Extracellular ubiquitination and proteasome-mediated degradation of the ascidian sperm receptor

Abstract

The ubiquitin–proteasome system is essential for intracellular protein degradation, but an extracellular role of this system has not been known until now. We have previously reported that the proteasome is secreted into the surrounding seawater from sperm of the ascidian (Urochordata) Halocynthia roretzi on sperm activation, and that the sperm proteasome plays a key role in fertilization. Here, we show that a 70-kDa component (HrVC70) of the vitelline coat is the physiological substrate for the ubiquitin–proteasome system during fertilization of H. roretzi. A cDNA clone encoding the HrVC70 precursor (HrVC120) was isolated, and a homology search revealed that HrVC120 contains 13 epidermal growth factor-like repeats and a mammalian zona pellucida glycoprotein-homologous domain. HrVC70 functions as a sperm receptor. We demonstrate that HrVC70 is ubiquitinated both in vitro and in vivo. The immunocytochemical localization of multiubiquitin chains in the vitelline coat and the inhibitory effect of monoclonal antibodies against the multiubiquitin chains on fertilization strongly support the role of the ubiquitin–proteasome system in ascidian fertilization. Taken together, these results indicate that the ubiquitin–proteasome system is responsible for extracellular degradation of the sperm receptor HrVC70 and, consequently, for sperm penetration of the vitelline coat during fertilization.

Fertilization is a key event in sexual reproduction, creating a new individual with novel genomic information. In animal reproduction, species-specific binding of sperm to the proteinaceous egg coat, called the vitelline coat in marine invertebrates or the zona pellucida in mammals, is particularly important for successful fertilization. Because the egg coat is a potential barrier to sperm-egg fusion, sperm must use a lytic agent (lysin) to penetrate it (1, 2). In mammals, it has long been believed that the sperm acrosomal trypsin-like protease acrosin is a zona lysin, which digests zona pellucida proteins to enable sperm to penetrate through the zona pellucida (3). However, recent studies by using acrosin-knockout mice revealed that acrosin is not essential for fertilization or sperm penetration through the zona pellucida (4). Rather, it is currently thought that acrosin is involved in the dispersal of acrosomal proteins during acrosome reaction (5) and that a sperm protease(s) other than acrosin is the actual zona-lysin in mammalian fertilization (6).

Ascidians (Urochordata) occupy a phylogenetic position between invertebrates and segmented vertebrates. They are hermaphrodites that usually release sperm and eggs simultaneously during the spawning season. Several ascidians, including Ciona intestinalis (7) and Halocynthia roretzi (8), strictly prohibit self-fertilization. Because self–nonself recognition in fertilization is accomplished by interaction between the sperm and vitelline coat (7, 8), the sperm–lysin system seems to be triggered after the sperm recognizes the vitelline coat as nonself.

We have previously reported that two trypsin-like sperm proteases, acrosin and spermosin (9–14), and sperm proteasomes (15, 16) play key roles in sperm penetration through the vitelline coat of the ascidian H. roretzi. We provided evidence that the 930-kDa proteasome, which has a 26S-proteasome-like subunit composition, functions as a lysin that directly degrades the vitelline coat (16): the 26S-like proteasome was found in the supernatant obtained by centrifugation of the alkaline seawater-treated sperm and exhibited a vitelline coat-degrading activity. The treatment of H. roretzi sperm with alkaline seawater mimics the sperm activation and corresponds to the acrosome reaction in mammals and sea urchins, strongly suggesting that the proteasome is secreted to the surrounding seawater on sperm activation in H. roretzi. In this study, we explore the vitelline coat component that is digested by the sperm proteasome during sperm penetration through the vitelline coat. The 70-kDa vitelline coat component called HrVC70 is ubiquitinated and subsequently degraded by the sperm 26S-like proteasome. We also report that HrVC70 is, to our knowledge, a novel sperm receptor and provide evidence that the ubiquitin–proteasome system functions extracellularly in ascidian fertilization.

Materials and Methods

Biologicals.

The solitary ascidian (Urochordata) H. roretzi type C (17) was used in this study. Sperm and eggs were obtained as described previously (9, 10). Vitelline coats were isolated from the frozen-thawed mature oocytes by homogenization and repeated washing with 5-fold diluted artificial seawater on a nylon mesh (pore size 60 μm). The isolated vitelline coats were labeled with [¹²⁵I]NaI by using chloramine T.

Assay for Degradation of Vitelline Coats.

H. roretzi sperm was homogenized in 2-fold volume of 50 mM Tris⋅HCl, pH 8.0, containing 0.1% Triton X-100 with Teflon homogenizer. After centrifugation (10,000 × g, 30 min), the resulting supernatant was used as the sperm extract. The ¹²⁵I-labeled vitelline coat, which was suspended in 12 μl of 50 mM Tris⋅HCl, pH 8.0, containing 10 mM CaCl₂ and 50 mM MgCl₂, was incubated at 37°C for 30 min in the presence or absence of the sperm extract (2 and 4 μl), followed by SDS/PAGE (12.5% gel) and autoradiography. The amount of radiolabeled HrVC70 was quantified by using a Fuji Bio Imaging Analyzer BAS2000.

Immunologicals.

The isolated vitelline coats were subjected to SDS/PAGE, and the 70-kDa band (HrVC70) was excised. Small pieces of the excised gel were suspended in PBS, mixed with an equal volume of Freund complete adjuvant, and used to immunize rabbits. The IgG fraction was obtained from antiserum by Protein A-Sepharose chromatography. The antibody was adsorbed to HrVC70-blotted membrane and eluted with 0.1 M glycine⋅HCl (pH 3). Alternatively, VC70 peptide (amino acid residues 226–240: CEITGGLHKGGVYTG) was crosslinked to keyhole limpet hemocyanin, emulsified with TiterMax Gold (CytRx, Norcross, GA), and used to immunize rabbits. The anti-HrVC70 antibody was affinity purified with Protein A-Sepharose and VC70-peptide-immobilized agarose (Affi-Gel 10, Bio-Rad) beads. A monoclonal antibody FK2 (IgG), specific for multiubiquitin chains but not for free ubiquitin, was prepared as reported previously (18). Western blotting was carried out by a standard procedure, and the bands were detected with the enhanced chemiluminescence reagent (Amersham Pharmacia).

Cloning of HrVC120 cDNA.

RNA was isolated from H. roretzi gonads by a method (19) using acid guanidium-phenol-chloroform (AGPC method), and poly(A)+ RNA was prepared by using Oligotex-dT30 Super (Roche Diagnostics). A λgt11 cDNA library was constructed with the SuperScript Choice System for cDNA Synthesis (GIBCO/BRL) according to the manufacturer's protocol. The N-terminal 38-aa sequence of HrVC70 was determined by an Applied Biosystems Procise 492 protein sequencer. A primer pair consisting of a reverse primer (Pri-a) (5′-GCAGCCAGGCAGCGGCAGATGTACCA-3′) specific to the residues 30–38 of HrVC70 and a forward primer (5′-GATTGGTGGCGACGACTCCTG-3′) specific to the sequence in λgt11 was used to amplify related clones from the H. roretzi gonad cDNA library. PCR (30 cycles; denaturation at 94°C for 1 min, annealing at 50°C for 2 min, and elongation at 72°C for 3 min) was carried out with AmpliTaq DNA polymerase (Perkin–Elmer). A PCR product (about 300 bp) thus obtained was subcloned in a pGEM T-vector (Promega), and the nucleotide sequence was determined. The sequence 50 bp upstream of initiation methionine was used as the second forward primer (Pri-b) (5′-CGGAATAGCCTTGTGTTGACTTTG-3′). The probe for screening the cDNA library was prepared by PCR under the same conditions as described above with a digoxigenin DNA-labeling kit (Roche Diagnostics) by using Pri-a and Pri-b as reverse and forward primers, respectively. Ten positive clones were obtained from the H. roretzi gonad cDNA library by the plaque-hybridization method. One clone contained the N-terminal amino acid sequence and the 3′-end poly(A)+ tail and had a 4-kb insert. Its nucleotide sequence was determined by an Applied Biosystems DNA sequencer, model 377.

Northern Blotting.

Poly(A)+ RNA (3 μg), from gonad, muscle, branchial basket, intestine, hepatopancreas, and hemocytes with Oligotex dT30 Super, was electrophoresed on a 1% agarose gel. After washing the gel with 20× SSC (1× SSC = 0.15 M sodium chloride/0.015 M sodium citrate, pH 7), the RNA was blotted onto a Hybond N+ membrane (Amersham Pharmacia) for 16 h. The membrane was pretreated with hybridization buffer [50% formamide, 5 × standard saline phosphate/EDTA (SSPE) (0.18 M NaCl/10 mM phosphate, pH 7.4/1 mM EDTA)/0.5% SDS/5 × Denhardt's solution/0.5 mg of yeast tRNA in 10 ml] at 42°C for 1 h and then hybridized at 42°C for 16 h with a full-length cDNA of HrVC120 labeled with ³²P (7.5 × 10⁵ cpm/ml) by using a Takara (Tokyo) BcaBEST labeling kit. This membrane was subsequently washed with 1 × SSPE/0.5% SDS (50, 55, 60, and 65°C for 20 min each), 0.5× SSPE/0.5% SDS (65°C, 10 min), 0.1× SSPE/1% SDS (65°C, 20 min), followed by exposure to x-ray film.

Preparation of Glutathione S-Transferase (GST)-Fusion Proteins and Site-Directed Mutagenesis.

The HrVC120 coding sequence was amplified from the cDNA by PCR with a BamH1 terminus primer on the 5′-end and a Xho1 primer on the 3′-end. The resulting fragment was digested with BamH1 and Xho1 and ligated in-frame into pGEX-6P-1 (Amersham Pharmacia). Point mutations in the GST-fusion protein were generated by site-directed mutagenesis by using a QuickChange kit (Stratagene). The sequences of all constructs were confirmed by using an Applied Biosystems 377 DNA sequencer. E. coli BL21 cells carrying ligated plasmid were grown to stationary phase in 10 ml of LB medium containing 50 μg/ml of ampicillin and transferred to 200 ml of LB medium at 37°C for 3 h. After adding 1 mM isopropyl β-D-thiogalactoside, the culture was incubated for 2 h. The cells were sonicated in lysis buffer (20 mM Tris⋅HCl, pH 8.0, containing 1 mM EDTA and 100 mM NaCl), and bacterial debris were removed by centrifugation. The resulting lysate was incubated at 4°C for 1 h with 50% slurry of glutathione-Sepharose beads. After washing with 20 mM Tris⋅HCl, pH 8.0, by centrifugation, the GST-fusion protein was eluted from the beads with elution buffer (50 mM Tris⋅HCl, pH 8.0, containing 10 mM glutathione).

Results and Discussion

To clarify the role of extracellular sperm proteasomes in fertilization, we first investigated their mode of proteolytic action on the H. roretzi vitelline coat. The isolated radiolabeled vitelline coat was digested with crude sperm proteasome extract. A 70-kDa major component of the vitelline coat (HrVC70) was found to be degraded (Fig. 1A). Previous results indicated that the 26S-like proteasome purified from the ascidian sperm (16) does not efficiently degrade the HrVC70 molecules, suggesting that ubiquitination may be necessary for degradation of HrVC70 by the purified proteasome. To assess this possibility, HrVC70 was ubiquitinated by ubiquitin-conjugating enzymes purified from a rabbit reticulocyte lysate (18) and then digested with the purified sperm 26S-like proteasome in the presence of ATP. The degradation of HrVC70 molecules was specifically monitored by Western blotting by using the affinity-purified anti-HrVC70 IgG (Fig. 1B). As expected, ubiquitinated HrVC70 molecules were detected as high-molecular-weight bands on SDS/PAGE, and a 45-kDa band was newly formed (Fig. 1B Right). Furthermore, the 45-kDa band was recognized by FK2 antibody, which is a multiubiquitin chain-specific monoclonal antibody (Fig. 1C). The 45-kDa-protein band was blotted onto a nitrocellulose membrane and the band excised and subjected to protein sequence analysis. The N-terminal 20-aa sequence was identical to that of ubiquitin. These results suggest that the 45-kDa protein observed in the right lane in Fig. 1B is a ubiquitinated fragment of the HrVC70 molecule (Ub-VC70 fragment in Fig. 1 B and C) formed as an intermediate during proteasome degradation. Because the sequence of HrVC70 was undetectable by N-terminal amino acid sequence determination of the 45-kDa fragment, it seems likely that this 45-kDa fragment is multi- rather than monoubiquitinated. In the experiment of Western blotting with FK2 antibody, many high-molecular-weight ubiquitinated proteins were detected even in the preparation of E1/2/3 plus ubiquitin. Therefore, we could not specifically visualize the high-molecular-weight ubiquitinated VC70 bands by Western blotting (data not shown).

Figure 1.

Degradation of the 70-kDa component (HrVC70) of the H. roretzi vitelline coat by the ubiquitin–proteasome system. (A) HrVC70 was specifically degraded by the ascidian sperm extract. The ¹²⁵I-labeled vitelline coat (VC) was incubated with or without the sperm extracts (2 and 4 μl), followed by SDS/PAGE and autoradiography (Left). Note that the amount of HrVC70 is decreased by addition of the sperm extract in a concentration-dependent manner (Right). A small amount of 45-kDa band was observed in the isolated VC preparation probably because of possible contamination of a small quantity of nonself sperm during the collection process of eggs from H. roretzi, a hermaphroditic animal. (B) HrVC70 was degraded in the presence of 2 mM ATP by the prior addition of ubiquitin (Ub) and ubiquitin-conjugating enzymes [E1, E2, and E3 (E1/2/3)] purified from rabbit reticulocyte lysate followed by the addition of the purified 26S-like proteasome (26S). HrVC70 and its degradation fragments were clearly detected by Western blotting (WB) by using an antibody specific to HrVC70. A 66-kDa faint band is an artifact or a nonspecific band observed in the E1/2/3 preparation. (Ub)n-VC70, high-molecular-weight ubiquitinated HrVC70; Ub-VC70 fragment, ubiquitinated 45-kDa fragment. (C) The same samples as in B were subjected to Western blotting (WB) with the FK2 monoclonal antibody. The 45-kDa fragment was immunoreacted with FK2.

To elucidate the structure of HrVC70, a physiological substrate of the proteasome, we isolated a cDNA clone encoding HrVC70 from the H. roretzi gonad λgt11 cDNA library by using a DNA probe, which corresponds to the N-terminal amino acid sequence (38 residues) of HrVC70. The isolated cDNA clone consisted of 3,633 nucleotides and had a poly(A)+ tail. A single ORF encoded the 120-kDa HrVC70 precursor (HrVC120) of 1,162 amino acids (Fig. 2A). The deduced amino acid sequence contains a sequence (21–58) that corresponds to the N-terminal amino acid sequence of HrVC70, suggesting that the mature protein begins with the valine residue at position 21. The first 20 residues appear to represent a typical hydrophobic signal sequence. A homology search revealed that HrVC120 contains 13 epidermal growth factor (EGF)-like repeats (even though it has incomplete homology in the 13th EGF module), a mammalian zona pellucida glycoprotein (ZP)-homologous domain, and a single transmembrane domain in the C-terminal region (Fig. 2B). As in the case of mammalian zona pellucida glycoproteins, HrVC120 contains a furin cleavage site (see Fig. 2A, indicated by green; Arg¹⁰⁴⁴-Lys¹⁰⁴⁵-Arg¹⁰⁴⁶-Arg¹⁰⁴⁷). This protein also contains four potential N-glycosylation sites (magenta) at the C-terminal side and five potential O-fucosylation sites (blue; consensus sequence, Cys-X-X-Gly-Gly-Ser/Thr-Cys) located between the second and third Cys residues in EGF domains 3, 5, 7, 9, and 11 (20). We propose that the HrVC70 molecule is generated by the action of a trypsin-like protease at the C-terminal side of the Arg⁶⁶⁸ residue located between the 12th and 13th EGF-like repeats in HrVC120 for the following reasons. First, the C-terminal sequence determination by a Hewlett–Packard N&C protein sequencer revealed that the C-terminal residue is Arg, and that Cys/Ser or Gly are penultimate residues. Second, the molecular mass of HrVC70 was determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis to be ≈68,800 Da, which is almost identical to the calculated value (M_r = 68,943) of the protein consisting of 12 EGF-like repeats (from Val²¹ to Arg⁶⁶⁸). Third, the amino acid composition of the isolated HrVC70 was almost identical to the calculated amino acid composition of the protein comprising 648 residues (data not shown).

Figure 2.

Structure and expression of the HrVC70 precursor protein HrVC120. (A) Deduced amino acid sequence of HrVC120. An N-terminal sequence of HrVC70 determined by Protein Sequencer (brown), a C-terminal Arg residue of HrVC70 (purple), potential O-fucosylation sites (blue), potential N-glycosylation sites (magenta), and a potential furin cleavage site (green) are also indicated. (B) The domain structure of HrVC120. TM domain, transmembrane domain. (C) Northern blotting of VC120. Gn, gonad; Ms, muscle; Bb, branchial basket; In, intestine; Hp, hepatopancreas; Hc, hemocytes.

Northern blot analysis was carried out by using full-length HrVC120 cDNA as a probe (Fig. 2C). A 3.4-kb mRNA for HrVC120 was detected in the gonad but not in muscles, branchial basket, intestine, hepatopancreas, or hemocytes, indicating a high level of expression of HrVC120 mRNA in only the gonad.

We observed that even- and odd-numbered EGF-like repeats appear to be more evolutionarily related to one another: EGF-5, -7, and -9 show a high level of homology, whereas EGF-6, -8, and -10 are more highly related to one another (Fig. 3A). These observations led us to speculate that two ancestral EGF-like domains may have been duplicated during evolution to make up the current 12 EGF-like repeat structure in HrVC70. In addition, a psi-blast homology search revealed that a domain in HrVC120 (residues 773-1032) bears a significant similarity to components of the mammalian zona pellucida (i.e., 19% identity with human sperm receptor ZP3) (Fig. 3B). The N-terminal three Cys residues in the ZP domain of HrVC120 occupy conserved positions as in mammalian and frog ZPCs, whereas no appreciable homology is detected at the positions of Cys residues in its C-terminal region. The position of a single transmembrane domain is conserved among ZP3 homologs including HrVC120. Further study is needed to determine whether the ZP domain functions independently after processing and what its role is.

Figure 3.

(A) Similarity of the EGF-like repeats in HrVC70. Consensus sequences in the odd- (CONS-5, 7, 9) and even-numbered EGF-like repeats (CONS-6, 8, 10) in HrVC70 are indicated. Two Lys residues in HrVC70 are shown by blue marks, and cysteine residues are indicated by magenta. (B) Alignment by clustal w of the ZP domain in HrVC120 with ZP domain-containing molecules including human sperm receptor ZP3, mouse ZP3, and Xenopus ZP3 homologue (gp43). (C) Binding of H. roretzi sperm to the HrVC70-immobilized agarose beads. HrVC70 isolated from the vitelline coat with 1 mM HCl or the extract with 50 mM 4-morpholinepropanesulfonic acid (pH 8.0) as a control was coupled to Affi-Gel-10 beads according to the manufacturer's protocol (≈0.5 mg HrVC70/ml beads). The HrVC70-immobilized agarose beads or the control beads in 500-μl suspension were incubated with sperm suspension (100 μl) for 2 h at 13°C. After repeated washing with artificial seawater, the number of sperm bound to one HrVC70-immobilized agarose bead or the control bead was counted in the presence of 4′,6-diamidino-2-phenylindole under a fluorescence microscope with UV excitation.

It is well known that transmembrane proteins containing EGF-like repeats are involved in cell–cell interaction and recognition (21). In particular, Notch and Delta/Serrate, both of which have EGF-like repeats in their extracellular regions, play a pivotal role in cell–cell interaction during neuronal differentiation in Drosophila. In this context, we examined the binding activity of HrVC70 toward H. roretzi sperm. First, we found that the HrVC70 was specifically extracted from the vitelline coat by treatment with 1 mM HCl (pH 3) but not with 50 mM 4-morpholinepropanesulfonic acid (Mops) (pH 8.0), as revealed by SDS/PAGE followed by Western blotting (date not shown). Next, the HrVC70 in the HCl extract and also the Mops extract (control) were immobilized on N-hydroxysuccinimide-activated agarose beads (Affi-Gel 10, Bio-Rad), and both beads were tested for the ability to bind sperm. The number of 4′,6-diamidino-2-phenylindole-labeled sperm bound to each HrVC70-immobilized agarose bead (≈0.5 mg/ml gel) was counted under a fluorescence microscope and found to be significantly higher than those of control beads (Fig. 3C) and of BSA-immobilized beads (data not shown). These results indicate that HrVC70 has sperm receptor activity. In Caenorhabditis elegans, the sperm membrane protein Spe-9, which has 10 EGF-like repeats in its extracellular region, has been reported to be essential for fertilization (22), but its precise role is unknown. Several mutants with amino acid substitutions in EGF modules display a defect in sperm–egg interaction. By analogy with the Notch/Delta interaction, we hypothesize that the physiological sperm ligand for HrVC70 is a Spe-9-like transmembrane protein possessing several EGF-like repeats.

The HrVC70 has only two Lys residues (Lys²³⁴ and Lys⁶³⁶; blue marks in Fig. 3A). One (Lys²³⁴) is located in the linker region between EGF-4 and -5 domains, whereas the other (Lys⁶³⁶) is located in the region between the third and fourth Cys residues in EGF-12 domain. We attempted to express several GST-fusion proteins carrying HrVC70 and its fragments to test their ubiquitination in vitro. Although the whole HrVC70-containing GST-fusion protein was unusable because of low solubility, we succeeded in expressing two GST-fusion proteins carrying an HrVC70 fragment (residues 227–270), derived from EGF-4 and -5 domains, and its lysine-to-arginine mutant. The fusion protein was purified by glutathione-Sepharose chromatography followed by H. roretzi sperm exudate in the presence of ATP/Mg. As shown in Fig. 4, the wild-type GST-fusion protein (GST-VC70_227–270 WT) was efficiently multiubiquitinated, but the Lys-substituted mutant [GST-VC70_227–270 (K234R)] was not. These results clearly indicate that the HrVC70, a main component of the vitelline coat, is multiubiquitinated in vitro at least via Lys²³⁴ by a putative ubiquitin-conjugating enzyme system in the sperm exudate.

Figure 4.

In vitro ubiquitination of GST-HrVC70 fragment fusion proteins by H. roretzi sperm exudate. The GST-fusion proteins carrying the HrVC70 fragment (GST-VC70_227–270 WT) and its Lys-to-Arg mutant [GST-VC70_227–270 (K234R)] were incubated with the sperm exudate in the presence of 5 mM ATP, ¹²⁵I-ubiquitin, 10 mM MgCl₂, and 1 mM MG115 (proteasome inhibitor). After incubation, the reaction mixtures were subjected to SDS/PAGE, followed by autoradiography.

The next most important question is whether HrVC70 is ubiquitinated under physiological conditions in vivo. To assess this issue, the unfertilized eggs were frozen before or 5 min after the addition of the nonself-sperm suspension. These unfertilized and fertilized eggs were thawed and the vitelline coats isolated and subjected to SDS/PAGE followed by Western blotting by using the anti-HrVC70 and FK2 antibodies. High-molecular-weight bands caused by ubiquitinated HrVC70 molecules were detected only in the fertilized eggs (Fig. 5) under our experimental conditions, indicating that HrVC70 is ubiquitinated on sperm–egg interaction under in vivo conditions. Furthermore, the observation that the ubiquitinated proteins consisted mainly of high-molecular-weight forms of HrVC70 (Fig. 5B) strongly suggests that HrVC70 is the preferential substrate ubiquitinated after insemination among various vitelline coat components.

Figure 5.

In vivo ubiquitination of HrVC70 in the vitelline coat of H. roretzi eggs on insemination with sperm. (A) The vitelline coats (VCs) were isolated from the unfertilized and fertilized (5 min after insemination) eggs, and then subjected to SDS/PAGE, followed by Western blotting (WB), by using anti-HrVC70-peptide antibody. (B) The same membrane in A was reprobed with FK2 antibody. (Ub)n-VC70, high-molecular-weight ubiquitinated HrVC70.

To obtain further evidence that in situ ubiquitination takes place during fertilization of H. roretzi, immunocytochemistry of the unfertilized and fertilized eggs was carried out by using FK2 monoclonal antibody. As shown in Fig. 6, strong FK2 immunofluorescence caused by the presence of ubiquitin conjugates was observed on the vitelline coat after, but not before, insemination. These results clearly show that the vitelline coat proteins are ubiquitinated on fertilization in H. roretzi. To examine whether this ubiquitination is indispensable for fertilization, we tested the effect of the FK2 antibody on fertilization and found that the FK2 antibody potently inhibited fertilization in a concentration-dependent manner (14 and 67% inhibition at 1.5 and 4.5 mg/ml of FK2 IgG, respectively). This strongly suggests that the extracellular formation of multiubiquitin chains catalyzed by a ubiquitin-conjugating enzyme system, probably derived from the sperm, is essential for ascidian fertilization.

Figure 6.

Immunocytochemistry of the H. roretzi vitelline coat before (1, 3) and after insemination (2, 4) by using the FK2 antibody as a primary antibody and the FITC-conjugated anti-mouse IgG goat antibody as a secondary antibody. Upper (1, 2) and Lower (3, 4) represent the fluorescence and bright field, respectively, under a fluorescence microscope. Note that the vitelline coat in fertilized eggs showed a strong fluorescence because of ubiquitinated proteins (2). No appreciable fluorescence was observed with a control IgG in either case (data not shown). It is evident that in situ ubiquitination takes place during fertilization. VC, vitelline coat; F, follicle cells; PS, perivitelline space. (Bar = 10 μm.)

With respect to the origin and extracellular role of ubiquitin, it has been reported that hairy cell leukemia cells secrete ubiquitin into the surrounding medium or serum, which in turn elicits growth suppression and apoptosis in human hematopoietic cells possibly through proteasome-mediated degradation of STAT3 (23). In connection with this, it should be noted that ubiquitin-crossreactive protein, which comprises two ubiquitin-like domains, is secreted from human monocytes and lymphocytes and modulates the immune system as a cytokine (24). In addition, there is an intriguing report that free ubiquitin is detected in human seminal plasma at a high concentration (2–19 μg/ml) (25). Although the local concentration of ubiquitin at the site of sperm–egg interaction in H. roretzi has yet to be determined, it is possible that free ubiquitin is secreted from somatic cells such as follicle cells as well as from germ cells. Further studies on the production of ubiquitin in the case of H. roretzi fertilization, as well as the elucidation of the ubiquitin-conjugating enzyme system in the sperm exudate, are necessary to define the extracellular roles of the ubiquitin–proteasome system in ascidian fertilization.

In conclusion, we propose that a main component of the vitelline coat, HrVC70, exhibits sperm receptor activity and is multiubiquitinated and then degraded by the sperm ubiquitin–proteasome system during H. roretzi fertilization. These results open a new gate for understanding the role of the ubiquitin–proteasome system in sperm–egg interaction.

Abbreviations

EGF

epidermal growth factor

GST

glutathione S-transferase

ZP

zona pellucida glycoprotein