1 Department of Environmental Biology and Chemistry, Faculty of Science, Toyama University, 3190 Gofuku, Toyama 930-8555, Japan
2 Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, 3-7-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
3 Division of Biological Sciences, Graduate School of Science, Hokkaido University, Kita-ku, Kita 10-jyo, Nishi 8-chome, Sapporo 060-0810, Japan
* Present address: Department of Pharmacology, National Defense Medical College, Namiki, Tokorozawa 359-8513, Japan
Author for correspondence (e-mail: kuroda{at}sci.toyama-u.ac.jp)
Accepted July 27, 2001
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SUMMARY |
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Key words: cGMP, cADP-ribose, IP3, Ca2+ transient, Fertilization, Sea urchin eggs
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INTRODUCTION |
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Partly because sea urchin eggs do contain both the RyR (McPherson et al., 1992) and the IP3R (Parys et al., 1994), it was supposed that both must be important in fertilization, perhaps being independently responsible for the Ca2+ transient (Galione et al., 1993a; Lee et al., 1993). Restricting evidence, however, has come from several experiments that suggest a primary role for only the IP3 system. First, in eggs preloaded with heparin, a potent competitive inhibitor of the IP3-induced Ca2+ release (Ghosh et al., 1988), the fertilization-induced Ca2+ transient was delayed and reduced in both velocity and amplitude (Mohri et al., 1995). Furthermore, intracellular injection of the tandem SH2 domains of bovine PLC produced a concentration-dependent inhibition of the Ca2+ release (Carroll et al., 1999; Shearer et al., 1999). As for the RyR system, inhibition of either PKG or ADP-ribosyl cyclase failed to block the Ca2+ transient in heparin-loaded eggs (Lee et al., 1996). These discoveries were interpreted to mean that the IP3 system must be the primary mechanism underlying the Ca2+ transient at fertilization of sea urchin eggs. However, another line of evidence has recently shown that nitric oxide (NO) was produced in sperm during their acrosome reaction and in eggs during fertilization, and intracellular injection of NO synthase caused the egg activation (Kuo et al., 2000). NO is expected to stimulate the production of cGMP by activating soluble guanylate cyclase (Garthewaite, 1991), and then cGMP is expected to stimulate the production of cADPR (Galione et al., 1993b).
Thus, the roles of the IP3 system and RyR pathway in the Ca2+ transient, especially in its initiation, are still controversial. As the signal transduction process at fertilization must have a spatial structure, the simultaneous measurements of cGMP, cADPR and IP3 with micrometer spatial resolution inside the eggs are the best way for precise analysis of regulatory pathways. However, a detailed description of the time courses of changes in cGMP, cADPR and IP3 contents compared with that of [Ca2+]i during fertilization would be still needed for revealing the initiating event of the Ca2+ transient at fertilization, although biochemical analysis on the contents of second messenger candidates using population of eggs cannot give any spatial information. No one has simultaneously measured the contents of these second messenger candidates in the eggs during fertilization, except for the observations that the cGMP concentration in eggs was higher 20-60 seconds after fertilization than it was before insemination (Ciapa and Epel, 1996), and that the IP3 content increased slowly after insemination relative to the [Ca2+]i rise (Lee and Shen, 1998). In the experiments described below, we have carried out these critical measurements, assaying cGMP, cADPR and IP3 simultaneously throughout the first 150 seconds after fertilization under the presence or absence of a specific inhibitor of soluble guanylate cyclase, 6-anilino-5, 8-quinolinedione (LY83583) (Pandol and Schoeffield-Payne, 1990) and compared these time courses with those of the [Ca2+]i change to reveal the sequential relationship among them.
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MATERIALS AND METHODS |
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Assays for cGMP, IP3 and cADPR
Each egg suspension was mixed with an equal volume of sperm suspension, and when necessary, eggs was preincubated in stdASW containing 200 µM LY83583 (Research Biochemicals International, Natick, MA) for 15-30 minutes at 20°C (A. crassispina) or
17°C (H. pulcherrimus) before insemination. For assays of ionophore-triggered eggs, each suspension was diluted with an equal volume of ASW containing 20 µg/ml of a Ca2+ ionophore, A23187 (Wako Pure Chemicals, Osaka, Japan). At appropriate intervals thereafter, aliquots (0.2-0.5 ml for experiments on cGMP and IP3; 10 ml for experiments on all the three) of the egg suspension were withdrawn and injected into one-fifth the volume of 21% (w/v) ice-cold perchloric acid. Immediately after each series of sampling, the appearance of the fertilization envelope was examined, and the batch to be processed further was selected only if the envelope formation was greater than 95%, except for the experiments in 0CaASW containing A23187. The stopped mixture was kept on ice for 30 minutes with occasional mixing, and then centrifuged at >104 g and 4°C for 15 minutes. The precipitate (acid-insoluble fraction) from each aliquot was assayed for the total protein via the Coomassie Blue reaction (Coomassie Protein Assay Reagent, Pierce Chemical, Rockford, IL). From this measurement, the number of eggs in each aliquot was calculated, using the value for the protein content of single egg cells determined on the same batch of eggs (0.02-0.04 µg/egg). The supernatant fluid (acid-soluble fraction) was neutralized with a mixture of 2.4 M KOH, 240 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (Hepes), and 6 or 40 mM ethylene glycol bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), and the precipitates of potassium perchlorate were removed by centrifugation. Aliquots of this neutralized acid-soluble fraction were used to assay cGMP and IP3.
IP3 levels were determined by isotope dilution, using a Biotrak assay system TRK 1000 (Amersham Pharmacia Biotech, Buckinghamshire, UK). cGMP levels were determined by enzyme immunoassay via a Biotrak RPN 226 kit (Amersham Pharmacia Biotech). In a few experiments, cGMP was determined by radioimmunoassay using a Biotrak TRK 500 kit (Amersham Pharmacia Biotech). Because radioassays routinely gave values 4.5±0.8 times (mean±s.d., n=21) as large as the cGMP values obtained by enzyme assays on the same samples, we normalized the values obtained by radioassays to the values calculated by the enzyme assays, in order to avoid the confusion between both assays. Because salt concentrations in the IP3 assay and the presence or absence of A23187 in the cGMP radioassay significantly affected the assay values, standard solutions of IP3 and cGMP were prepared in the same media as the experimental samples, which were processed in parallel with the experimental samples but without eggs or sperm.
For cADPR measurements, large amounts of gametes were required. Our measurements of cADPR, which required to mix large volumes of egg and sperm suspension, lead to uncertainty in synchrony of fertilization, especially in A. crassispina. cADPR in each acid-soluble fraction was concentrated by adsorbing to acid-washed charcoal (Norit X Plus, American Norit Company, Amersfoort, The Netherlands), and eluting from charcoal with pyridine water (Tsuboi and Price, 1959). By this method, more than 60% of cADPR was recovered except samples containing sperm alone (45%), as checked in preliminary experiments using [32P]cADPR. Each eluate was subsequently lyophilized, then dissolved in ultra-pure water, and incubated with bovine alkaline phosphatase (Type II-G, Sigma Chemical Co., St. Louis, MO) and venom phosphodiesterase (Crotalus adamanteus venom, Worthington Biochemical, NJ), in order to destroy the nucleotides that crossreacted with the anti-cADPR antiserum. cADPR was then quantified by radioimmunoassay with anti-cADPR antiserum, as described previously (Takahashi et al., 1995). By ratioing the total amounts of cGMP, cADPR and IP3 in each experimental sample to the total egg number, the individual contents per egg were obtained. However, the contents of each compound per egg were probably not absolute values. Averaged intracellular concentrations of the individuals can be obtained by dividing the contents per egg by the egg volume (4x1010 l for A. crassispina or 5x1010 l for H. pulcherrimus) estimated from the measured diameter.
Guanylate cyclase assay
The method of Harumi et al. (Harumi et al., 1992) was used with slight modifications. Briefly, dry sperm and wet-packed dejellied unfertilized eggs were homogenized in a medium containing 1% (w/v) Nonidet-P40 (BDH Laboratory, Poole, UK), 0.5 M glycerol, 1 mM EGTA, 1 mM dithiothreitol (DTT; Wako), 0.5 mM phenylmethylsulfonyl fluoride (PMSF; Sigma), 10 µM leupeptin (Peptide Institute, Osaka, Japan), 10 µM pepstatin A (Peptide Institute) and 20 mM Hepes buffered to pH 7.0 with Tris, with 10 strokes of a glass-Teflon homogenizer on ice, and then stirred for 60 minutes at 0°C. For fertilized eggs, the dejellied eggs were inseminated in the same way as for the experiments on cGMP, cADPR and IP3 measurements, and after 30 seconds the suspension was poured into a fivefold volume of ice-cold stdASW, and centrifuged immediately at 50 g and 4°C for 20 seconds. The resultant pellet was homogenized as for sperm and unfertilized eggs.
The assay medium contains 0.5% Nonidet-P40, 0.25 M glycerol, 5 mg/ml bovine serum albumin (fraction V, Sigma), 4 mM MnCl2, 15 mM sodium phosphocreatine (Sigma), 30 units/ml creatine phosphokinase (Sigma), 1 mM 3-isobutyl-1-methylxanthine (Research Biochemicals International, Natick, MA), 1 mM DTT, 0.5 mM PMSF, 10 µM leupeptin, 10 µM pepstatin A and 40 mM Hepes buffered to pH 7.5 with Tris, with or without 200 µM LY83583. The reaction was started by the addition of 5'-GTP (Sigma), incubated at 20°C for 10 minutes, and then product cGMP was quantified by radioimmunoassay in the same way as for the experiments on cGMP during fertilization.
DNA sequencing
Total RNA, which had been extracted from the unfertilized eggs of A. crassispina according to Seimiya et al. (Seimiya et al., 1997), was used as the template to synthesize the first strand of cDNA using an oligo (dT) primer according to the manufacturers protocol (SuperScript Preamplification System for First Strand cDNA Synthesis, Gibco BRL, Grand Island, NY). The cDNA fragments encoding guanylate cyclase(s) were amplified using PCR with the following degenerate oligonucleotide primers: P2, 5'-GAYATHGTNGGNTTYAC-3'; P6, 5'-GTRTTNACNGTRTCNCC-3'; and P7, 5'-ARRCARTANCKNGGCAT-3', which were synthesized based on the amino acid sequences of three conserved regions (DIVGFT, DTVNTA and MPRYCL) in known membrane guanylate cyclases as described previously (Seimiya et al., 1997). The cDNA fragments in pBluescript vectors (Stratagene, La Jolla, CA) were used directly to determine the nucleotide sequences as a template by the dideoxy chain termination procedure (Sanger et al., 1977) with an Applied Biosystems 373A sequencer, and analyzed on a DNASIS software (Hitachi Software Engineering, Tokyo, Japan).
Microinjection and measurements of [Ca2+]i
[Ca2+]i was measured with a Ca2+-sensitive fluorescent dye, indo-1 (Dojindo, Kumamoto, Japan). Each egg cell was fastened to a poly-L-lysine (Mr=1-4x103; Sigma) coated glass coverslip at the bottom of 150 µl of stdASW in a Lucite-frame chamber, mounted on the stage of a TMD epifluorescence microscope (Nikon, Tokyo, Japan). The dye was pressure-injected into eggs at a final concentration of 100 µM, from a 10-mM stock solution containing 100 mM potassium aspartate and 10 mM Hepes, titrated to pH 7.0 with Tris, according to the method of Hiramoto (Hiramoto, 1974). Then, 150 µl 5000-fold diluted sperm suspension or 20 µg/ml Ca2+-ionophore, bromo-A23187 (Molecular Probes, Eugene, OR), was added into the chamber. Experiments on H. pulcherrimus were performed at 16-20°C and on A. crassispina at 20-25°C. For measuring the fluorescence of indo-1, selected wave lengths were 355 nm (bandpass) at the excitation filter, 380 nm (cutoff) at the dichroic mirror, and simultaneously 405 nm (bandpass) and 485 nm (bandpass) at the emission detectors, which were Hamamatsu R647-01 photomultipliers (Hamamatsu Photonics, Hamamatsu, Japan). Fluorescence from the whole egg was measured, and [Ca2+]i was calculated from the ratio of 405 nm fluorescence intensity to the corresponding 485 nm intensity (Grynkiewicz et al., 1985).
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RESULTS |
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Demonstration of IP3 production under artificial [Ca2+]i elevation
The temporal relationship between IP3 and [Ca2+]i (Fig. 1, Fig. 2) suggested the large increase of IP3 to be secondary to the [Ca2+]i increase rather than causal to it. Several reports (Ciapa and Whitaker, 1986; Swann and Whitaker, 1986; Ciapa et al., 1992) have already presented the hypothesis that Ca2+ causes the IP3 production. Therefore, that inference was examined by determining IP3 and cGMP contents in unfertilized H. pulcherrimus eggs in which [Ca2+]i had been elevated artificially by a Ca2+ ionophore, A23187. Fig. 4 displays the resultant rise in [Ca2+]i, the corresponding effect on IP3 and, as a control, the lack of effect on cGMP. [Ca2+]i began to increase a couple of seconds after addition of A23187 into the medium, but the rate and extent of rise depended on the extracellular Ca2+ ion concentration. Clearly, the artificial elevation of [Ca2+]i was accompanied by the production of a large amount of IP3 for eggs in stdASW (Fig. 4A), although for eggs in 0CaASW, A23187 caused only a very small increase of [Ca2+]i and the production of a small amount of IP3 (Fig. 4B). In P. depressus, also, IP3 production was induced with a longer lag period (data not shown).
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cGMP is produced by egg soluble guanylate cyclase
Fig. 1, Fig. 2 provide no information on the source of cGMP for the first rise. That source could be either the egg itself or the sperms acrosomal reaction, as sperm-activating peptides such as speract and resact residing in the jelly coat of eggs are known to induce the cGMP production in sperm (Hansbrough and Garbers, 1981). In order to test the latter possibility, we measured cGMP production by sperm inseminated into a suspension of egg jelly coats at the same density as into egg suspensions. The result showed that in both species of A. crassispina and H. pulcherrimus, cGMP produced by sperm was below the limit of detection (data not shown). It indicates that the initial rise in cGMP normally observed in sea urchin eggs at fertilization (Fig. 1, Fig. 2) represents an egg-cell reaction rather than a sperm reaction.
In order to determine which of the two main types of guanylate cyclase, soluble or membrane bound, was functional immediately after fertilization, effects of a specific inhibitor of soluble guanylate cyclase, LY83583 (Pandol and Schoeffield-Payne, 1990), on guanylate cyclase activity was examined on separate homogenates of sperm, unfertilized eggs and fertilized eggs of P. depressus. The results are summarized in Table 1. Sea urchin sperm are known to possess abundant membrane-bound guanylate cyclase (Radany et al., 1983; Harumi et al., 1992), and in these experiments (Table 1) the guanylate cyclase activity of sperm homogenates was indeed high, and proved almost insensitive to LY83583. Homogenates of unfertilized eggs, on the other hand, showed a very low guanylate cyclase activity, and equivalent fertilized egg homogenates displayed a reliable level about 10-fold higher that of unfertilized eggs. For both types of egg homogenates, the guanylate cyclase activity was 65% inhibited by 200 µM LY83583. The possibility that contaminating sperm enzyme might vitiate the measurements on fertilized eggs is unlikely, because the sperm guanylate cyclase activity is insensitive to LY83583. These results suggest that soluble guanylate cyclase within unfertilized eggs is probably activated upon fertilization. The existence of soluble guanylate cyclase within the eggs before fertilization is supported by the finding that the deduced amino acid sequence for one of cDNA fragments obtained from unfertilized A. crassispina eggs was highly homologous to the amino acid sequence of the catalytic domain of the soluble guanylate cyclase of rat kidney (Yuen et al., 1990) as shown in Fig. 5.
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DISCUSSION |
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A small increase of IP3 early in fertilization and the IP3 production induced by the [Ca2+]i elevation
One of the salient outcomes of the experiments reported above is the finding that the IP3 peak is reached considerably later than the [Ca2+]i peak (Fig. 1, Fig. 2). This large increase of IP3 after the Ca2+ peak (Fig. 1, Fig. 2) can be interpreted to indicate that the IP3 production is secondary to the [Ca2+]i increase rather than causal to it. Supporting this argument, the artificial [Ca2+]i elevation by A23187 actually induced IP3 production (Fig. 4), and the artificial diminution of Ca2+ transient by LY83583 accompanied a reduction of IP3 production (Fig. 8, Fig. 9).
However, the closer inspection of time courses of IP3 changes in the early phase of fertilization (Fig. 1, Fig. 2, Fig. 9) proves a small increase of IP3 (0.20.3 µM) before the onset of the explosive [Ca2+]i increase in both species of A. crassispina and H. pulcherrimus. Such a concentration of IP3 is enough to induce a [Ca2+]i increase comparable with the sperm-induced Ca2+ transient (Whalley et al., 1992) and enough to induce an elevation of the fertilization envelope (Whitaker and Irvine, 1984). We also confirmed those. Fig. 3 clearly shows that the early increase of IP3 was comparable with that of cGMP, although the onset of its increase was slightly later than cGMP. Thus, this early small increase in IP3 could induce the Ca2+ transients at fertilization, while the significance of the large increase of IP3 after the Ca2+ peak, in the egg activation, is unclear at present. Lee and Shen (Lee and Shen, 1998) have reported that the inhibition of IP3 production with an inhibitor against phospholipase C, U73122, completely abolished the Ca2+ transient. However, they later reported that U73122 blocked cGMP-induced Ca2+ release in sea urchin eggs (Lee et al., 1998). De Nadai et al. (De Nadai et al., 1998) have shown the presence of a Ca2+-sensitive phospholipase activity and PLC protein in sea urchin eggs. Carroll et al. (Carroll et al., 1999) and Shearer et al. (Shearer et al., 1999) have shown that the microinjection of the tandem SH2 domains of bovine PLC
, which is linked to a receptor or cytoplasmic tyrosine kinase, inhibits the Ca2+ release after fertilization of sea urchin eggs. These results could indicate that the early increase in IP3 results from PLC action and results in release of Ca2+ from the ER. IP3 production induced by the artificial [Ca2+]i elevation by A23187 in unfertilized H. pulcherrimus eggs (Fig. 4) is also probably due to this PLC action. In Xenopus laevis oocytes, however, the level of [Ca2+]i does not affect the IP3 production at fertilization (Stith et al., 1994), suggesting a species difference in the mechanism of the signal transduction at fertilization.
The RyR pathway involving cGMP and cADPR interacts with the IP3 system in the fertilization-induced Ca2+ transients
As for the RyR pathway via cGMP and cADPR, on the other hand, the above-mentioned experiments showed that the fertilization-induced increase in cGMP preceded the explosive [Ca2+]i rise in both species of A. crassispina and H. pulcherrimus (Fig. 1, Fig. 2, Fig. 3), that cADPR increases and decreases in parallel with cGMP at least during the first minute (Fig. 6), and that inhibition of cGMP production had a large effect on net synthesis of cADPR (Fig. 8), the IP3 production (Fig. 8, Fig. 9) and the [Ca2+]i increase (Fig. 7, Fig. 9). These findings suggest that cGMP could play a key role in the Ca2+ transients at fertilization of sea urchin eggs. cADPR also seems to contribute to the fertilization-induced Ca2+ transients, because cADPR increased in fertilized eggs to the averaged concentration of 35 nM in H. pulcherrimus and
80 nM in A. crassispina, estimated from the recovery of charcoal procedure (
60%) and the cell volume. Several investigators have reported that 10-150 nM cADPR induces the Ca2+ release in Lytechinus pictus eggs (Dargie et al., 1990; Galione et al., 1993a; Lee et al., 1993; Buck et al., 1994). Dargie et al. (Dargie et al., 1990) have also mentioned that cADPR induced the cortical reaction above 45 nM. Incidentally, the increase in cADPR at fertilization is remarkable in being one of a very few examples to show net synthesis of cADPR in response to extracellular stimuli (Clementi et al., 1996; Takasawa et al., 1998; Guse et al., 1999). However, cGMP injection will not induce the [Ca2+]i increase in the unfertilized eggs when injected at the peak concentration (
25 nM by enzyme assay and
110 nM by radioassay), which was estimated from the values, 9.4±1.5 amoles/egg (n=3) in A. crassispina or 12±1 amoles/egg (n=3) in H. pulcherrimus. Whalley et al. (Whalley et al., 1992) have reported that the final concentrations of more than 5 µM are required for cGMP to induce the Ca2+ transients, and we could barely induce at 10 µM. The above cGMP concentrations are averaged for the entire cytoplasm of the egg. Therefore, if cGMP were to play some role in the initiation of Ca2+ release from the ER, cGMP production is required to be localized strictly perhaps around the sperm entry site. Such a possibility is suggested by the recent results of Kuo et al. (Kuo et al., 2000), who showed that NO was produced in sperm and eggs during fertilization, that intracellular injection of NO synthase caused the egg activation, and that NO synthase accounted for 0.4% of sperm soluble proteins. As NO is expected to activate soluble guanylate cyclase in eggs (Garthewaite, 1991), the cGMP production is likely to begin around the sperm entry site, which in turn could induce the cADPR production so that Ca2+ is finally released from the ER, as suggested by Willmott et al. (Willmott et al., 1996). Released Ca2+ could stimulate the Ca2+-sensitive PLC
, which in turn accelerates the Ca2+ release from the ER via the IP3 increase. A second peak of the cGMP production after fertilization (Fig. 1, Fig. 2, Fig. 6, Fig. 8, Fig. 9) is probably related to NO synthesis by egg itself (Kuo et al., 2000).
Other evidence against the role of RyR pathway is that the inhibition of soluble guanylate cyclase, such that the cGMP increase did not occur (Fig. 8, Fig. 9), did not abolish the [Ca2+]i increase (Fig. 7, Fig. 9), and did not lengthen the latent periods for the [Ca2+]i rise (Fig. 7, Fig. 9). These findings imply that the RyR pathway is not solely responsible for the induction of Ca2+ transients. An essential point for the initiation of the [Ca2+]i increase must exist elsewhere. One possibility is a putative receptor that directly links to the IP3 system, including Src tyrosine kinase and PLC. An intriguing alternative hypothesis has been suggested by Kuo et al. (Kuo et al., 2000): NO activates Src tyrosine kinase in addition to guanylate cyclase and in turn PLC
is activated. Thus, our results could not reveal the initiating event of the Ca2+ transients at fertilization of sea urchin eggs, but revealed the interaction of the RyR pathway with the IP3R pathway.
Conclusion
The experiments described above showed that cGMP began to increase first, followed by cADPR and IP3, all before the Ca2+ transients, and suggest that the RyR pathway involving cGMP and cADPR contributes to the Ca2+ transients by stimulating the IP3 production during fertilization of sea urchin eggs.
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ACKNOWLEDGMENTS |
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