Store-operated calcium channel regulates the chemotactic behavior of ascidian sperm

Abstract

The sperm-activating and -attracting factor released from the eggs of the ascidians Ciona intestinalis and Ciona savignyi requires extracellular Ca²⁺ for activating sperm motility and eliciting chemotactic behavior of the activated sperm toward the egg. Here, we show that modulators of the store-operated Ca²⁺ channel, SK&F96365, Ni²⁺, 2-aminoethoxydiphenylborane, and thapsigargin inhibit the chemotactic behavior of the ascidian sperm; on the other hand, blockers of voltage-dependent Ca²⁺ channels did not inhibit the chemotaxis, even though they inhibited the sperm activation operated by voltage-dependent Ca²⁺ channels. The blockers of store-operated Ca²⁺ channels also inhibited the asymmetrical flagellar beating and turning movements of the ascidian sperm, which are typical signs of sperm chemotaxis. Depletion of internal Ca²⁺ stores by thapsigargin induced capacitative Ca²⁺ entry into the sperm, which was blocked by SK&F96365. These results suggest that the intracellular Ca²⁺ concentration increase through the store-operated Ca²⁺ channels induces asymmetrical flagellar movements to establish the chemotactic behavior of the sperm.

It is now well known that many cells, e.g., leukocytes, starved amoebae of Dictyostelium discoideum, bacteria, and migrating axon cells during development, can detect an extracellular chemical gradient and migrate toward the source of the chemicals, that is, exhibit chemotactic behavior. There is a variety of factors inducing chemotaxis of cells, e.g., food, pathogens, partner cells, etc., and chemotactic behavior of cells is one of the important communication systems between separated cells. Chemotactic behavior of the sperm toward an egg during fertilization in animal species is a typical case, first reported in hydrozoans (1). This phenomenon is now widely known to occur, from sponges to humans (2–4), and much effort has been devoted to clarifying the mechanisms underlying sperm chemotaxis.

Extracellular Ca²⁺ is generally accepted as being an important factor in the induction of chemotactic behavior of sperm in many plant and animal species (5–8). In the case of the sea urchin, Arbacia punctulata, a peptide named resact in the jelly layer of the egg has been shown to have sperm-activating and -attracting activities (7). The peptide increases the intracellular Ca²⁺ concentration ([Ca²⁺]_i) via cAMP-gated Ca²⁺ channels (9, 10), which, in turn, induces asymmetrical flagellum movements (5, 11, 12) that result in reorientation of the sperm swimming direction (13). The role of extracellular Ca²⁺-induced asymmetrical flagellar movement in inducing sperm chemotaxis has also been reported in hydrozoan species (14). Ascidian sperm chemotactic behavior is also characterized by abrupt turning movements of the sperm (15, 16). Furthermore, it has been shown that the sperm-attracting activity in the ascidians Ciona intestinalis and Ciona savignyi does not originate from the overall surface of the egg coats, such as follicle cells, but from the vegetal pole of the egg (17). We also demonstrated that there is no species specificity in sperm chemotaxis between C. intestinalis and C. savignyi, and that a single molecule, named the sperm-activating and -attracting factor (SAAF) (8, 17), induces both sperm activation and attraction. Recently, we determined the chemical structure of SAAF as 3,4,7,26-tetrahydroxycholestane-3,26-disulfate, a novel sulfated steroid (18).

In contrast to the finding that SAAF-dependent sperm activation is caused by Ca²⁺ influx through voltage-dependent Ca²⁺ channels and cAMP synthesis (8, 19), we show here that capacitative Ca²⁺ entry mediates asymmetrical flagellar movement that regulates chemotaxis.

Materials and Methods

Materials.

Specimens of the ascidians C. intestinalis and C. savignyi were collected from Onagawa Bay, Tokyo Bay, Japan, and Gulf of Napoli, Italy. These were kept in an aquarium under continuous light exposure to prevent spontaneous spawning. Semen and eggs were obtained from the gonoducts by dissection and kept on ice and at 18°C, respectively. SAAF was partially purified from the eggs as described (8). Artificial seawater (ASW) used for the experiments contained 462 mM NaCl, 9 mM KCl, 11 mM CaCl₂, 48 mM MgCl₂, and 10 mM Hepes (pH 8.2); Ca²⁺-free seawater (CaFSW) contained 462 mM NaCl, 9 mM KCl, 59 mM MgCl₂, and 10 mM Hepes (pH 8.2). With Mediterranean Ciona, we used ASW containing 500 mM NaCl, 10 mM KCl, 10 mM CaCl₂, 50 mM MgSO₄, and 10 mM Hepes (pH 8.2).

Thapsigargin was purchased from Molecular Probes, SK&F96365 from Biomol (Plymouth Meeting, PA), 2-aminoethoxydiphenylborane (2-APB) from Tocris Cookson (Bristol, U.K.), and Calcium Channel Blocker EconoKit from Alamone Labs (Jerusalem).

Analysis of Sperm Chemotaxis.

Semen was diluted ≈2,000 times in ASW containing 1 mM theophylline (Sigma) and incubated for 1 min on ice to activate the sperm. SAAF was packed at the tip of a glass capillary and inserted into ASW or CaFSW with 1 mM EGTA containing sperm, and the sperm movements were observed as described (17). To examine the effects of Ca²⁺ channel blockers, theophylline-activated sperm were incubated with the respective blockers at appropriate concentrations for 3 min before observations were conducted.

The accumulation index of the sperm expressed the time course of the changes of the density ratio from the inner to the outer area of a circle with a radius of 100 μm drawn around the capillary tip containing SAAF. The index value was expressed as 1 when the sperm did not exhibit any chemotactic behavior toward the capillary tip. The sperm trajectories were analyzed as follows: the positions of the sperm heads at intervals of 1/30 or 1/6 sec were plotted by hand on a transparency from images recorded in a videocassette recorder. The data were inputted into a personal computer by using an image scanner, and the sperm trajectories were described in coordinates with the capillary tip located at the origin of the coordinates, by using the software datapicker (shareware made by M. Aotsuka, http://hp.vector.co.jp/authors/VA019223/). To determine the index of the sperm chemotaxis, we calculated the distance between the capillary tip and the sperm (D), the displacement angle of the sperm direction (θ), and the linear equation chemotaxis index (LECI), by using the software excel (Microsoft), as described (18).

Observation of Sperm Flagellar Waveform.

Sperm and the glass capillary containing SAAF were prepared as described above. Waveforms of flagellar movements of sperm around the capillary tip were observed under a phase-contrast microscope (Nikon, Optiphot) with a ×20 objective lens (Olympus, Tokyo). The waveforms were recorded onto a personal computer at 20-msec intervals for 1 msec, by using a high-speed charge-coupled device camera (HAS-200, Ditect, Tokyo) and a video card (HAS-PCI, Ditect). The position of each sperm was analyzed by using an image-analyzing program (Dip-motion 2D, Ditect).

Measurement of [Ca²⁺]_i.

Semen was diluted in 10 volumes of CaFSW containing 0.025% Cremophor EL and 10 μM fluo-3 AM (Molecular Probes) and incubated for 2 h with gentle shaking at 18°C in the dark. After incubation, 5 ml of CaFSW was added, and the solution was centrifuged at 1,200 ×g for 10 min. The sperm pellet was resuspended in 1 ml of CaFSW and incubated for 1 h at 18°C to hydrolyze the AM esters of the dye. The sperm were then washed by centrifugation and suspended again in 10 volumes of CaFSW. Twenty to forty microliters of the suspension containing the dye-loaded sperm was mixed with 1 ml of either CaFSW, 2CaASW (ASW in which the concentration of Ca²⁺ was reduced to 2 mM), or 2CaASW containing 25 μM SK&F96365, and the fluorescence was monitored at 18°C at wavelengths of 485 nm (excitation)/530 nm (emission) with a fluorescence spectrophotometer (Hitachi, Tokyo, 650–10S).

Results

Voltage-Dependent Ca²⁺ Channels Are Not Involved in Sperm Chemotaxis.

Activation of Ciona sperm by SAAF requires the presence of extracellular Ca²⁺, and Ca²⁺ entry is mediated by voltage-dependent Ca²⁺ channels (VDCC) (8). Thus, we first examined the role of VDCC in sperm chemotaxis. To examine the effects of inhibitors on chemotaxis, we measured the time course of changes of the density ratio from the inner to the outer area of a circle with a radius of 100 μm drawn around the capillary tip. Sperm of C. intestinalis were suspended in CaFSW and activated with 1 mM theophylline. The capillary containing SAAF was inserted 1 min after the theophylline activation and then, 2 min after the theophylline activation, Ca²⁺ was added at the final concentration of 10 mM. In the absence of Ca²⁺, the sperm did not exhibit any chemotactic behavior (Fig. 1). Nifedipine, a specific blocker of the L-type calcium channel, which is the most common VDCC in non-neuronal cells, had no effect on sperm chemotaxis in the presence of 10 mM Ca²⁺ (Fig. 1a). Flunarizine, which blocks both L-type and T-type Ca²⁺ channels as well as sperm activation (8), also did not affect sperm chemotaxis (Fig. 1b). Furthermore, the N-, P-, and Q-type Ca²⁺ channel blocker, ω-conotoxin (Fig. 1c), and other voltage-dependent channel blockers, namely, ω-agatoxin TK, calcicludine, calciseptine, ω-conotoxin GVIIA, ω-conotoxin MVIIA, ω-conotoxin SVIB, FS-2, FTX-3.3, sFTX-3.3, PLTX-II, TaiCatoxin and waglerine, also had no effect on sperm chemotaxis (data not shown). These suggest that VDCC are not involved in sperm chemotaxis.

Figure 1.

Effects of Ca²⁺ channel blockers on sperm accumulation. Semen of C. intestinalis was diluted in CaFSW containing 1 mM theophylline and (a) ○, vehicle (0.5% DMSO) (n = 3); ●, 100 μM nifedipine (n = 4); □, ASW (n = 26); (b) ○, ASW (n = 5); ●, 10 μM flunarizine (n = 3); (c) ○, ASW (n = 3); ●, 1 μM ω-conotoxin MVIIC (n = 3); □, 10 μM ω-conotoxin MVIIC (n = 3); (d) ○, ASW (n = 4); ●, 1 μM SK&F96365 (n = 4); □, 5 μM SK&F96365 (n = 4); ■, 10 μM SK&F96365 (n = 3); (e) ○, ASW (n = 3); ●, 1 mM Ni²⁺ (n = 3); □, 5 mM Ni²⁺ (n = 3), on a glass slide at 0 min. A glass capillary plugged with SAAF at its tip was inserted into the sperm suspension at 1 min, and CaCl₂ was added up to 10 mM at 2 min after the sperm dilution (arrow). Data are expressed as mean ± SEM.

SK&F96365 and Ni²⁺ Inhibit Sperm Chemotaxis.

SK&F96365, a blocker of receptor- and store-operated Ca²⁺ channels (SOC) (20), inhibited the chemotaxis of C. intestinalis sperm: 1 μM or more SK&F96365 prevented accumulation of sperm around the capillary tip, in both the presence and absence of extracellular Ca²⁺ (Fig. 1d). Furthermore, the nonspecific Ca²⁺ channel antagonist, Ni²⁺, which inhibits SOC at higher concentrations (20), suppressed sperm accumulation at 5 mM (Fig. 1e). These results show that SOC may be involved in the chemotactic behavior of the sperm.

Next, we precisely examined the effects of SOC on sperm chemotaxis. We quantified the chemotaxis value by using the LECI, as described (17, 18). The chemotactic movement of the sperm toward SAAF was observed in ASW (Fig. 2 a and d). However, the sperm showed only circular movements in the presence of SK&F96365 and Ni²⁺ (Fig. 2 b, c, e, and f). The LECI of sperm that were incubated with 1 μM SK&F96365 was 0.95 ± 3.1 μm/sec, and that of control sperm was 24.2 ± 4.7 μm/sec (Fig. 2g). SK&F96365 also affected sperm motility. Although the movement velocity of control C. intestinalis sperm was 276.3 ± 5.8 μm/sec, it was reduced to 103.8 ± 13.0 μm/sec in the presence of 1 μM SK&F96365. At lower concentrations, SK&F96365 had no effect on either sperm chemotaxis or movement velocity. At higher concentrations, sperm motility was suppressed so that the effect on chemotaxis could not be observed. Below 25 μM, SK&F96365 had no effect on SAAF-induced sperm activation (see Fig. 7 and supporting text, which are published as supporting information on the PNAS web site, www.pnas.org). Furthermore, 0.2 mM Ni²⁺ had no effect on sperm chemotaxis, but higher concentrations of Ni²⁺ inhibited sperm chemotaxis (Fig. 2g). The LECI decreased to around one-half of that in control sperm in the presence of 0.5 and 1 mM Ni²⁺ and to one-sixth of that in the control sperm in the presence of 5 mM Ni²⁺. However, the velocity of sperm was not changed by any concentration of Ni²⁺.

Figure 2.

Effects of Ca²⁺ channel blockers on chemotaxis of C. intestinalis sperm. In a–c, the trajectories of sperm suspended in ASW (a), or ASW containing 1 μM SK&F96365 (b) and 5 mM NiCl₂ (c) are shown. The capillary tip containing SAAF was set as the origin of the coordinates. Each figure shows the typical trajectories of two sperm (black and white circles). The arrowhead indicates a chemotactic turn. Sperm chemotaxis was seen only in ASW. In d–f, changes in the distances between the capillary tip (origin) and the sperm (D) during sperm movement in ASW (d), or ASW containing 1 μM SK&F96365 (e) and 1 mM NiCl₂ (f) are shown. The line and formula show the linear equation for each spermatozoon. The negative value of the coefficients of the equation yields the LECI. (g) The average LECI of sperm suspended in ASW (control), SK&F96365 and NiCl₂ is shown. Data are expressed as mean ± SEM (n = 15–32).

Capacitative Ca²⁺ Entry and Sperm Chemotaxis.

The results described above suggest that a SOC may participate in the chemotaxis of ascidian sperm. We examined whether capacitative Ca²⁺ entry is present in the ascidian sperm using thapsigargin, a specific blocker of the sarco/endoplasmic reticulum Ca²⁺-ATPase, which causes Ca²⁺ depletion from internal stores and leads to Ca²⁺ entry (21). When fluo-3-loaded C. intestinalis sperm were suspended in CaFSW and the [Ca²⁺]_i in the sperm was measured, no [Ca²⁺]_i increase was observed (Fig. 3a). In ASW containing 2 mM Ca²⁺, thapsigargin induced increase in the [Ca²⁺]_i (Fig. 3b); i.e., the capacitative Ca²⁺ entry through SOC occurs in the Ciona sperm. Furthermore, SK&F96365 blocked the capacitative Ca²⁺ entry induced by thapsigargin (Fig. 3c). Thapsigargin-induced Ca²⁺ release from internal stores, usually observed in other cells, could not be detected in our experiments. This may be due to the highly reduced capacity of internal store in the spermatozoa.

Figure 3.

Capacitative Ca²⁺ entry in the sperm of C. intestinalis. Five micromol thapsigargin (Tg) was added to the sperm suspension (arrow), and changes of the [Ca²⁺]_i in the sperm were measured by using fluo-3. One micromol ionomycin (im) was added 5 min after the addition of thapsigargin (arrowhead). (a) Spermatozoa suspended in CaFSW. No increase of [Ca²⁺]_i was observed. (b) Spermatozoa suspended in 2CaASW. Thapsigargin-induced [Ca²⁺]_i increase, i.e., capacitative Ca²⁺ entry, was observed. (c) Spermatozoa in 2CaASW containing 25 μM SK&F96365. Capacitative Ca²⁺ entry was blocked by SK&F96365.

Next, we observed the effects of thapsigargin on sperm chemotaxis. At 2.5 μM thapsigargin, no change in sperm velocity was observed, and chemotaxis was partially blocked: the LECI was reduced to almost one-fourth of that in the control sperm (Fig. 4). At a higher concentration (5 μM), sperm chemotaxis was completely blocked: the sperm showed only circular movements, and the LECI became almost zero (−1.5 ± 3.8 μm/sec; Fig. 4). At this concentration, the sperm velocity was decreased to 89.8 ± 10.6 μm/sec. The effective dose of thapsigargin in this study was higher than that typically needed for mammalian somatic cells but similar to the values reported for gametes [IC₅₀ of rat sperm = 0.5 μM (22); mouse sperm, 0.77 μM (23); mouse eggs >1 μM (24)]. These results suggest that SOC may be involved in the chemotaxis of ascidian sperm. Thapsigargin and SK&F96365 had similar inhibitory effects on sperm chemotaxis, though thapsigargin must activate SOC pathways, and SK&F96365 is thought to inhibit them. These discrepant results may be because the open/closed regulation of SOC may regulate sperm chemotaxis; that is, thapsigargin inhibited the sperm chemotaxis because it clamped open-state on SOC.

Figure 4.

Effect of thapsigargin and 2-APB on sperm chemotaxis. In a–c, the trajectories of sperm suspended in ASW containing 0.5% DMSO (a), 5 μM thapsigargin (b), or 50 μM 2-APB (c). Capillary tip containing SAAF was set as the origin of the coordinates. Each figure shows the typical trajectories of two sperm (black and white circles). In d–f, changes of D in ASW containing 0.5% DMSO (d), 5 μM thapsigargin (e), or 50 μM 2-APB (f). Line and formula show linear equation of each sperm. (g) The average of LECI of C. intestinalis sperm suspended in ASW containing thapsigargin or vehicle (0.5% DMSO), and that of C. savignyi sperm suspended in ASW or 50 μM 2-APB. Data are expressed as mean ± SEM (n = 7–34).

Effects of 2-APB on Sperm Chemotaxis.

2-APB, originally characterized as a cell-permeable inhibitor of inositol 1,4,5-trisphosphate (IP₃)-induced Ca²⁺ release (IICR) (25), also acts as an inhibitor of SOC (26–28). To confirm the involvement of SOC in chemotaxis of the ascidian sperm, we observed the effects of 2-APB on the sperm chemotaxis of C. savignyi. In the presence of 50 μM 2-APB, the sperm velocity was not influenced (213.2 ± 10.8 μm/sec, control; 216.1 ± 5.0 μm/sec), but chemotaxis was blocked: the LECI was almost zero (−4.9 ± 2.4 μm/sec; Fig. 4). At a higher concentration (500 μM), sperm motility was completely suppressed. These results suggest that SOC or IICR may be involved in the chemotaxis of the ascidian sperm. The IC₅₀ of 2-APB for capacitative Ca²⁺ entry is ≈5 − 10 μM (27, 28), and that of IICR is 42 μM (25). Therefore, the inhibition of sperm chemotaxis by 2-APB at the dose used in these experiments (50 μM) may be mainly because of its inhibitory effect on the SOC.

Asymmetrical Flagellar Waveform and Turning Behavior in Sperm Chemotaxis.

Chemotaxis of the hydroid and ascidian sperm is characterized by abrupt changes of the swimming direction associated with asymmetrical flagellar movements (13, 16). These turning movements are considered important indicators of sperm chemotaxis. Our results show that Ni²⁺, SK&F96365, thapsigargin, and 2-APB inhibited sperm chemotaxis, and SK&F96365 and thapsigargin reduced sperm velocity. Thus, it is necessary to examine whether these blockers really blocked sperm chemotaxis. When examining the sperm trajectories and flagellar movements in detail, the displacement angle of the sperm direction (θ) was usually in the range of 0−30° in the absence of chemotactic behavior. However, θ briefly increased to over 60° when sperm exhibited chemotactic behavior (Fig. 5a) (18). Therefore, if θ increased above 60°, we defined it as “turn.” Turns were usually observed when sperm moved away from an attractant (Fig. 5a). The turns, identified by measuring θ, were also associated with asymmetrical flagellar movements (Fig. 6), therefore turns and asymmetrical flagellar movement are considered to be correlated. When the frequency of the turns was examined, normal sperm exhibiting chemotactic behavior showed 0.84 ± 0.16 turns per sec (C. intestinalis) or 1.73 ± 0.44 turns per sec (C. savignyi); the blockers decreased the frequency of turns to one-fifth to one-half of that in the control sperm (Fig. 5 b and c). Furthermore, the blockers also reduced the number of sperm that showed turns. In the absence of inhibitors, the number of sperm not showing turns was 1/7 (14%) and 2/14 (14%) with and without 0.5% DMSO, respectively. However, the number of sperm not showing turns increased to 13/30 (43%) in the presence of 1 μM SK&F96365, 14/20 (70%) in 5 mM NiCl₂, and 9/15 (60%) in 5 μM thapsigargin. These results suggest that the inhibitors block sperm turns, that is, sperm chemotaxis.

Figure 5.

Turning movements during sperm chemotaxis. (a) Changes in three parameters of a spermatozoon of C. intestinalis that showed chemotactic behavior in ASW (i), or inhibition of chemotactic behavior in the presence of 5 mM NiCl₂ (ii). The points at which θ rose above 60°, i.e., when the turns occurred just after the peak of dD/dt (arrow), coincided with the decrease of dD/dt (i). In the presence of 5 mM Ni²⁺, sperm did not exhibit any chemotactic behavior and no change in θ was observed (ii). (b) Frequency of the turning movements of C. intestinalis sperm in the presence of 1 μM SK&F96365 (n = 30), 5 mM NiCl₂ (n = 20), 5 μM thapsigargin (n = 15), 0.5% DMSO (n = 7), and control (n = 14). (c) Frequency of the turning movements of C. savignyi sperm in the presence of 50 μM 2-APB (n = 11) and control (n = 7). Data are expressed as mean ± SEM.

Figure 6.

Pattern of flagellar beating of C. intestinalis sperm during chemotactic behavior (a) and changes of θ in one cycle of sperm movements (b) in ASW. The trajectory of the observed spermatozoon is shown in c. Images of sperm were collected every 40 msec and are ordered left to right. Each image of sperm in a corresponds to the point of the open circle in b and c. The point at which θ rose above 60° is shown as a frame (a) and arrows (b and c). Asymmetrical flagellar beating started at the point at which θ rose above 60°, i.e., the turning movement, and lasted for 320 msec (eight pictures).

Discussion

The requirement of extracellular Ca²⁺ for chemotaxis has been generally known. However, the Ca²⁺-dependent cell signaling underlying the regulation of the chemotactic behavior of sperm, e.g., the pathway from reception of attractants to Ca²⁺ entry, is still unclear. Resact, a sperm-activating peptide derived from egg jelly of the sea urchin, has a sperm-attracting activity (7). The peptide binds to the receptor, which is guanylyl cyclase (29), and seems to induce an increase in [Ca²⁺]_i through modulation of the cGMP and cAMP levels (10). However, there has been no study for sperm chemotaxis that allowed discrimination between the pathway contributing sperm activation and sperm attraction in the species, making accurate understanding of the mechanism on the sperm chemotaxis difficult. Spermatozoa of the ascidians C. intestinalis and C. savignyi require cAMP and extracellular Ca²⁺ for activation, but the chemotactic behavior of the sperm is not influenced by changes in the cAMP levels (8). This allows discriminating sperm attraction from sperm activation in these species by using sperm whose motility has already been activated by the treatment of theophylline.

Using theophylline-treated sperm, we show here that a SOC participates in the chemotactic behavior of the ascidian sperm. Generally, SOC is localized on the plasma membrane and mediates Ca²⁺ entry when the internal stores are depleted due to the action of IP₃ or other Ca²⁺-releasing signals (capacitative Ca²⁺ entry) (30, 31). Capacitative Ca²⁺ entry replenishes the internal Ca²⁺ stores and is mediated by a signal transduction process (32). In regard to sperm function, a SOC is known to be involved in the acrosome reaction (23, 33–35). However, there is as yet no evidence suggesting that the channel is involved in sperm motility, although sperm activation and chemotaxis usually require extracellular Ca²⁺ (36, 37). The chemotactic behavior of the Ciona sperm also requires extracellular Ca²⁺ (8). In the sperm of another ascidian, Ascidia ceratodes, Ca²⁺ is released from the internal Ca²⁺ stores, and the IP₃ receptor seems to be involved in the activation of sperm motility, even though localization of the internal Ca²⁺ stores in the sperm is still obscure (38). These facts suggest that Ca²⁺ release from the internal stores is involved not only in sperm activation but also in sperm chemotaxis. It is likely that SAAF induces IP₃-induced Ca²⁺ release, and the depletion of the internal Ca²⁺ stores promotes capacitative Ca²⁺ entry, inducing sperm chemotaxis toward egg at fertilization in the ascidians.

Spermatozoa of many animals have only a small amount of cytoplasm and usually lack an endoplasmic reticulum, raising the question of where the internal Ca²⁺ stores are localized in the sperm. In the mammalian sperm, IP₃ receptor is located on the acrosomal vesicle, which is considered as the internal Ca²⁺ stores (21, 39). On the other hand, whereas the spermatozoon has a large mitochondrion that has been proposed as internal Ca²⁺ stores (38), the IP₃ receptor has never been found in the mitochondria of any cells. The sperm of some teleost species have vesicular structures in the region connecting the sperm head and tail (40, 41). It is still unclear whether Ca²⁺ is stored in the acrosomal vesicle or some vesicular structure in the Ciona sperm. However, the small number of Ca²⁺ ions released from the small stores may elevate the [Ca²⁺]_i enough for regulating sperm movement, because the cell has only a small volume of cytoplasm.

Two patterns of sperm behavior have been designated as the signs of sperm chemotaxis. One is observed in the hydrozoan Siphonophore, whose radius of curvature of sperm trajectories reduces with increasing concentration of the chemoattractant (14). In this case, Ca²⁺ ionophores can induce the reduction of the radius of curvature (14). In other hydrozoa and ascidians, an abrupt turning movement is observed in sperm exhibiting chemotactic behavior (13, 15, 17, 42), and during the turning movement, the sperm show temporal and intensive asymmetrical flagellar movements (Fig. 6) (13, 16). This asymmetrical pattern of flagellar movement was induced in the demembranated sperm of the sea urchin when the Ca²⁺ concentration in the reactivating medium was increased (5, 43); furthermore, the asymmetrical flagellar beating was shown to be regulated by calmodulin (12). Similar Ca²⁺-induced asymmetrical flagellar beating was observed in Ciona sperm (44). Therefore, the turning movement observed in the ascidian may be derived from the Ca²⁺-induced asymmetrical movements of the sperm flagella and is considered responsible for the chemotactic behavior of the sperm. Namely, increase in the asymmetry of flagellar movements may increase the curvature of the sperm swimming trajectory and cause reorientation of the sperm swimming direction.

Speract and resact, the sperm-activating peptides of sea urchins, could induce a rise in the [Ca²⁺]_i, and speract may be responsible for the asymmetrical flagellar beating (10). However, it has remained unclear whether the increase in [Ca²⁺]_i is a cue of chemotaxis in the sea urchin, because speract has no sperm-attracting activity (7). Furthermore, whereas resact is considered to be responsible for inducing chemotaxis of the sea urchin sperm, asymmetrical flagellar movements of the sperm in resact-induced chemotaxis are not clear. In the present study, asymmetrical flagellar beating of the ascidian sperm was noted when the sperm detected a decrease in the concentration of the attractant during chemotactic behavior (Fig. 6). Furthermore, SK&F96365, Ni²⁺, thapsigargin, and 2-APB, reagents related to SOC, reduced the chemotactic turn and chemotaxis index, LECI, in the ascidian sperm. These results suggest that capacitative Ca²⁺ entry mediates asymmetrical flagellar movements when sperm move away from the source of SAAF, a sperm attractant, and when the concentration of the SAAF surrounding them decreases. These asymmetrical movements may cause a turning movement and chemotaxis of the sperm. In other words, chemotactic behavior of the Ciona sperm may be determined by a turn caused by the opening of a SOC and subsequent increase in the [Ca²⁺]_i with decreasing SAAF concentration.

Abbreviations

SOC

store-operated Ca²⁺ channel

IP₃

inositol 1,4,5-trisphosphate

SAAF

sperm-activating and -attracting factor

ASW

artificial seawater

CaFSW

calcium-free seawater

2-APB

2-aminoethoxydiphenylborane

LECI

linear equation chemotaxis index

[Ca²⁺]_i

intracellular Ca²⁺ concentration