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J. Biol. Chem., Vol. 277, Issue 4, 2763-2772, January 25, 2002

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Molecular Characterization of the Starfish Inositol 1,4,5-Trisphosphate Receptor and Its Role during Oocyte Maturation and Fertilization^*

Hirohide Iwasaki^§¶, Kazuyoshi Chiba, Tsuyoshi Uchiyama^§, Fumio Yoshikawa^**, Fumiko Suzuki, Masako Ikeda, Teiichi Furuichi^**, and Katsuhiko Mikoshiba^§

From the  Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan, the ^§ Division of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8039, Japan, the  Department of Biology, Ochanomizu University, 2-1-1 Ootsuka, Bunkyo-ku, Tokyo 112-8610, Japan, ^** Laboratory for Molecular Neurogenesis, Brain Science, Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan, and the  Department of Biosciences, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan

Received for publication, September 13, 2001, and in revised form, October 25, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The release of calcium ions (Ca²⁺) from their intracellular stores is essential for the fertilization of oocytes of various species. The calcium pools can be induced to release Ca²⁺ via two main types of calcium channel receptor: the inositol 1,4,5-trisphosphate receptor (IP₃R) and the ryanodine receptor. Starfish oocytes have often been used to study intracellular calcium mobilization during oocyte maturation and fertilization, but how the intracellular calcium channels contribute to intracellular calcium mobilization has never been understood fully, because these molecules have not been identified and no specific inhibitors of these channels have ever been found. In this study, we utilized a novel IP₃R antagonist, the "IP₃ sponge," to investigate the role of IP₃ during fertilization of the starfish oocyte. The IP₃ sponge strongly and specifically competed with endogenous IP₃R for binding to IP₃. By injecting IP₃ sponge into starfish oocyte, the increase in intracellular calcium and formation of the fertilization envelope were both dramatically blocked, although oocyte maturation was not blocked. To investigate the role of IP₃R in the starfish oocyte more precisely, we cloned IP₃R from the ovary of starfish, and the predicted amino acid sequence indicated that the starfish IP₃R has 58-68% identity to mammalian IP₃R types 1, 2, and 3. We then raised antibodies that recognize starfish IP₃R, and use of the antibodies to perform immunoblot analysis revealed that the level of expression of IP₃R remained unchanged throughout oocyte maturation. An immunocytochemical study, however, revealed that the distribution of starfish IP₃R changes during oocyte maturation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A dramatic transient release of Ca²⁺ from internal stores has been demonstrated during fertilization (1-3). This rise in Ca²⁺ is essential to the stimulation of many subsequent events in egg activation, including exocytosis of cortical granules (which establish polyspermy blocks), and reentry of the egg into the meiotic cell cycle. Ca²⁺ can be released from internal stores via two main types of calcium channel receptors: (i) the inositol 1,4,5-trisphosphate receptor (IP₃R),¹ which is responsive to the second messenger, inositol 1,4,5-trisphosphate (IP₃), and (ii) the ryanodine receptor (RyR), which is insensitive to IP₃ but can be stimulated by calcium or other agents, including ryanodine, caffeine, and the naturally occurring metabolite cyclic adenosine diphosphate-ribose. Nicotinic acid adenine dinucleotide phosphate is also known to release Ca²⁺ from internal stores in sea urchin eggs (4, 5) and starfish eggs (6), but the molecular mechanism is still unknown. Several observations suggest that IP₃ mediates Ca²⁺ release during fertilization: (i) phosphatidylinositol lipid turnover and IP₃ levels increase rapidly during fertilization (7-11), (ii) IP₃ injection of unfertilized eggs and IP₃ application to endoplasmic reticulum isolated from eggs causes calcium release (12-18), (iii) injection of phospholipase C- Src homology 2 domain into oocyte inhibits Ca²⁺ release during fertilization by inhibiting the generation of IP₃ (19), and (iv) injection of oocytes with heparin, a nonspecific competitive inhibitor of IP₃R, causes abnormal fertilization-induced calcium dynamics in a dose-dependent fashion (20-24). However, the effects of heparin are difficult to interpret because of its lack of specificity. In some studies, heparin has been found to reduce ryanodine-induced (25) and cyclic adenosine diphosphate-ribose-induced (26) Ca²⁺ release, and heparin has been reported to inhibit cortical granule exocytosis even after a Ca²⁺ rise of near normal amplitude has occurred (27). In addition, frog eggs injected with ~300 µg/ml heparin have been reported to often exhibit splotchy pigmentation characteristic of unhealthy eggs (23).

Monoclonal antibody 18A10, which binds to the carboxyl terminus of IP₃R and specifically inhibits IP₃-induced Ca²⁺ release (IICR), provides a more specific approach. In hamster eggs, complete inhibition was obtained with more than 100 µg/ml IP₃R antibody 18A10, whereas 200~800 µg/ml heparin only partially inhibited Ca²⁺ release during fertilization (28). However, 18A10 does not always cross-react with IP₃Rs from other species, because it was raised against mouse IP₃R1 (mIP₃R1) and its epitope is not conserved well among all the reported IP₃Rs of different species and types. In fact, we have confirmed by immunoblot analysis that 18A10 does not recognize starfish IP₃R (data not shown).

In this study, we used a novel IP₃R antagonist, the "IP₃ sponge," which we recently developed to identify the physiological role of IP₃R in starfish oocytes (29).² IP₃R is composed of three major functional domains: an N-terminal ligand-binding domain, a modulatory domain in the middle, and a transmembrane domain near the C terminus (30, 31). In our previous study, we found that amino acids (aa) 226-578 of mIP₃R1 are the core region for IP₃ binding and that the bacterially expressed aa 224-604 has markedly higher binding affinity than aa 1-604 (29).² Although the IP₃ binding core region of aa 224-604 was not efficiently expressed as a soluble active form, this problem was overcome by N-terminal fusion of aa 224-604 with glutathione S-transferase. The resulting fusion protein was called "G224." G224 showed high IP₃ binding activity, approximately ~1000 times greater than that of endogenous IP₃R in mouse cerebellar microsomes (see Fig. 1 and "Experimental Procedures").² G224 strongly, specifically, and dose-dependently competed with endogenous IP₃R for binding to IP₃. We therefore decided to call G224 the "IP₃ sponge," because it blocked IICR by absorbing IP₃.

Fully grown immature starfish oocytes have a large nucleus called the "germinal vesicle" (GV) and are arrested at the first meiotic prophase. The prophase arrest is released in response to a starfish maturation-inducing hormone, 1-methyladenine (1-MA), which acts on the oocyte surface (32). At 20 °C, germinal vesicle breakdown (GVBD; nuclear disassembly), a sign of resumption of meiosis reinitiation, occurs ~20 min after application of 1-MA. After GVBD, oocytes acquire increased sensitivity to sperm and IP₃, which results in a release of calcium from intracellular stores and a rise in intracellular free calcium (16). We expressed IP₃ sponge in Escherichia coli and injected it into immature starfish oocytes to investigate the role of IP₃R during oocyte maturation and fertilization. Moreover, to identify the role of IP₃R in starfish more precisely, we cloned the starfish IP₃R cDNA and used the predicted amino acid sequence of starfish IP₃R to raise antibodies that recognize the receptor and to investigate the level of expression of IP₃R during oocyte maturation. We also examined immunocytochemistry to investigate changes in localization of the IP₃R in oocytes during maturation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Preparation of Gametes-- Starfish Asterina pectinifera were collected on the Pacific coast of Honshu and kept in a laboratory aquarium supplied with circulating sea water at 17 °C. In this paper we use the term "oocyte" to refer to fully grown immature oocytes at meiotic prophase and "egg" to refer to the maturing oocytes after GVBD stimulated with 1-MA to resume meiosis. 1-MA was obtained from Sigma and used at 1-2 µM. Isolated ovaries were incubated in ice-cold calcium-free sea water to remove follicle cells, and the oocytes released were washed twice in ice-cold calcium-free sea water and stored in artificial sea water at 20 °C. Sperm were collected from an excised testis and stored on ice without dilution. Except as noted, the sperm were diluted with artificial sea water (10⁶ times volume of the testis extract) before addition to the chamber containing the eggs.

Microinjection of IP₃ Sponge into Immature Oocytes-- Preparation of the IP₃ sponge was described elsewhere (29).² Briefly, to express N-terminal fused proteins of residues 224-604 of mIP₃R1 with the glutathione S-transferase gene, the cDNA that encodes aa 224-604 of mIP₃R1 was amplified by PCR by using pET-1-604 (29) as a template and subcloned into pGEX-2T (Amersham Biosciences, Inc.). Site-directed mutagenesis of K508A was introduced by two-step PCR, as described elsewhere (33). Expression of recombinant proteins in E. coli was carried out by the low temperature method (33), and purification of these proteins with glutathione-Sepharose CL4B beads was done according to the manufacturer's method (Amersham Biosciences). The purified proteins were then applied to a PD-10 column containing Sephadex G-25 M (Amersham Biosciences) preequilibrated with elution buffer (88 mM NaCl, 1 mM KCl, 10 mM Hepes-KOH, pH 7.2). Protein concentration was determined using a kit (Bio-Rad) with bovine serum albumin as the standard and adjusted to a concentration of 2 mg/ml with elution buffer. The volume injected was 5% of the oocyte volume (oocyte volume was ~3 nl). The concentrations of IP₃ sponge, GST-m30, and GST in cytoplasm were ~1.5, 1.5, and 3 µM, respectively. The injection was made with a micropipette with an oil-filled constriction connected to a syringe (34, 35). The oocytes were held between two coverslips separated by two pieces of double-stick tape during the microinjection and the observation period (36). IP₃ was dissolved in 100 mM potassium aspartate, 10 mM Hepes, pH 7.0, at a series of concentrations ranging from 10⁷ to 10⁴ M. The injected volume was 1% of the cell volume.

Calcium Measurement-- The fluorescence from an oocyte injected with 1 mM Fura2-dextran was collected with a ×20, 0.5 N.A. objective and focused onto a photomultiplier (Nikon, P1) mounted on an inverted fluorescence microscope; excitation filters at 360 ± 10 and 380 ± 10 nm, dichroic beam splitter at 400 nm, and long pass emission filters at 420 and 450 nm were used. Fura-2 fluorescence was observed at 20 °C by exciting Fura-2 at 380 nm. Fura-2 fluorescence induced by excitation at 360 nm was observed both at the beginning and at the end of the observation period to calibrate bleaching of the dye.

Molecular Cloning of Starfish IP₃R-- Total RNA from starfish ovary was purified on an RNeasy column (Qiagen-GmbH, Hilden, Germany). Reverse transcription was performed using SuperScript II (Invitrogen). The degenerate oligonucleotide primers used were as follows: 5'-1 (5'-GTT(T/C)CAIGC(T/C)GA(A/G)CA(A/G)GA(A/G)AA(A/G)TT-3') and 3'-1a (5'-G(T/C)TTIGC(A/G/T)AT(A/G)TA(T/C)TC(T/C)TG(A/G)TT(T/C)TT-3') plus 3'-1b (5'-G(T/C)TTIGC(A/G/T)AT(A/G)TG(T/C)TC(T/C)TG(A/G)TT(T/C)TT-3'); 5'-2 (5'-GAA(A/G)AA(T/C)GTITA(T/C)ACIGA(A/G)AT(T/C/A)AA(A/G)TG-3') and 3'-2 (5'-G(A/G)TA(A/G/T)AT(T/C)TC(T/C)TTCA(T/C)TCIAC(T/C)TCIGT(A/G)TC-3'); 5'-3 (5'-GGA(A/G)TA(T/C)TG(T/C)CA(A/G)GGICCITG(T/C)CA(T/C)GA(A/G)AA(T/C)CA-3' and 3'-3 (5'-G(A/G)TCIGC(A/G)AAIGT(A/G)TC(A/G/T)AT(A/G/T)ATIACICC(A/G)AA-3'), as described in the legend to Fig. 6A.

PCR was performed using AmpliTaq DNA polymerase (PerkinElmer Life Sciences). The thermocycler was programmed for 94 °C denaturation for 2 min followed by 40 cycles of 94 °C denaturation for 45 s, 50 °C annealing for 45 s, and 72 °C extension for 1 min. The final cycle was followed by an additional extension at 72 °C for 6 min. These products were subcloned into the T/A vector pCR2.1 (Invitrogen, Carlsbad, CA) and sequenced with the dideoxy method.

Other pairs of primers used to clone the residual parts of IP₃R were 5'-0.5 (5'-CCACCAAA(A/G)AA(A/G)TT(C/T)AGAGA-3') and 3'-0.5 (5'-CCTCAGGAAGATGTAGCTCT-3'), 5'-1.5 (5'-GCATTCTCAGGCTGTCACAG-3') and 3'-1.5 (5'-TCCAGTGGTAGCAGGGAGTG-3'), and 5'-2.5 (5'-GGAGGATGCCTACATCAACT-3') and 3'-2.5 (5'-TTGATGTCGTTCAGTATGAG-3'), as described in the legend to Fig. 6A.

PCR was performed using Platinum Pfx DNA polymerase (Invitrogen). The thermocycler was programmed for 94 °C denaturation for 2 min followed by 40 cycles of 94 °C denaturation for 15 s, 50 °C annealing for 30 s, and 68 °C extension for 3 min. The final cycle was followed by an additional extension at 68 °C for 7 min. These products were subcloned into the EcoRV site of pBlueScript KS () (Toyobo, Osaka, Japan).

5'- and 3'-untranslated region were isolated with the 5'-RACE System for Amplification of cDNA Ends, version 2.0 (Invitrogen) and the 3'-RACE System for Amplification of cDNA Ends (Invitrogen), respectively. The gene-specific primers used were as follows: 5'-RACE gene-specific primer-1, 5'-CTCTTGCTCAGCATGGAAGA-3'; 5'-RACE gene-specific primer-2, 5'-TCTTCTCTAGCAAGGCTGGT-3'; 3'-RACE gene-specific primer-1, 5'-TACCAACACCTGGGTGCATA-3'; 3'-RACE gene-specific primer-2, and 5'-TGCTCTGACAGCCTGAGAAT-3', as described in the legend to Fig. 6A.

Antibody Production-- Rabbit polyclonal antibody was raised against a synthetic oligopeptide corresponding to amino acid residues 595-604 of mIP₃R1 (LAEDTITALLHNNRK) (37). The peptide was coupled via an additional N-terminal cysteine to keyhole limpet hemocyanin with the cross-linking reagent m-maleimidobenzoyl-N-hydroxysuccinimide ester. Mice (C3HB6, female, 6 weeks) were used to raise monoclonal antibody against a synthetic peptide based on the sequence of amino acid residues 2684-2698 of starfish IP₃R (INLLSSPSIPQMGGP). The peptide was coupled via an additional N-terminal cysteine to mouse albumin with the cross-linking agent m-maleimidobenzoyl-N-hydroxysuccinimide ester. Splenocytes from immunized mice were fused to PAI (BALB/c mouse-derived myeloma) to produce a hybridoma. The hybridoma cells were screened by enzyme-linked immunosorbent assay, and positive cells were diluted to a single clone. Antibody classes were identified with a mouse monoclonal antibody isotyping kit RPN29 (Amersham Biosciences).

SDS-PAGE and Immunoblot Analysis-- A 30-fold volume of SDS sample buffer was added to pellets of defolliculated immature oocytes and mature eggs, and the specimens were heated for 15 min at 55 °C. Samples of the specimens were subjected to 5% SDS-PAGE according to Laemmli's method (38). After SDS-PAGE, the separated proteins in the gel were electrophoretically transferred to Immobilon-P (Millipore Corp., Bedford, MA). The membrane was incubated in 5% skim milk (Snow Brand, Sapporo, Japan) dissolved in PBS-Tween 20 (0.1%) for 2 h, and then incubated with monoclonal antibody (culture supernatant of hybridoma) overnight at 4 °C. After washing the membrane with PBS-Tween 20, it was exposed to the secondary antibody (1:1000; horseradish peroxidase-conjugated anti-mouse immunoglobulin from donkey; Amersham Biosciences), and the labeled bands were identified on x-ray film using enhanced chemiluminescence (ECL; Amersham Biosciences). The density of each band was analyzed by LAS-1000 (Fujifilm, Tokyo, Japan).

Immunocytochemistry and DAPI Staining-- Oocytes were treated with 0.02% Pronase (Kaken Seiyaku, Tokyo, Japan) at 20 °C in artificial sea water and washed several times with ice-cold Ca²⁺-free artificial sea water to remove the vitelline membrane. The oocytes were then fixed with 4% paraformaldehyde for 1 h at room temperature and permeabilized with PBS-Triton X-100 (0.1%). Sperm nuclei were stained by staining the inseminated eggs with 0.1 µg/ml DAPI for 30 min at room temperature and washed twice with PBS. The starfish IP₃R was immunostained by using the polyclonal antibody (1:300 dilution) described above as the primary antibody subsequently after blocking with 2% of normal goat serum (Vector Laboratories, Burlingame, CA). Rhodamine-conjugated anti-rabbit IgG (from goat) (Cappel, Organon Teknika Corp., West Chester, PA) was used as the secondary antibody. After washing with PBS, the specimens were mounted in Vectorshield (Vector Laboratories, Burlingame, CA). Specimens were observed by confocal microscopy, TCS NT (Leica Microsystems Heidelberg, Mannheim, Germany).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

IP₃ Sponge Inhibits IP₃-induced Ca²⁺ Release-- The "IP₃ sponge" is a truncated form of IP₃R that consists of the ligand binding domain fused to GST (Fig. 1). The IP₃ sponge has been found to exhibit high IP₃ binding affinity, approximately ~1000 times higher than that of endogenous IP₃R in mouse cerebellar microsomes (Fig. 1),² and thus it blocked IP₃-induced Ca²⁺ release (IICR) by absorbing IP₃. GST-m30 is a mutant of IP₃ sponge in which Lys-508 is substituted for Ala, and it displays low affinity for IP₃, approximately ~0.25 times lower than that of endogenous IP₃R in mouse cerebellar microsomes (Fig. 1).² Accordingly, GST and GST-m30 can be used as negative controls of IP₃ sponge.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Construction of IP3 sponge and its binding affinity to IP3. At the top is a schematic representation of the N-terminal region of mouse IP3R1, with the IP3 binding core (aa 226-578) indicated by a solid line. G224 (the IP3 sponge) is a protein in which GST has been fused to the truncated mutant of mIP3R1 (aa 224-604). GST-m30 is a K508A mutant of G224. GST was also used as a negative control. The binding affinity to IP3 was calculated by Scatchard plot analysis of inhibition of specific [3H]IP3 (9.6 nM) binding to GST-fused IP3 binding proteins by cold IP3 (28).

First, we examined whether IP₃ sponge inhibited IICR in starfish oocyte by injecting various concentrations of IP₃ into oocyte (Fig. 2). After injecting more than 10⁶ M IP₃ (10⁸ M IP₃ in cytoplasm, after 1% injection) into mature eggs that have been injected with 3 µM GST (equal to 0.1 mg/ml in cytoplasm), a Ca²⁺ peak was observed, and a fertilization envelope was formed. By contrast, after injecting less than 10⁶ M IP₃, no fertilization envelope formation or Ca²⁺ peak was observed in eggs that had been injected with 1.5 µM IP₃ sponge (equal to 0.1 mg/ml in cytoplasm). However, after injecting more than 10⁴ M IP₃ (10⁶ M IP₃ in cytoplasm, after 1% injection), both a calcium rise and a fertilization envelope were observed in eggs injected with IP₃ sponge. At 10⁵ M IP₃, an increase in Ca²⁺ was not observed in most cases, but in some cases, a Ca²⁺ peak was observed after a delay (about 5 min), or, in some cases, the baseline increased gradually and reached a plateau within 5 min (data not shown). In these cases, a fertilization envelope was observed after measurement of intracellular Ca²⁺. Thus, injection of a high dose of IP₃ overcame the inhibitory effects of IP₃ sponge, suggesting that the effect of IP₃ sponge was specific.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Effect of microinjection of oocytes with IP3 on calcium release in response to IP3. A, calcium release in response to IP3 injection in eggs injected with GST or IP3 sponge. Immature oocytes were injected with GST or IP3 sponge, and [Ca2+]i was measured at 1 h after 1-MA stimulation. The IP3 concentrations indicated were those in the pipettes from which injections of 1% of the cell volume were made. The traces show the fluorescence ratio (F360/F380) emitted by Fura-2 dextran. a-d, GST-injected eggs. Eggs a, b, and c formed fertilization envelopes; egg d did not. e-g, IP3 sponge-injected eggs. Egg e formed a fertilization envelope. B, calcium release by eggs in response to IP3 injection. The x axis indicates the IP3 concentrations in the pipettes, from which injections of 1% of the cell volume were made. The y axis indicates the (F360/F380)peak  (F360/F380)baseline at each concentration of IP3. Data are the means ± S.E. of eggs (n = 6) injected with GST () or IP3 sponge () (*, p < 0.1; **p < 0.05; t test).

Role of IP₃R during Oocyte Maturation and Fertilization-- Next, we used IP₃ sponge to investigate the physiological roles of IP₃R during starfish oocyte maturation and fertilization.

When immature oocytes injected with 1.5 µM IP₃ sponge were treated with 1-2 µM 1-MA to induce maturation, 85% (90 of 106) of oocytes underwent GVBD, similar to results with control oocytes injected with 3 µM GST (94%; 138 of 147) or 1.5 µM (equal to 0.1 mg/ml in cytoplasm) GST-m30 (96%; 67 of 70) did (Figs. 3 and 4). Thus, the IP₃ sponge did not inhibit 1-MA-induced oocyte maturation.

View larger version (116K): [in this window] [in a new window]   Fig. 3.   Microinjection of oocytes with IP3 sponge prevents formation of fertilization envelope. Oocytes injected with GST (A and C) or IP3 sponge (B and D) were treated with 1-MA and then inseminated. A and B, bright field images. Both oocytes injected with GST (A) and IP3 sponge (B) underwent GVBD. By contrast, oocytes injected with IP3 sponge lacked the ability to develop a fertilization envelope (B), although GST-injected eggs developed a normal fertilization envelope. C and D, the nuclei of the eggs stained with DAPI. A single sperm nucleus entered the GST-injected eggs (C), whereas several sperm nuclei entered IP3 sponge-injected eggs (D). Scale bar, 100 µm.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Microinjection of oocytes with IP3 sponge prevents formation of fertilization envelope: Statistical analysis. Oocytes were injected with IP3 sponge, treated with 1-MA, 2 µM, and inseminated. GST and GST-m30 were utilized as negative controls. The y axis shows the percentages of oocytes that underwent normal GVBD in response to 2 µM 1-MA (left) or the percentages of eggs that developed a normal fertilization envelope when inseminated (right). The number of oocytes that underwent normal GVBD was calculated 1 h after stimulation with 1-MA. The number of eggs that developed a normal fertilization envelope was counted 15 min after insemination. Each bar shows the results for oocytes injected with GST (open bar), GST-m30 (dotted bar), or IP3 sponge (filled bar).

After GVBD, these eggs injected with recombinant proteins were inseminated with sperm diluted to 10⁶. IP₃ sponge did not inhibit sperm penetration, since DAPI staining of these eggs showed that sperm nuclei had entered into the cells preinjected with IP₃ sponge (Fig. 3D). Polar body extrusion was also unaffected by the IP₃ sponge (Fig. 3B). However, the formation of a fertilization envelope was clearly inhibited in eggs injected with IP₃ sponge (13%; 12 of 90), whereas those injected with control proteins developed a fertilization envelope (GST: 94%, 129 of 138; GST-m30: 90%, 60/67) (Figs. 3 and 4).

Since the intracellular calcium rise during fertilization is known to be involved in the formation of the fertilization envelope, the IP₃ sponge probably inhibited IICR. Indeed, when the concentration of intracellular calcium ion was measured with Fura-2 dextran, the transient increase in Ca²⁺ during fertilization was greatly reduced of the eggs preinjected with IP₃ sponge (Fig. 5B), whereas GST had no such inhibitory effect (Fig. 5A). When treated with sperm diluted to 10⁴, a delay of the Ca²⁺ peak or a gradual base-line elevation was observed in the oocyte that had been injected with IP₃ sponge. These results may have occurred because the IP₃ sponge in oocytes was saturated with IP₃, thereby diminishing the inhibitory effect of IP₃ sponge.

View larger version (10K): [in this window] [in a new window]   Fig. 5.   Microinjection of oocyte with IP3 sponge inhibits elevation of intracellular calcium levels during fertilization. Oocytes were injected with GST (A) or IP3 sponge (B) and treated with 1-MA to induce maturation. After GVBD, the eggs were inseminated. The intracellular calcium level was monitored by using Fura-2 dextran. The x axis indicates the time course, and the y axis indicates the fluorescence ratio (F360/F380). The data shown represent the results of typical experiments repeated at least five times. The arrows indicate the time when sperm was added to the chamber. A, in the eggs injected with GST, the intracellular calcium level rose normally during fertilization. B, in the oocyte-injected IP3 sponge, the intracellular calcium elevation during fertilization was inhibited.

Primary Structure of Starfish IP₃R-- To investigate the roles of IP₃R in starfish oocyte further, we cloned the IP₃R from starfish ovary by RT-PCR. Degenerate oligonucleotides encoding the sequence conserved among all of the reported IP₃Rs of different species and types were used as the primers, and they are shown in Fig. 6A. Although we used degenerate primers encoding the sequence conserved among all of the reported IP₃Rs of different species and types, the sequences of the PCR products were identical to the sequence shown in Fig. 6B, and there were no products with different sequences. The 9374-nucleotide sequence of the cloned starfish IP₃R contained a single open reading frame of 8094 bp encoding a protein of 2698 amino acids (Fig. 6B), and the calculated molecular mass of starfish IP₃R was 308 kDa.

View larger version (107K): [in this window] [in a new window]   Fig. 6.   Primary structure of starfish IP3R. A, primers used for the amplification of starfish IP3R by RT-PCR. LBD, ligand binding domain; MD, modulatory domain; CHD, channel domain. B, predicted amino acid sequence (in single-letter code) of starfish IP3R (top) and alignment with hIP3R1 (SI+, SII, SIII+) is shown. C, phylogenic tree of IP3Rs generated using the ClustalW program. D, Kyte-Doolittle hydrophobicity profile of starfish IP3R generated with a window size of 10 amino acids.

Sequence alignment of starfish IP₃R with known full-length human IP₃R sequences showed that starfish IP₃R most closely resembled IP₃R1. The amino acid sequence of starfish IP₃R was 68% identical to human IP₃R1 (hIP₃R1) (39), 62% identical to hIP₃R2 (40), and 58% identical to hIP₃R3 (40). When compared with the IP₃R of other species, starfish IP₃R was found to be 68% identical to mIP₃R1 (41), 67% identical to Xenopus IP₃R (42), 58% identical to Drosophila IP₃R (43), and 41% identical to C. elegans IP₃R (44) (Fig. 6C).

The structure of IP₃R can be divided into three functionally different domains: an NH₂-terminal ligand-binding domain, a long modulatory domain presumed to span the cytoplasm, and a transmembrane domain near the C terminus.

Ligand Binding Domain-- The N-terminal amino acid residues (aa 1-604 of mIP₃R1) reported as the IP₃-binding domain within the large cytoplasmic portion of the IP₃Rs show significant sequence similarities with the different species and receptor types. This region of starfish IP₃R (aa 1-593) also displays high similarity to human IP₃R (75% to type 1, 70% to type 2, 67% to type 3). It is especially noteworthy that all three basic amino acid residues (Arg-265, Lys-508, and Arg-511) in mIP₃R1 that are known to have a crucial role in IP₃ binding are conserved in starfish IP₃R residues (Arg-265, Lys-497, and Arg-500 of starfish IP₃R) (33). An alternative splicing region in the ligand binding domain, the SI region, has been reported in mouse, rat, and human IP₃R1 (45). However, all of the reported IP₃Rs except for the ones from these animals lack this region, suggesting that IP₃Rs are generally spliced at SI. Starfish IP₃R, shown in Fig. 6, also lacks this region.

Modulatory Domain-- The modulatory domain of starfish IP₃R (aa 594-2224) shows 67% similarity to hIP₃R1, 60% to hIP₃R2, and 54% to hIP₃R3, and it contains several sites that are thought to modulate IP₃ function in channel opening, including cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase, and ATP. A PKA phosphorylation site is present at positions 1833-1836. This site can also be phosphorylated by cGMP-dependent protein kinase. Both kinases are known to modulate the function of IP₃R (46-52). ATP is known to significantly potentiate in IP₃Rs from cerebellum (53-55). Two consensus Gly-rich sequences (GXGXXG) for nucleotide binding were found in the mIP₃R1 (aa 1773-1778 (or 1775-1780)) and aa 2016-2021), but only one of them, corresponding to aa 2016-2021 in the mIP₃R1, was conserved in the sequence of starfish IP₃R (aa 1958-1964 in starfish IP₃R). Several consensus sites of protein kinase C have also been found throughout the sequence of starfish IP₃R. Thus, starfish IP₃R can be regulated by these factors described above.

Putative Channel Domain-- The putative channel domain of starfish IP₃R (aa 2225-2698) showed 59% similarity to hIP₃R1, 58% to hIP₃R2, and 55% to hIP₃R3. Hydropathy profiles (Fig. 6D) suggested that starfish IP₃R contains six potential membrane-spanning segments, M1-M6, near the C terminus. These six hydrophobic regions were thought to form part of the channel region (56). In the putative large luminal loop regions, the putative pore between M5 and M6, hIP₃R1 and IP₃R2 have two potential N-glycosylation sites, whereas hIP₃R3 has only one. However, neither of these N-glycosylation sites has been conserved in starfish IP₃R. The similarities between sequences in this region described above were lower than in other parts of the IP₃R, because the similarity between the regions between M5 and M6 was extremely low. There was absolutely no sequence similarity between the 10 C-terminal amino acids of starfish IP₃R and mIP₃R1, and as a result, a monoclonal antibody, 18A10, which recognizes the C terminus of mIP₃R1, does not cross-react with starfish IP₃R.

Expression of IP₃R during Oocyte Maturation-- From quantitative injections of IP₃, it was found that 100-fold less IP₃ was sufficient to release the same amount of Ca²⁺ in mature starfish eggs than in immature oocytes (16). However, the mechanism of increase in sensitivity to IP₃ has remained elusive. To investigate whether the sensitization of starfish oocytes to IP₃ is due to the elevation of the expression level of IP₃R, we examined the expression level of IP₃R by immunoblot analysis.

We raised a polyclonal antibody against a synthetic oligopeptide having the highly conserved peptide sequence of the IP₃ binding domain. This region was also conserved in starfish IP₃R. Immunoblot analysis using the antibody yielded a single band with a smaller molecular weight than that of mIP₃R1 (Fig. 7A). We also raised a monoclonal antibody against the 15-amino acid C-terminal sequence of starfish IP₃R, and it detected a single band having exactly the same molecular size as obtained with the polyclonal antibody (Fig. 7A). The monoclonal antibody did not recognize mIP₃R1 (data not shown). These findings are consistent with the fact that there is no similarity between mIP₃R1 and starfish IP₃R in this region. Preabsorption of antibody with antigen diminished the band, suggesting that this antibody specifically recognized the starfish IP₃R.

View larger version (12K): [in this window] [in a new window]   Fig. 7.   Immunoblot analysis of starfish IP3R during oocyte maturation. A, immunoblot of starfish IP3R. C, the microsomal fraction of mouse cerebellum; S, starfish oocytes. The microsomal fraction of mouse cerebellum was used as the positive control (lane 1). The IP3R was probed with polyclonal antibody that recognizes the ligand binding domain of IP3R. The size of the starfish IP3R was smaller than that of the mouse one (lane 2). A band of the same size was detected by using a monoclonal antibody that recognizes the C terminus of starfish IP3R (lane 3). B, the level of expression of IP3R during oocyte maturation was detected by using polyclonal antibody (a) and monoclonal antibody (b). The number above each lane indicates the time after 1-MA treatment when each sample was collected and treated with SDS-PAGE sample buffer. In this experiment, GVBD occurred 15 min after the application of 1-MA. C, densitometric analysis showing the relative intensity of the band corresponding to starfish IP3R. The intensities are plotted as multiples of intensity relative to immature oocytes before stimulation with 1-MA. The density of each band was analyzed by using LAS-1000.

Next we used these antibodies to perform immunoblot analysis as a means of determining the level of expression of IP₃R during oocyte maturation. Oocytes were collected at various times after 1-MA treatment and treated with SDS-PAGE sample buffer. Samples loaded in each lane were prepared so that they contained approximately the same numbers of oocytes. Fig. 7B shows that the level of expression of IP₃R during oocyte maturation. Densitometric analysis confirmed that the level of expression of IP₃R did not change significantly during oocyte maturation (Fig. 7C).

Localization of IP₃R during Oocyte Maturation-- We then investigated the distribution of IP₃R during oocyte maturation. Immunocytochemistry was performed with the polyclonal antibody against the IP₃ binding domain, and signals were detected by confocal microscopy. In immature oocytes, IP₃R distributed throughout the cytoplasm but not in the GV (Fig. 8A). Localization of the IP₃R was then examined at various stages of oocyte maturation. When the GV was disintegrating 20 min after 1-MA treatment, a large patch of signal was observed at the site where the GV had been (Fig. 8B). After GVBD (40 min after 1-MA treatment), the signal patch dispersed, and signals were evenly distributed throughout the egg (Fig. 8C). These changes of localization were observed using more than 100 eggs from several animals. The signals were diminished by preincubation with primary antibody to the synthetic peptide used as antigen (Fig. 8D), suggesting that the signals were specific to the IP₃R.

View larger version (81K): [in this window] [in a new window]   Fig. 8.   Immunocytochemistry of starfish IP3R during oocyte maturation. Starfish IP3R was immunocytochemically characterized by confocal microscopy. Polyclonal antibody was used as the primary antibody, and rhodamine-conjugated IgG (anti-rabbit IgG) was used as the secondary antibody. A, an immature oocyte. Signals are distributed throughout the cytoplasm and absent in the GV. B, an oocyte undergoing GVBD (20 min after application of 1-MA). The signals are concentrated in the area where a GV had been present in the immature oocyte. C, a mature egg. Signals are redistributed throughout the cytoplasm. D, no signals were observed when primary antibody was preincubated with antigen. Scale bar, 100 µm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Microinjection of starfish eggs with IP₃ sponge did not inhibit either sperm penetration during fertilization or polar body extrusion, but it did block the increase in [Ca²⁺]_i and formation of the fertilization envelope. Since IP₃ sponge exhibited high affinity for IP₃, the inhibition of the calcium rise is probably due to the IP₃ sponge absorbing IP₃. Indeed, more than 10 times as much IP₃ was required to achieve the same calcium release in a IP₃ sponge-injected egg as in a control egg. These results clearly indicate that IICR is the major contributor to the increase in [Ca²⁺]_i during the fertilization of starfish eggs. Oocyte maturation, however, was not inhibited by the injection of IP₃ sponge, indicating that IICR is not essential for meiosis reinitiation, although a transient Ca²⁺ increase has been reported to occur in response to 1-MA treatment (57, 58).

The contribution of IICR during fertilization of hamster eggs has also been clearly demonstrated by using 18A10, the specific antibody against the IP₃R1 (28). However, the antibody did not recognize starfish IP₃R, and it cannot inhibit IICR completely when the cells of interest express multiple types of IP₃R. IP₃ sponge can be applied to cells of various species, including those whose IP₃R has not yet been identified and to the cells in which multiple types of IP₃R are expressed. GST or GST-m30 can be used as a negative control for IP₃ sponge. Thus, the IP₃ sponge may open the way to understanding the mechanisms of calcium dynamics in various cells from different species.

We cloned IP₃R from starfish ovary by RT-PCR. Of all of the types of human IP₃R known, starfish IP₃R was closest to IP₃R1. Although the degenerate primers used in this study were designed within the regions that are highly conserved among all types of IP₃R, all of the clones amplified by RT-PCR were identical. These findings suggest that the IP₃R shown in Fig. 6 is dominantly expressed in starfish ovary, and starfish may have only one type of IP₃R, the same as Drosophila. None of the products had sequences similar to those of IP₃R2 or IP₃R3. The PCR product shown in Fig. 6, however, cannot be classified simply as IP₃R1, because some of the residues in the sequence of the starfish IP₃R are not conserved in IP₃R1 but are conserved in hIP₃R2 or IP₃R3. Therefore, the sequence of the starfish IP₃R should be interpreted as a mixture of the three types of mammalian IP₃R rather than as IP₃R1.

The sensitivity of starfish oocytes to IP₃ is known to increase during maturation (16). The results presented here show that the level of expression of IP₃R remained unchanged throughout starfish oocyte maturation. Therefore, the sensitization of IICR during starfish oocyte maturation was not due to an increase in level of expression of IP₃R. A similar change has been seen during mouse (59) and Xenopus (60) oocyte maturation, but it has been reported that the number of IP₃R increases during oocyte maturation in these species. However, the increases in IP₃R number reported in these studies are too small (1.2× in Xenopus oocyte (60) and 1.8× in mouse oocytes (59)) to account for the dramatic change of sensitization of oocyte to IP₃ during oocyte maturation. Therefore, it may be that in all of these species, modulation of the IP₃ receptor is more important than receptor number in explaining the IP₃ sensitivity change. Seen from this point of view, the starfish is a particularly valuable "model system" to study a general phenomenon. One of the possibilities is that starfish IP₃R is modulated during maturation. Indeed, there are many consensus sequences of various kinases or substances, including PKA, cGMP-dependent protein kinase, protein kinase C, and ATP. Interestingly, cAMP decreases during starfish oocyte maturation (61), and there is a putative PKA phosphorylation site in the sequence of starfish IP₃R. Thus, PKA-dependent phosphorylation of IP₃R may modulate the function of IP₃R in immature oocytes. However, the effect of PKA on IP₃R is controversial. By the study using purified mIP₃R1 reconstituted into lipid vesicles, PKA phosphorylation enhances the function of IP₃R1 (46). There are several lines of evidence that phosphorylation by PKA increases IICR in platelets (47), hepatocytes (48), and cerebellar membranes (49). By contrast, Supattapone et al. (50) and Quinton and Dean (51) reported that phosphorylation by PKA of cerebellar membranes or platelet membranes substantially reduced the potency of IP₃ in releasing Ca²⁺. Therefore, the real nature of IP₃R regulation by PKA has remained elusive in starfish oocyte. It is also possible that other kinases such as protein kinase C (62) and protein kinase C-related kinase 2 (63) phosphorylate starfish IP₃R. Such modifications may be responsible for the sensitization of IP₃R during oocyte maturation. Because the starfish oocyte undergoes a rapid and large change in sensitivity to IICR in response to application of a hormone, it provides an excellent system for future studies of IP₃ receptor modulation.

In immature oocytes, IP₃R signals were distributed throughout the cytoplasm, but none were detected in the GV (Fig. 8A). When the GV was disintegrating after 1-MA treatment, the IP₃Rs were concentrated at the site where the GV had been located (Fig. 8B), whereas after GVBD, they were evenly distributed throughout the egg (Fig. 8C). A structural change in the endoplasmic reticulum, which presumably contains an IP₃-sensitive calcium store, has been observed during starfish oocyte maturation and fertilization by using DiI (64) and a green fluorescence protein targeted to the lumen of the endoplasmic reticulum (65, 66). In addition, translocation of IP₃Rs from the perinuclear region to the cortex has been reported in studies in mouse (59) and Xenopus (60) oocyte. These structural changes in the endoplasmic reticulum or in the distribution of IP₃Rs may contribute to the sensitization of IICR during oocyte maturation.

	ACKNOWLEDGEMENTS

We thank Drs. T. Inoue, T. Michikawa, M. Hattori (University of Tokyo), A. Mizutani (RIKEN), and J. Hirota (Rockefeller University) for valuable discussions, Dr. M. Hino-Yamamoto (RIKEN) for cloning starfish IP₃R, Dr. M. Hoshi (Keio University) for the monoclonal antibody, and M. Iwai (University of Tokyo) for excellent technical support.

	FOOTNOTES

^* This work was supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AB071372.

^¶ To whom correspondence should be addressed. Tel.: 81-48-467-9745; Fax: 81-48-467-9744; E-mail: hiwasaki@ims.u-tokyo.ac.jp.

Published, JBC Papers in Press, October 30, 2001, DOI 10.1074/jbc.M108839200

² T. Uchiyama, F. Yoshikawa, A. Hishida, T. Furuichi, and K. Mikoshiba, submitted for publication.

	ABBREVIATIONS

The abbreviations used are: IP₃R, inositol 1,4,5-trisphosphate receptor; IP₃, inositol 1,4,5-trisphosphate; IICR, IP₃-induced Ca²⁺ release; GV, germinal vesicle; GVBD, germinal vesicle breakdown; 1-MA, 1-methyladenine; GST, glutathione S-transferase; RyR, ryanodine receptor; mIP₃R1, mouse IP₃R1; PBS, phosphate-buffered saline; DAPI, 4',6-diamidino-2-phenylindole; hIP3R1, -2, and -3, human IP₃R1, -2, and -3, respectively; PKA, cAMP-dependent protein kinase; RT, reverse transcription; RACE, rapid amplification of cDNA ends; aa, amino acids.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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