Introduction
Extracellular Ca2+ is generally accepted as an important factor inducing changes in sperm essential for fertilization, such as chemotactic behaviour and the acrosome reaction. In the echinoderms, peptides released from the jelly coat of eggs play a pivotal role as a signal for such changes. For example, resact is a chemoattractant for sperm of the sea urchin Arbacia punctulata and binds to the receptor guanylyl cyclase on the plasma membrane of sperm flagella (Ward et al., Reference Ward, Brokaw, Garbers and Vacquier1985). The stimulation of sperm by resact evokes changes in intracellular concentrations of cGMP, cAMP and Ca2+, and in the membrane potential (for review see Darszon et al., Reference Darszon, Nishigaki, Wood, Trevino, Felix and Beltran2005). In the starfish Asterias amurensis, asterosap serves as a sperm attractant (Bohmer et al., Reference Bohmer, Van, Weyand, Hagen, Beyermann, Matsumoto, Hoshi, Hildebrand and Kaupp2005), and as a factor for triggering the acrosome reaction in concert with two other jelly components: acrosome reaction-inducing substance (ARIS, a proteoglycan-like molecule; Koyota et al., Reference Koyota, Wimalasiri and Hoshi1997; Gunaratne et al., Reference Gunaratne, Yamagaki, Matsumoto and Hoshi2003) and Co-ARIS (a group of sulfated steroidal saponins; Nishiyama et al., Reference Nishiyama, Matsui, Fujimoto, Ikekawa and Hoshi1987) (Hoshi et al., Reference Hoshi, Nishigaki, Ushiyama, Okinaga, Chiba and Matsumoto1994). Asterosap is a group of equally active isoforms of sperm-activating peptide (Nishigaki et al., Reference Nishigaki, Chiba, Miki and Hoshi1996) that transiently increases the intracellular concentration of cGMP ([cGMP]i), the intracellular pH (pHi) and the intracellular concentration of Ca2+ ([Ca2+]i) via the activation of asterosap receptor, guanylyl cyclase (Nishigaki et al., Reference Nishigaki, Chiba and Hoshi2000; Matsumoto et al., Reference Matsumoto, Solzin, Helbig, Hagen, Ueno, Kawase, Maruyama, Ogiso, Godde, Minakata, Kaupp, Hoshi and Weyand2003). However, the signalling pathway leading to such changes has not been elucidated.
Na+/Ca2+ exchange across the plasma membrane is an important determinant of Ca2+ entry (Li et al., Reference Li, Matsuoka, Hryshko, Nicoll, Bersohn and Burke1994). The process is catalysed by the Na+/Ca2+ exchanger complex that electrogenically exchanges Na+ and Ca2+ across the plasma membrane in either the Ca2+-efflux (forward) or Ca2+-influx (reverse) mode, depending on the electrochemical gradients of the substrate ions. Detailed studies on the function and structure of the complex have revealed that it consists of two types of proteins: Na+/Ca2+ exchanger (NCX), which catalyses the exchange of three sodium ions for one calcium ion (Blaustein & Lederer, Reference Blaustein and Lederer1999; Fujioka et al., Reference Fujioka, Komeda and Matsuoka2000), and K+-dependent Na+/Ca2+ exchanger (NCKX), which exchanges four sodium ions for one calcium and one potassium ion (Schnetkamp, Reference Schnetkamp1995). Six subfamilies of NCKX (NCKX1―6) have been cloned from different animals. Among them NCKX1 was first cloned from bovine retinal rods (Reilander et al., Reference Reilander, Achilles, Friedel, Maul, Lottspeich and Cook1992), and then from those of dolphin (Cooper et al., Reference Cooper, Winkfein, Szerencsei and Schnetkamp1999), chicken (Prinsen et al., Reference Pintado, Herrero, Garcia and Montiel2000), rat (Poon et al., Reference Poon, Leach, Li, Tucker, Schnetkamp and Lytton2000) and human (Tucker et al., Reference Tucker, Winkfein, Cooper and Schnetkamp1998). Subsequently, NCKX2 was demonstrated in rat brain (Tsoi et al., Reference Tsoi, Rhee, Bungard, Li, Lee, Auer and Lytton1998) and in retinal cone cells of chicken and human (Prinsen et al., Reference Prinsen, Szerencsei and Schnetkamp2000), and NCKX3 (Kraev et al., Reference Kraev, Quednau, Leach, Li, Dong, Winkfein, Perizzolo, Cai, Yang, Philipson and Lytton2001) and NCKX4 (Li et al., Reference Li, Kraev and Lytton2002) in rodents and human. More recently NCKX5 (Schnetkamp, Reference Schnetkamp2004) and NCKX6 (Cai & Lytton, Reference Cai and Lytton2004) have also been cloned and characterized from human and mouse, respectively. NCKX1―2 play a significant role in regulation of [Ca2+]i during phototransduction, while NCKX3―6 are expressed in a variety of tissues, indicating their broad roles in the homeostasis of [Ca2+]i. Recently a sea urchin homologue of NCKX (suNCKX) was cloned and found to play a major role in keeping sperm [Ca2+]i low during swimming (Su & Vacquier, Reference Su and Vacquier2002).
In this paper we present the evidence that the asterosap-induced transient elevation of [Ca2+]i is mediated by sfNCKX, an NCKX named after cloning of the cDNA from starfish testes.
Materials and methods
Animal
Starfish, Asterias amurensis, were collected from several locations in Japan and in Tasmania. Those from Tasmania are the offspring of invaders most probably from Tokyo Bay and show no significant differences from the Japanese animals (Byrne et al., Reference Byrne, Morrice and Wolf1997). Sperm were obtained in the form of ‘dry sperm’ by cutting the testes, and kept on ice until use.
Materials
Artificial seawater (ASW) contained 430 mM NaCl, 9 mM CaCl2, 9 mM KCl, 23 mM MgCl2, 25 mM MgSO4 and 10 mM EPPS (N-2-hydroxyethyl-piperazine-N’-3-propane sulfonic acid, pH 8.2). Low Ca2+ ASW was also treated as ASW but had 1 mM CaCl2. K+-divalent cation free seawater (KFSW) contained (in mM): 480 NaCl and 10 EPPS, pH adjusted to 8.2 with NaOH. Li+/K+ was buffer prepared as (in mM): 480 LiCl, 10 KCl, 1 EGTA and 20 EPPS, pH 8.2. Li+ buffer was prepared as Li+/K+ buffer but omitting KCl. Pluronic F-127 and nifedipine were from Sigma (St Louis, MO), and Fluo-4 AM from Nacalai Tesque (Kyoto, Japan). Nitrendipine from Research Biochemical (Natick, MA), 2-(2-(4-nitrobenzyloxy)-phenyl)-isothiourea methanosulfonate (KB-R7943 mesylate, KB) from Tocris (Bristol, UK) and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) from Sigma were dissolved in dimethylsulfoxide (DMSO) for the stocks. The remaining reagents used were of the highest available quality. The synthetic asterosap isoform, P15 (Nishigaki et al., Reference Nishigaki, Chiba, Miki and Hoshi1996), was dissolved in ASW at a concentration of 1μM before use.
[Ca2+]i measurement
Dry sperm were diluted 10-fold in low Ca2+ ASW and incubated with 10μM Fluo-4 AM plus 0.1 mM EDTA (ethylenediamine-N,N,N’,N’-tetraacetic acid), and 0.5% Pluronic F-127, and incubated for 2 h at 16 °C. The sperm were washed once to remove excess Fluo-4 AM (and free Fluo-4 that might exist in the medium) by centrifugation (1000 g for 5 min at 4 °C), resuspended in the original volume of low Ca2+ ASW, and kept on ice in the dark until use. In the K+-dependency experiments, sperm were loaded into KFSW following the same protocol as described above. For [Ca2+]i measurements, 20μl of the loaded sperm were diluted in 1.5 ml of ASW in a round cuvette at 16 °C under constant stirring. The fluorescence intensity was recorded on a Spectrofluorophotometer RF-540 (Shimadzu Science, Tokyo, Japan) with excitation at 494 nm and emission at 516 nm.
Cloning of the full-length K+-dependent Na+/Ca2+ exchanger in starfish, sfNCKX
We screened a starfish testes cDNA library with a PCR-amplified cDNA fragment employing a pair of degenerate primers [5′-GTNGCNGGNGCNACNTT(C/T) ATG-3′ and 5′-GCN(C/G)(T/A)NCCNAC(T/G/A)ATNGTNCC-3′]. We designed primers from a conserved region sequence of suNCKX (Su & Vacquier, Reference Su and Vacquier2002). The PCR was performed under the following conditions: 95 °C for 5 min, followed by 30 cycles at 95 °C for 30 s, 53 °C for 30 s and 74 °C for 1 min, and 74 °C for 10 min. The resulting fragment was subcloned into pGEM-T Easy vector (Promega) according to the manufacturer's instructions. The inserted DNA fragment was fully sequenced on both strands using an automated DNA sequencer (3100 Genetic Analyzer, Applied Biosystems). To obtain the full-length cDNA we used the 5′/3′-RACE system from Clontech according to the manufacturer's instructions. The resulting fragments were combined by subcloning into pGEM-T vector and sequenced as indicated above.
Sequence analysis
The complete nucleotide and deduced amino acid sequences were analysed using commercial software (GENETYX-MAC v.11.2, Software Development, Japan). Homologues of sfNCKX were constructed at Genome Net server (Bio-informatics Center, Kyoto University, Japan; http://www.genome.ad.jp). Glycosylation sites were detected using the Scan Prosite web site http://kr.expasy.org/cgi-bin/scanprosite. The signal sequence was predicted using the web site http://psort.nibb.ac.jp/cgi-bin/runpsort.pl. GenBank accession numbers for NCKX sequences are: sea urchin (suNCKX), AY077699; chicken rod (cNCKX1), AF177984; rat brain (rNCKX2), AF021923; partial human (hNCKX3), AF169257; human (hNCX1), M91368; guinea pig (gNCX1), U04955; bovine (bNCX1), L06438; mouse (mNCX1), U70033; and human (hNCX2), AB029010. The GenBank accession number of the sfNCKX is AB188343.
Southern blot analysis
Genomic DNA from A. amurensis testes was isolated, digested with restriction enzymes, separated by 0.7% agarose gel electrophoresis and then transferred to a nitrocellulose membrane (Pall Biodyne B, Pensacola, USA). The membrane was prehybridized for 2 h at 65 °C in 6× SSC, 0.5% SDS and 5× Denhardt's solution, 0.12 mg/ml salmon sperm DNA and 4 mM EDTA (pH 7). The full-length sfNCKX was labeled with [α-32P]dCTP by random priming (Amersham Biosciences). The probe was added to the hybridization mixture and the incubation was continued for 18 h at 65 °C. After terminating hybridization the membranes were washed once (1 h) with 2× SSC, 0.1% SDS and once (1 h) with 0.2× SSC, 0.1% SDS at 65 °C. After washing, the blots were analysed using a BAS 5000 Bio-Image Analyzer (Fuji Photo Film).
Northern blot analysis
The RNA (7.5μg of total RNA) from the testes and ovaries of A. amurensis was denatured and separated by electrophoresis on a 1% agarose gel containing formaldehyde. The separated RNA was transferred onto a nitrocellulose membrane (Pall Biodyne B, Pensacola, USA). The Northern blot hybridization was carried out as follows: hybridization ― 4× SSC, 0.12 mg/ml salmon sperm DNA, 0.2% SDS, 5× Denhardt's solution, 50% formaldehyde at 42 °C for 16 h; washing ― 2× SSC―0.1% SDS at 50 °C for 10 min, 0.2× SSC―0.1% SDS at 50 °C for 1 h, repeated once. The 3′ region of the cDNAs was labelled with [α-32P]dCTP (3000 Ci/mmol) using a random prime labelling system (Amersham Biosciences) for hybridizing probes. After washing, the blots were analysed using a BAS 5000 Bio-Image Analyzer (Fuji Photo Film).
Results
Ca2+ channel inhibitors do not have significant effects on asterosap-induced changes in [Ca2+]i
The presence of extracellular Ca2+ is required for the activation of A. amurensis sperm by asterosap. It is known that store-operated Ca2+ channels (SOC) are not involved in the asterosap-induced elevation of [Ca2+]i (Kawase et al., Reference Kawase, Minakata, Hoshi and Matsumoto2005). We thus examined whether antagonists against voltage-dependent Ca2+ channels (VDCC) affected the asterosap-induced elevation of [Ca2+]i. Nifedipine and nitrendipine, potent and specific blockers of the L-type Ca2+ channel (Su & Vacquier, Reference Su and Vacquier2002; Rodriguez & Darszon, Reference Rodriguez and Darszon2003), affected the asterosap-induced elevation of [Ca2+]i only slightly, if at all, when the sperm had been incubated with these drugs for 3 min prior to the asterosap treatments (Fig. 1). The results indicate that the VDCC does not play an important role in the asterosap-induced elevation of [Ca2+]i. It is also reported that CCCP prevents mitochondrial Ca2+ uptake by collapsing the transmitochondrial membrane potential (Babcock et al., Reference Babcock, Herrington, Goodwin, Park and Hille1997). However, pretreatments of sperm with 5μM CCCP for 3 min did not reduce the asterosap-induced [Ca2+]i increase (data not shown). Our observations suggest the presence of a distinct signalling pathway for the asterosap-induced [Ca2+]i changes.
Figure 1 Effect of Ca2+ channel blockers on [Ca2+]i elevation induced by asterosap. Fluo-4 loaded sperm were diluted in a cuvette containing 1.5 ml of ASW. At the time shown by the white arrowhead, different concentrations of Ca2+ channel blockers (A, nifedipine; B, nitrendipine) were added. Following a 3 min incubation of loaded sperm in ASW containing different concentrations of the blockers, asterosap (shown by the black arrowhead) was added. Experiments were repeated three times.
Asterosap induces [Ca2+]i elevation through an NCKX
An NCX is involved in the [Ca2+]i changes induced by speract in sea urchin sperm (Schackmann & Chock, Reference Schackmann and Chock1986). Speract signalling causes K+ efflux through cGMP-dependent K+ channels, resulting in a decrease in sperm membrane potential (E m), namely hyperpolarization. The hyperpolarization activates Na+/H+ exchange, adenylyl cyclase and a hyperpolarization-activated and cyclic nucleotidegated K+ channel. These changes lead to increases in pHi, [cAMP]i and Na+ influx. Furthermore, hyperpolarization enhances Na+/Ca2+ exchange to keep [Ca2+]i low. Also, the increase in [Na+]i and depolarization of the membrane potential lead to reversal of the NCX (Darszon et al., Reference Darszon, Nishigaki, Wood, Trevino, Felix and Beltran2005).
In A. amurensis, asterosap activates a K+ channel of sperm plasma membrane and causes a transient hyperpolarization, which then activates Na+/H+ exchange and eventually leads to an increase in pHi (Nishigaki et al., Reference Nishigaki, Chiba and Hoshi2000). Recently Matsumoto et al. (Reference Matsumoto, Solzin, Helbig, Hagen, Ueno, Kawase, Maruyama, Ogiso, Godde, Minakata, Kaupp, Hoshi and Weyand2003) reported that asterosap activates a cGMP pathway and thereby increases the [Ca2+]i. Thus it is of much interest to ask which exchanger is involved in the asterosap-induced increase in [Ca2+]i. Pretreatments of sperm with KB-R7943 (KB), a potent inhibitor of the reverse mode of the NCX (Iwamoto et al., Reference Iwamoto, Watano and Shigekawa1996; Watano et al., Reference Watano, Kimura, Morita and Nakanishi1996), inhibited the asterosap-induced transient increase in [Ca2+]i only partially at 1μM but significantly at 5μM (Fig. 2). The results suggest that the asterosap-induced transient influx of Ca2+ is caused by the reverse mode of an NCKX.
Figure 2 KB inhibits the asterosap-induced Ca2+ elevation by the NCKX. Fluo-4 loaded sperm were diluted in a cuvette containing 1.5 ml of ASW. At the time indicated by the white arrowhead, a different concentration of KB was added. Following a 2 min incubation, asterosap (indicated by the black arrowhead) was added. KB blocks the asterosap-induced Ca2+ elevation in a dose-dependent manner. Data shown in the figure are representative of at least six different batches of sperm.
sfNCKX activity is dependent on K+
We used Fluo-4 for fluorescent calcium imaging to examine the transport function of sfNCKX. When we studied K+-dependent Ca2+ influx, the medium was changed from one containing 480 mM sodium to one containing 480 mM lithium, solution in the presence or absence of potassium (Tsoi et al., Reference Tsoi, Rhee, Bungard, Li, Lee, Auer and Lytton1998; Cooper et al., Reference Cooper, Winkfein, Szerencsei and Schnetkamp1999; Prinsen et al., Reference Prinsen, Szerencsei and Schnetkamp2000; Poon et al., Reference Poon, Leach, Li, Tucker, Schnetkamp and Lytton2000; Kraev et al., Reference Kraev, Quednau, Leach, Li, Dong, Winkfein, Perizzolo, Cai, Yang, Philipson and Lytton2001). This manoeuvre will reverse the sodium gradient and remove sodium competition at the outwardly facing calcium binding sites, and it should therefore favour calcium entry, employing the reverse mode of the exchanger. If sfNCKX function requires the co-transport of potassium with calcium, however, there should be no chance of an increase in [Ca2+]i until potassium is present in the medium. To examine the [Ca2+]i changes, loaded sperm in KFSW were suspended in Li+ or Li+/K+ buffer. Addition of 10 mM CaCl2 to Li+ or Li+/K+ buffer resulted in a greater Ca2+ increase in the presence of K+. We investigated the idea that sfNCKX is K+-dependent and that Ca2+ can be induced through the reverse mode of NCX (Fig. 3A).
Figure 3 Dependence of sfNCKX activity on K+. (A) K+-dependent Ca2+ influx of sfNCKX. Loaded sperm in KFSW were added to Li+/K+ or Li+ buffer and left to equilibrate for 2 min. An addition of 10 mM Ca2+ (white arrowhead) indicates the K+ dependence of sfNCKX. Note that if this gene is present in starfish sperm, Ca2+ will move into the cells by reversing the Na+ movement in the presence of K+. (B) KB blocks K+-dependent Ca2+ influx. Loaded sperm were diluted in Li+ buffer and 10 mM CaCl2 added to it. Following a 2 min incubation in buffer containing different concentrations of KB (in μM), KCl (10 mM) was added (white arrow). Experiments were repeated three times.
KB is a potent inhibitor of NCX. Fig. 3A shows that we found sfNCKX causes an increase in [Ca2+]i in the presence of K+. Accordingly, we used KB to monitor the dependence of sfNCKX on K+. A final concentration of 10μM KB was required to inhibit the K+-dependent Ca2+ influx (Fig. 3B). The above findings ratify the sfNCKX activity in starfish sperm.
Cloning of sfNCKX
We isolated an NCKX clone from the testes cDNA library of A. amurensis. The full-length cDNA of 3169 bp showed a 1848 bp open reading frame encoding a 616 amino acid protein with a calculated molecular mass of 67.7 kDa. It displays a significant sequence similarity to the known NCKXs (Tsoi et al., Reference Tsoi, Rhee, Bungard, Li, Lee, Auer and Lytton1998; Prinsen et al., Reference Prinsen, Szerencsei and Schnetkamp2000; Kraev et al., Reference Kraev, Quednau, Leach, Li, Dong, Winkfein, Perizzolo, Cai, Yang, Philipson and Lytton2001; Su & Vacquier, Reference Su and Vacquier2002), and was thus designated sfNCKX (Fig. 4). It contains 12 transmembrane domains (TM1―12), and a potential cleavage site for signal peptidase is present on TM1, at the position analogous to the known sites of NCKXs (Kraev et al., Reference Kraev, Quednau, Leach, Li, Dong, Winkfein, Perizzolo, Cai, Yang, Philipson and Lytton2001; Li et al., Reference Li, Kraev and Lytton2002; Schnetkamp, Reference Schnetkamp2004; Cai & Lytton, Reference Cai and Lytton2004). It shares a few more features with them ― a cytoplasmic amino-terminus, conserved N-linked glycosylation sites, a long cytoplasmic loop between TM6 and TM7, and a short cytoplasmic carboxyl-terminus (Tsoi et al., Reference Tsoi, Rhee, Bungard, Li, Lee, Auer and Lytton1998; Prinsen et al., Reference Prinsen, Szerencsei and Schnetkamp2000; Kraev et al., Reference Kraev, Quednau, Leach, Li, Dong, Winkfein, Perizzolo, Cai, Yang, Philipson and Lytton2001; Su & Vacquier Reference Su and Vacquier2002) ― suggesting further similarities to other NCKXs. There are 10 protein kinase C (PKC) phosphorylation sites: two in the cytoplasmic amino-terminus, one in the inside loop between TM2 and TM3, and the rest in the cytoplasmic loop between TM6 and TM7. The highest level of sequence identity among different NCKXs occurs between TM3 and TM4 and between TM8 and TM10 (Fig. 4). A BLAST search has revealed that 46% of the amino acid sequence is identical to, and 59% of the sequence is similar to, that of the suNCKX.
Figure 4 Alignment of NCKX proteins obtained from starfish, sea urchin, chicken, rat and human. The shaded regions indicate identical sequences, or at least three similar residues in all sequences. Locations of the putative transmembrane segments (TM1―TM12) are over-lined. An asterisk indicates potential sites of protein kinase C phosphorylation. Other potential sites for signal peptidase cleavage (SPC ↓) and four N-linked glycosylation (●) are also shown. Dashes indicate gaps introduced to maximize the alignment.
Although suNCKX contains an unusual His-rich region in its intracellular loop, sfNCKX does not contain such a His-rich region. The relationships between NCKX and NCX sequences of different animals are shown in Fig. 5. The analysis reveals that sfNCKX groups with NCKX and is separated from NCX.
Figure 5 Phylogeny of sfNCKX. A rootless phylogenetic tree was constructed by the neighbour-joining method. Bootstrap percentage values (1000 replicates) are shown over the corresponding nodes. All branch lengths are proportional to the distances between sequences. Protein IDs in GenBank are given in Materials and Methods. b, c, g, h, m, r, sf and su denote bovine, chicken, guinea pig, human, mouse, rat, starfish and sea urchin, respectively.
To confirm the copy number of sfNCKX in the A. amurensis genome, we carried out Southern blot analysis (Fig. 6A). This sfNCKX cDNA sequence contains two BglII sites and there are no EcoRI, SalI or XhoI sites. The samples digested with EcoRI (lane 1), SalI (lane 2) and XhoI (lane 3) show one band of hybridization, and that with BglII (lane 4) shows two bands. Simple patterns of hybridizing fragments suggest that sfNCKX is a unique and single-copy gene in the A. amurensis genome.
Figure 6 (A) Southern blot analysis of the sfNCKX gene. Samples of the A. amurensis DNA (10μg/lane) were digested with EcoRI (lane 1), SalI (lane 2), XhoI (lane 3) or BglII (lane 4). Size markers in kilobases are indicated on left. (B) Tissue distribution of sfNCKX transcripts. RNA (7.5μg of the total RNA) was isolated from testes and ovaries, and analysed by Northern blotting at higher stringency from starfish NCKX.
The transcripts of the tissue distribution of sfNCKX are shown in Fig. 6B. Approximately 2.6 kb was found to be abundantly expressed in the testes but not in the ovaries, which corresponds to the size of the cloned cDNA.
Discussion
The main finding of this study is that an NCKX, most probably by the reverse mode of sfNCKX, plays a pivotal role in the asterosap-induced transient increase in [Ca2+]i in A. amurensis sperm. An NCKX mechanism is found in a number of species and in a variety of tissues. This exchanger can run in either direction; however, the exchanger usually operates to pump Ca2+ out of the cell (forward mode) in most systems. In previous studies on the K+ dependence of NCKX expressed in cultured cells (Tsoi et al., Reference Tsoi, Rhee, Bungard, Li, Lee, Auer and Lytton1998; Kraev et al., Reference Kraev, Quednau, Leach, Li, Dong, Winkfein, Perizzolo, Cai, Yang, Philipson and Lytton2001), the dependence of the reverse mode NCX on external K+ was observed. In Fig. 3, we investigated whether Ca2+ influx can be induced through the reverse mode of NCX in starfish sperm and its dependence on external K+. However, we summarize our results sequentially here. First, using nifedipine and nitrendipine, our data suggested that VDCC does not play an important role in the asterosap-induced transient elevation of [Ca2+]i. In addition, SOC-like channels are not involved (Kawase et al., Reference Kawase, Minakata, Hoshi and Matsumoto2005). Secondly, by using KB, we showed that an NCKX is involved in this process. Thirdly, we cloned, named and characterized an NCKX gene, sfNCKX.
The NCX electrogenically exchanges three Na+ for one Ca2+ and can function to cause Ca2+ accumulation (reverse mode) or Ca2+ extrusion (forward mode) depending on the concentrations of each ion on either side of the membrane and on the membrane potential (Blaustein & Lederer, Reference Blaustein and Lederer1999). Speract signalling causes an increase in [Na+]i; it is possible that this process, along with membrane depolarization, favours the influx of Ca2+ via the NCX during the stimulus (Darszon et al., Reference Darszon, Nishigaki, Wood, Trevino, Felix and Beltran2005). It has been hypothesized that NCKX contributes to asterosap-induced Ca2+ accumulation, but it has been difficult to test this hypothesis rigorously given the lack of specific inhibitors. The use of KB, a selective inhibitor of reverse NCX, allowed us to evaluate the role of this exchanger in the [Ca2+]i changes caused by asterosap. KB exerts a preferential effect on reverse-mode NCX activity (Iwamoto et al., Reference Iwamoto, Watano and Shigekawa1996; Watano et al., Reference Watano, Kimura, Morita and Nakanishi1996). It inhibits [Ca2+]i accumulation into rat cardiomyocytes and some other NCX1-expressing cells with an IC50 of 1.2―2.4μmol/l (Iwamoto et al., Reference Iwamoto, Watano and Shigekawa1996). We found a significant inhibition of the transient increase in [Ca2+]i in a concentration-dependent manner when sperm were pretreated with KB for 2 min (Fig. 2). KB at 5μM significantly inhibited the [Ca2+]i increase. Therefore, we assume that KB can also inhibit an NCKX complex as we found in starfish sperm. Su & Vacquier Reference Su and Vacquier(2002) reported that higher concentrations of KB (>10μM) alter the resting sperm [Ca2+]i. KB at <5μmol/l has been used as a fairly selective blocker for reverse-mode NCX activity in isolated cardiomyocytes of guinea pig (Watano et al., Reference Watano, Harada, Harada and Nishimura1999). In other cell types, such as bovine adrenal chromaffin cells (Pintado et al., Reference Pintado, Herrero, Garcia and Montiel2000), KB has recently been reported to inhibit neuronal nicotinic acetylcholine receptors (IC50 0.3―6.5μmol/l).
Although sperm-specific ion channels also regulate cation flux in the mammalian sperm flagellum, the regulation mechanism remains unknown. The sperm activating peptide-activated signalling pathways are known in sea urchin and starfish with some points of similarity. For example, resact, speract and asterosap activate the cGMP-signalling pathway and thereby increase the [Ca2+]i. In the sea urchin Strongylocentrotus purpuratus, speract also transiently increases [cGMP]i causing a transitory hyperpolarization, which is mediated by cGMP-modulated K+-selective channels that activate the Na+/H+ exchanger and then stimulate other ion transporters (Darszon et al., Reference Darszon, Nishigaki, Wood, Trevino, Felix and Beltran2005). Our study indicates that the transient [Ca2+]i elevation in sperm occurs in response to asterosap, and is suggested to be caused by the function of NCKX (Fig. 2).
We have identified the sfNCKX molecule expressed in starfish testes, which shares a significant sequence similarity with other NCKX proteins. There are 12 TM segments, with an extracellular loop between TM1 and TM2, with conserved N-linked glycosylation sites, and a large cytoplasmic loop between TM6 and TM7, which is typical of other NCKX proteins (Fig. 4). This is notable because these regions are believed to play a central role in the formation of an ion-binding domain (Schwarz & Benzer, Reference Schwarz and Benzer1997; Su & Vacquier, Reference Su and Vacquier2002). The long central loop, which is presumed to be cytoplasmic based on a comparison between NCKX1 and NCX1, contains consensus sequences for several protein kinases. As the amino acid sequence of suNCKX contains a His-rich region in the intracellular loop, it has been thought that this region plays an important role in maintaining the pHi in sperm (Su & Vacquier, Reference Su and Vacquier2002). However, as the sfNCKX sequence does not have such a His-rich region, it is not certain whether this region is essential for sperm activation.
Taking the data presented in this paper together with our previous findings (Nishigaki et al., Reference Nishigaki, Chiba, Miki and Hoshi1996, Reference Nishigaki, Chiba and Hoshi2000; Matsumoto et al., Reference Matsumoto, Solzin, Helbig, Hagen, Ueno, Kawase, Maruyama, Ogiso, Godde, Minakata, Kaupp, Hoshi and Weyand2003; Kawase et al., Reference Kawase, Minakata, Hoshi and Matsumoto2005; Bohmer et al., Reference Bohmer, Van, Weyand, Hagen, Beyermann, Matsumoto, Hoshi, Hildebrand and Kaupp2005), we conclude that the main signalling pathway for asterosap to trigger chemotactic behaviour and/or the acrosome reaction in sperm is initiated by asterosap binding to, and activation of, the asterosap receptor (guanylyl cyclase), which is followed by a transient increase in [cGMP]i, activation of an NCKX, most probably the reverse mode of sfNCKX, and a transient increase in [Ca2+]i, in that order. Further details of the signalling pathway are currently being investigated.
Acknowledgements
We thank the Director and staff members of the Otsuchi Marine Research Center and the Misaki Marine Biological Station, the University of Tokyo for their help in collecting the starfish. M.S.I. extends his thanks to the Jinnai International Student Scholarship Foundation for its generous financial assistance. This research was partially supported by the Ministry of Education, Culture, Sports, Science and Technology, Grants-in-Aid for Scientific Research on Priority Area (A) (to M.M.) and Grant-in-Aid for the 21st Century Center of Excellence (COE) Program entitled ‘Understanding and Control of Life's Function via Systems Biology (Keio University)’ (to M.S.I).
