecules in pathways involving sperm protein phosphorylation. We have shown that phosphorylated PPP1CC_v2 is bound to protein YWHAZ in spermatozoa. In somatic cells, protein YWHA is known to bind a number of phosphoproteins involved in signaling and energy metabolism. Thus, in addition to PPP1CC_v2, it is likely that sperm contain other YWHA-binding proteins. A goal of the present study was to identify these sperm YWHA-binding proteins. The binding proteins were isolated by affinity chromatography with GST-YWHAZ followed by elution with a peptide, R-11, which is known to disrupt YWHA complexes. The YWHA-binding proteins in sperm can be classified as those involved in fertilization, acrosome reaction, energy metabolism, protein folding, and ubiquitin-mediated proteolysis. A subset of these putative YWHA-binding proteins contain known amino acid consensus motifs, not only for YWHA binding but also for PPP1C binding. Identification of sperm PPP1CC_v2-binding proteins by microcystin-agarose chromatography confirmed that PPP1CC_v2 and YWHA interactomes contain several common proteins. These are metabolic enzymes phosphoglycerate kinase 2, hexokinase 1, and glucose phosphate isomerase; proteins involved in sperm-egg fusion; angiotensin-converting enzyme, sperm adhesion molecule, and chaperones; heat shock 70-kDa protein 5 (glucose-regulated protein 78 kDa; and heat shock 70-kDa protein 1-like. These proteins are likely to be phosphoproteins and potential PPP1CC_v2 substrates. Our data suggest that in addition to potential regulation of a number of important sperm functions, YWHA may act as an adaptor molecule for a subset of PPP1CC_v2 substrates.     

Functional, biochemical, and chromatographic characterization of the complete [Ca2+]i oscillation-inducing activity of porcine sperm

A cytosolic sperm protein(s), referred to as sperm factor (SF), is delivered into eggs by the sperm during mammalian fertilization to induce repetitive increases in the intracellular concentration of free Ca2+ ([Ca2+]i) that are referred to as [Ca2+]i oscillations. [Ca2+]i oscillations are essential for egg activation and early embryonic development. Recent evidence shows that the novel sperm-specific phospholipase C (PLC), PLCzeta, may be the long sought after [Ca2+]i oscillation-inducing SF. Here, we demonstrate the complete extraction of SF from porcine sperm and show that regardless of the method of extraction a single molecule/complex appears to be responsible for the [Ca2+]i oscillation-inducing activity of these extracts. Consistent with this notion, all sperm fractions that induced [Ca2+]i oscillations, including FPLC-purified fractions, exhibited high in vitro PLC activity at basal Ca2+ levels (0.1-5 microM), a hallmark of PLCzeta. Notably, we detected immunoreactive 72-kDa PLCzeta in an inactive fraction, and several fractions capable of inducing oscillations were devoid of 72-kDa PLCzeta. Nonetheless, in the latter fractions, proteolytic fragments, presumably corresponding to cleaved forms of PLCzeta, were detected by immunoblotting. Therefore, our findings corroborate the hypothesis that a sperm-specific PLC is the main component of the [Ca2+]i oscillation-inducing activity of sperm but provide evidence that the presence of 72-kDa PLCzeta does not precisely correspond with the Ca2+ releasing activity of porcine sperm fractions.     

Recombinant mouse ZP3 inhibits sperm binding and induces the acrosome reaction.

Mammalian fertilization involves interactions of sperm surface receptors with ligands of the zona pellucida, an extracellular matrix surrounding the ovulated egg. In mouse, the zona is composed of three glycoproteins. One of them, ZP3, participates in primary sperm binding and in the subsequent triggering of the sperm's acrosome reaction. Considerable evidence suggests that carbohydrate determinants of ZP3 are responsible for binding to sperm and may be important for acrosomal exocytosis. A full-length cDNA encoding mouse ZP3 was assembled and cloned into expression vectors that contained either a cytomegalovirus (CMV) or a vaccinia (P11) promoter. Mouse L-929 cells were stably transformed with the pZP3-CMV constructs, and green monkey CV-1 cells were infected with a recombinant vaccinia virus containing ZP3. rZP3 was affinity purified from culture media and detected on Western blots as a single 60- to 70-kDa band, which differed in molecular weight from native ZP3 (mean, 83 kDa). Nevertheless, rZP3 is biologically active. rZP3 decreases sperm-zona binding with a potency equivalent to that of native zona pellucida and, like native ZP3, rZP3 triggers acrosomal exocytosis in capacitated mouse sperm. Thus, rZP3 isolated from both rodent and primate cells appears to contain those carbohydrate and protein structures necessary for ZP3's dual role in fertilization.     

Blood System Formation in the Urochordate Ciona intestinalis Requires the Variable Receptor vCRL1

Adaptive immune systems are present only in vertebrates. How do all the remaining animals withstand continuous attacks of permanently evolving pathogens? Even in the absence of adaptive immunity, every organism must be able to unambiguously distinguish "self" cells from any imaginable "nonself." Here, we analyzed the function of highly polymorphic gene vCRL1, which is expressed in follicle and blood cells of Ciona intestinalis, pointing to possible recognition roles either during fertilization or in immune reactions. By using segregation analysis, we demonstrate that vCRL1 locus is not involved in the control of self-sterility. Interestingly, genetic knockdown of vCRL1 in all tissues or specifically in hemocytes results in a drastic developmental arrest during metamorphosis exactly when blood system formation in Ciona normally occurs. Our data demonstrate that vCRL1 gene might be essential for the establishment of a functional blood system in Ciona. Presumably, presence of the vCRL1 receptor on the surface of blood cells renders them as self, whereas any cell lacking it is referred to as nonself and will be consequently destroyed. We propose that individual-specific receptor vCRL1 might be utilized to facilitate somatic self/nonself discrimination.     

Chymotrypsin-like enzymes are involved in sperm penetration through the vitelline coat of Ciona intestinalis egg

In Ciona intestinalis, sperm penetration through the egg vitelline coat is an essential event of fertilization. We investigated whether trypsin- and chymotrypsin-like enzymes are involved in this event. Inhibitors and peptide substrates for chymotrypsin-like enzymes blocked the overall process of fertilization in a concentration-dependent manner. The inhibitory activity was specifically exerted on the step of sperm penetration. Chymotrypsin-like protease activity was identified in spermatozoa with the fluorogenic synthetic substrate Suc-Ala-Ala-Phe-AMC, which was the most effective substrate in blocking sperm penetration. These data indicate that a chymotrypsin-like protease activity is a sperm lysin of Ciona intestinalis.     

Ascidian sperm penetration and the translocation of a cell surface glycosidase

Sperm bind to vitelline coat (VC) glycosides of ascidian eggs by means of a sperm surface glycosidase (Hoshi et al.: Zool Sci 2:65, 1985). In the genus Ascidia, N-acetylglucosamine (NAG) is the VC ligand. After initial binding by the tip of the head, sperm pass through the VC and perivitelline space leaving the single mitochondrion outside. This process can also be followed in vitro on a coverslip. Analysis of recorded video images shows that the sperm moves away from the anchored mitochondrion. Our model for sperm penetration suggests that mitochondrial translocation is responsible for driving the sperm into the egg. In the work presented here, we have demonstrated that ascidian sperm have N-acetyl-beta-D-glucosaminidase (NAGase) activity with an acidic pH optimum. This enzyme, which can be removed from the sperm with Triton X-100, binds to concanavalin A, demonstrating that it is glycosylated. Histochemical methods disclose that the enzyme is originally located at the tip of the head but subsequently remains with the surface overlying the mitochondrion during translocation. Fluorescent Con A was used as a second label for localization of the enzyme on the cell surface during translocation. Colocalization of both probes of the enzyme support a crucial facet of our model; the sperm surface VC binding site remains over the mitochondrion during translocation. This would couple mitochondrial translocation with sperm penetration and drive the sperm into the egg.     

Identification of sperm plasma membrane proteins exhibiting binding affinity for the ascidian egg coat

In the initial stage of ascidian fertilization sequential sperm–egg coat interactions assure successful species-specific fertilization. Sperm recognize, bind to, and then penetrate the egg investment that consists of follicle cells (FC) and an acellular vitelline coat (VC). To identify plasma proteins that recognize the egg coat, a membrane fraction was prepared from Phallusia mammillata sperm using nitrogen cavitation followed by three centrifugation steps. The purity of the membrane fractions was assessed by transmission electron microscopy and marker enzymes. Comparison of the electrophoretic pattern of sperm extracellular membrane domains labeled by radio-iodination or biotinylation and recorded by autoradiography or enhanced chemiluminescence, respectively, showed the non-radioactive procedure to be a convenient and efficient method. Isolated sperm membrane components were found to inhibit fertilization in a concentration-dependent manner and to bind mainly to the FC. Eggs were used as an affinity matrix to determine which of the solubilized sperm membrane proteins possess egg-binding activity. Three biotinylated proteins (66kDa, 120kDa and 140kDa) were found to bind to the VC. Assays probing heterospecific binding to Ascidia mentula eggs revealed that the 120kDa protein possesses species-specific binding activity. Thus, the current data suggest the 120 kDa sperm membrane protein as a candidate adhesion molecule with a possible role in gamete binding and species-specific recognition in P. mammillata .     

Classification,inhibition, and specificity studies of the vitelline coat lysin from toad sperm

The sonicated supernatant of the sperm of the toad, Bufo japonicus, can digest easily the vitelline coat (VC) of uterine eggs, and to a lesser extent the VC of coelomic eggs, but not that of activated eggs. The VC lysis and fertilization were competitively inhibited in the presence of t-butyloxycarbonyl-L-Gln-L-Arg-L-Arg-4-methylcoumaryl-7-amide (Boc-Gln-Arg-Arg-MCA), suggesting the involvement of proteases in the fertilization process. Starting from a sonicated supernatant, a potent VC lysin, possessing hydrolytic activity on Boc-Gln-Arg-Arg-MCA, was obtained by anion-exchange chromatography and gel filtration. The activity of the partially purified lysin was inhibited by diisopropyl fluorophosphate (DFP) and by such trypsin inhibitors as soybean trypsin inhibitor, leupeptin, and (p-amidinophenyl) methanesulfonyl fluoride hydrochloride, but not by chymostatin, E-64, and ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid. The molecular weight of the lysin was estimated to be 32K, based on the fluorographic image of ³H-DFP binding to the lysin on sodium dodecyl sulfate gel electrophoresis. The VC lysin was most active at pH 7.0–7.6 and under low ionic strength equivalent to fresh water. The release of the VC lysin was induced upon incubation of sperm with the contents of oviducal pars recta granules (PRG), which are known to induce the acrosome reaction. We conclude that the protease studied here represents the VC lysin of toad sperm that is involved in fertilization by digesting the VC of uterine eggs, probably released as a result of the acrosome reaction induced by PRG.