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Ca2+ spikes in the flagellum control chemotactic behavior of sperm

2005; Springer Nature; Volume: 24; Issue: 15 Linguagem: Inglês

10.1038/sj.emboj.7600744

1460-2075

Martín Bohmër, Qui Van, Ingo Weyand, Volker Hagen, Michael Beyermann, Midori Matsumoto, Motonori Hoshi, Eilo Hildebrand, U. Benjamin Kaupp,

Plant Reproductive Biology

Article7 July 2005free access Ca2+ spikes in the flagellum control chemotactic behavior of sperm Martin Böhmer Martin Böhmer Institut für Biologische Informationsverarbeitung, Forschungszentrum Jülich, Jülich, Germany Search for more papers by this author Qui Van Qui Van Institut für Biologische Informationsverarbeitung, Forschungszentrum Jülich, Jülich, Germany Search for more papers by this author Ingo Weyand Ingo Weyand Institut für Biologische Informationsverarbeitung, Forschungszentrum Jülich, Jülich, Germany Search for more papers by this author Volker Hagen Volker Hagen Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany Search for more papers by this author Michael Beyermann Michael Beyermann Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany Search for more papers by this author Midori Matsumoto Midori Matsumoto Center for Life Science and Technology, Graduate School of Science and Technology, Keio University, Yokohama, Japan Search for more papers by this author Motonori Hoshi Motonori Hoshi Center for Life Science and Technology, Graduate School of Science and Technology, Keio University, Yokohama, Japan Search for more papers by this author Eilo Hildebrand Eilo Hildebrand Institut für Biologische Informationsverarbeitung, Forschungszentrum Jülich, Jülich, Germany Search for more papers by this author Ulrich Benjamin Kaupp Corresponding Author Ulrich Benjamin Kaupp Institut für Biologische Informationsverarbeitung, Forschungszentrum Jülich, Jülich, Germany Search for more papers by this author Martin Böhmer Martin Böhmer Institut für Biologische Informationsverarbeitung, Forschungszentrum Jülich, Jülich, Germany Search for more papers by this author Qui Van Qui Van Institut für Biologische Informationsverarbeitung, Forschungszentrum Jülich, Jülich, Germany Search for more papers by this author Ingo Weyand Ingo Weyand Institut für Biologische Informationsverarbeitung, Forschungszentrum Jülich, Jülich, Germany Search for more papers by this author Volker Hagen Volker Hagen Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany Search for more papers by this author Michael Beyermann Michael Beyermann Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany Search for more papers by this author Midori Matsumoto Midori Matsumoto Center for Life Science and Technology, Graduate School of Science and Technology, Keio University, Yokohama, Japan Search for more papers by this author Motonori Hoshi Motonori Hoshi Center for Life Science and Technology, Graduate School of Science and Technology, Keio University, Yokohama, Japan Search for more papers by this author Eilo Hildebrand Eilo Hildebrand Institut für Biologische Informationsverarbeitung, Forschungszentrum Jülich, Jülich, Germany Search for more papers by this author Ulrich Benjamin Kaupp Corresponding Author Ulrich Benjamin Kaupp Institut für Biologische Informationsverarbeitung, Forschungszentrum Jülich, Jülich, Germany Search for more papers by this author Author Information Martin Böhmer1,‡, Qui Van1,‡, Ingo Weyand1, Volker Hagen2, Michael Beyermann2, Midori Matsumoto3, Motonori Hoshi3, Eilo Hildebrand1 and Ulrich Benjamin Kaupp 1 1Institut für Biologische Informationsverarbeitung, Forschungszentrum Jülich, Jülich, Germany 2Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany 3Center for Life Science and Technology, Graduate School of Science and Technology, Keio University, Yokohama, Japan ‡These authors contributed equally to this work *Corresponding author. Institut für Biologische Informationsverarbeitung, Forschungszentrum Jülich, 52425 Jülich, Germany. Tel.: +49 2461 614041; Fax: +49 2461 614216; E-mail: [email protected] The EMBO Journal (2005)24:2741-2752https://doi.org/10.1038/sj.emboj.7600744 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The events that occur during chemotaxis of sperm are only partly known. As an essential step toward determining the underlying mechanism, we have recorded Ca2+ dynamics in swimming sperm of marine invertebrates. Stimulation of the sea urchin Arbacia punctulata by the chemoattractant or by intracellular cGMP evokes Ca2+ spikes in the flagellum. A Ca2+ spike elicits a turn in the trajectory followed by a period of straight swimming ('turn-and-run'). The train of Ca2+ spikes gives rise to repetitive loop-like movements. When sperm swim in a concentration gradient of the attractant, the Ca2+ spikes and the stimulus function are synchronized, suggesting that precise timing of Ca2+ spikes controls navigation. We identified the peptide asterosap as a chemotactic factor of the starfish Asterias amurensis. The Ca2+ spikes and swimming behavior of sperm from starfish and sea urchin are similar, implying that the signaling pathway of chemotaxis has been conserved for almost 500 million years. Introduction Eggs attract sperm using chemical factors—a process called chemotaxis (Miller, 1985; Suzuki, 1995; Eisenbach, 2004; Eisenbach et al, 2004). In both mammals and marine invertebrates, the underlying cellular mechanism(s) and the behavioral strategy remain ill defined. Sperm swim on straight trajectories toward the source of chemoattractants and turn when swimming away from the source (Miller, 1985). It is believed that changes in the intracellular Ca2+ concentration ([Ca2+]i) control the swimming trajectory (Brokaw et al, 1974; Brokaw, 1979; Ward et al, 1985; Schackmann and Chock, 1986; Cook and Babcock, 1993b; Cook et al, 1994; Kaupp et al, 2003; Matsumoto et al, 2003; Spehr et al, 2003; Wood et al, 2003; Yoshida et al, 2003; Harper et al, 2004; Ishikawa et al, 2004). However, support for this hypothesis is indirect. In sperm, whose cell membrane has been disrupted by detergent, [Ca2+] of the medium controls the asymmetry of the flagellar beat. At low [Ca2+], flagella beat more symmetrically than at high [Ca2+] (Brokaw et al, 1974; Brokaw, 1979). Thus, when sperm swim up-gradient on a straight trajectory, [Ca2+]i is expected to be low, and when sperm swim down-gradient, [Ca2+]i should become high (Cook et al, 1994). However, these predictions seem to be incompatible with recent time-resolved studies (Kaupp et al, 2003; Solzin et al, 2004) of Ca2+ dynamics in sperm of the sea urchin Arbacia punctulata, a model system for sperm chemotaxis. Resact, the chemoattractant of A. punctulata, evokes a rapid and transient increase of [Ca2+]i in sperm suspensions (Kaupp et al, 2003; see, however, Nishigaki et al, 2004; Shiba et al, 2005). Stimulation of sperm with resact results in a transient increase of flagellar asymmetry and subsequent reorientation of the swimming direction ('turn'). Because both the Ca2+ entry and the motor response occur on a similar time scale, it is plausible that a rise of [Ca2+]i evokes a turn in the swimming trajectory. These results predict that sperm should turn when swimming toward the source of the chemoattractant. Another puzzle concerns the intracellular messengers involved in the opening of Ca2+-permeable channels (Kaupp et al, 2003; Kirkman-Brown et al, 2003; Eisenbach, 2004; Solzin et al, 2004). In one model (reviewed in Darszon et al, 1999, 2001), the increase of intracellular pH and cAMP concentration stimulates Ca2+ entry (Cook and Babcock, 1993a; Cook et al, 1994; Darszon et al, 1999, 2001; Wood et al, 2003). In a competing model, cGMP activates Ca2+ entry, either directly or indirectly, without an obligatory role for cAMP or protons (Kaupp et al, 2003). The different models may be reconciled by the finding that the Ca2+ entry stimulated by resact is composed of an early and a late component (Kaupp et al, 2003) that may be controlled by cGMP and cAMP, respectively. To gain further insight into the mechanisms that control sperm motility, we developed a sensitive laser-stroboscopic technique that allows the recording of changes in [Ca2+]i in the flagellum of swimming sperm. Moreover, we employed a caged form of resact to establish a defined gradient of resact and to follow the swimming trajectory and changes in [Ca2+]i during chemotaxis. Furthermore, we identified asterosap, a sperm-activating peptide of the starfish Asterias amurensis, as a chemoattractant. This enabled us to compare the signaling pathways and chemotactic behavior of two different species. We found that Ca2+ responses and swimming behavior of sperm from sea urchin and starfish are similar, suggesting that the signaling pathway and the mechanisms underlying chemotaxis have been preserved in species that diverged approximately 500 million years ago. Results Navigation of Arbacia sperm in a resact gradient We recorded the trajectories before and after stimulation of A. punctulata sperm by either extracellular resact or intracellular cGMP. These molecules were released from their caged forms by a flash of UV light. In a shallow observation chamber, sperm swim in circles at the water–glass interface (Miller, 1985; Ward et al, 1985; Cook et al, 1994; Kaupp et al, 2003). Upon release of cGMP, the waveform of the flagellar beat changed, and sperm temporarily left the circular swimming mode (Kaupp et al, 2003). The new trajectory was characterized by alternating periods of high curvature ('turns' or 'bends') and low curvature ('straight swimming') (Figure 1A). After several seconds (6.8±1.8 s, n=16 (mean±s.d., number of experiments)), sperm resumed their circular swimming mode, probably because the released cGMP has been degraded by phosphodiesterase activity (Kaupp et al, 2003). Initially, these circles were larger than before stimulation, but became gradually smaller until the original diameter had been reached. This behavior was faithfully reflected by the changes in the local curvature (1/r (μm−1)) of the swimming trajectory (Figure 1A, inset). Shortly after stimulation, the curvature steeply increased and then decreased below the value before stimulation. This spike-like change recurred several times before the curvature gradually returned to prestimulation values. The last stage reflects the transition from larger to smaller circles. Figure 1.Changes in swimming behavior of Arbacia sperm upon photolysis of caged cGMP and caged resact. (A) Swimming trajectory before (blue trace) and after (green trace) release of cGMP from DEACM-caged cGMP (red); the arrowhead indicates the start of the trajectory and small arrows indicate the direction of trajectory. The sections of the trajectory that represent a turn or bend are highlighted in black and by numbered arrows. The interval between consecutive dots is 80 ms. Scale bar, 50 μm. Inset: Changes in the local curvature (1/r) of the trajectory after stimulation by cGMP; arrow: UV flash. Negative values of the curvature indicate that the trajectory becomes 'concave' versus 'convex' with respect to the normal of the observation plane. The numbers denote peaks in the curvature and the corresponding sections of the trajectory. The dashed black line highlights the slow component of the decrease of curvature. (B) Trajectories (upper) and curvature (middle) of three sperm cells swimming in a resact gradient that was produced by local UV irradiation (square). Sperm were suspended in artificial sea water (ASW) containing caged resact (50 nM). Scale bar in the upper panel represents 100 μm. Horizontal bars in the middle panels indicate the duration of the UV irradiation. The lowest panel shows the trajectory of sperm no. 1 in the boxed time periods a–c. Numbers 1–3 refer to the peaks in curvature of box b. (C) Changes in swimming speed (black) and curvature (red) upon stimulation with cGMP in the absence of extracellular Ca2+. The decrease in curvature expected from an increase in swimming speed is shown in green. Inset: Trajectory before (blue) and after the flash (green). Scale bar, 50 μm. Download figure Download PowerPoint Furthermore, we examined the swimming pattern of sperm in a resact gradient. The gradient was established by diffusion of resact that had been photo-released in a given area of the chamber. After the release of resact, sperm accumulated in the irradiated area, while the surrounding area became depleted of sperm (Supplementary movie S1). Figure 1B (upper panels) shows the trajectories of three cells located outside of the irradiated area. Sperm left their circular trajectory several seconds after irradiation. The delay is qualitatively accounted for by diffusion of released resact. The trajectories were also characterized by consecutive periods of high and low curvature (Figure 1B, middle panel), which produced epicycloid-like movements, that is, cycles whose center moves on a curved or meandering trajectory toward the source. Similar epicycloid movements toward the source have been observed in all 39 analyzed cells. This pattern is characteristic of sperm from many different species (Miller, 1985). When sperm swim in a gradient, the changes in the curvature of trajectories were smaller and less regular than the changes evoked by cGMP. When sperm sensed the gradient, circles first became larger and then smaller again when sperm approached the source (Figure 1B, upper and lower panels). The changes in circle diameter probably reflect adaptation to persistent stimulation. Taking into account the changes in swimming velocity after stimulation (see below), the trajectory can be reconstructed from the curvature profile with high fidelity (not shown). The swimming pattern can be understood as the result of two different processes, spike-like increases in curvature (turns) superposed on a slow continuous decrease of curvature (straighter trajectory) that slowly returns to the prestimulus value. This pattern can be observed both upon activation by cGMP and resact (Figure 1A, inset, and Figure 1B, middle panel, dashed black lines). In the absence of extracellular Ca2+, the spike-like changes in curvature were abolished, whereas the slower continuous changes remained unaffected (Figure 1C). This experiment demonstrates that the curvature spikes require Ca2+, whereas the slower changes do not. Furthermore, motor responses were accompanied by an increase of swimming speed (see, for example, distances between sperm heads before and after the first turn in Figure 2A). The mean speed before and after stimulation was 197±59 and 284±105 μm s−1 (n=8), respectively (mean relative increase 1.44; P<0.01; unpaired, two-tailed t-test). The increase of swimming speed was largely independent of external Ca2+. The similar time course of both processes suggests that the slow decrease of curvature is caused by speeding of sperm (Figure 1C). In fact, a simple kinematic relation (1/r=ω/v, ω refers to angular frequency and v to speed; Gray, 1955) predicts a decrease in curvature due to an increase in speed (Figure 1C, green trace). Figure 2.Ca2+ dynamics in swimming Arbacia sperm induced by photolysis of caged cGMP. (A) Ca2+ fluorescence signals from a single cell along its trajectory after stimulation by cGMP (BECMCM-caged cGMP). The inset shows the trajectory and the direction of movements before (blue trace) and after (green trace) the stimulus (red dot); the arrowhead indicates the start. (B) Changes in fluorescence and curvature before, during, and after the four turns or bends in the trajectory of panel A. Changes in Ca2+ fluorescence (blue) and curvature (red) (lower panels) along segments 1–4 shown in the upper panels are also shown. Note the different scales of ordinates. The interval between consecutive images is 60 ms. Scale bars, 50 μm. Colors indicate a linear scale of fluorescence intensity: dark blue, 0 photons pixel−1; red, 450 photons pixel−1. Download figure Download PowerPoint Ca2+ spikes in swimming Arbacia sperm We examined the hypothesis that the succession of turns and linear swimming episodes is produced by the resact- and cGMP-induced dynamics of [Ca2+]i. More specifically, do sperm turn when [Ca2+]i is rising, and do trajectories become straighter when [Ca2+]i is declining? To this end, we loaded sperm with the fluorescent Ca2+ indicator dye Fluo-4 and with caged cGMP, and simultaneously recorded the swimming trajectory and changes in [Ca2+]i after the release of cGMP. Figure 2 and Supplementary movie S2 show the fluorescence images of a cell along the trajectory before and after stimulation. The flagellum of unstimulated sperm was nonfluorescent, whereas the head was fluorescent. The fluorescence of the head probably results from the Ca2+-filled acrosome and mitochondria. The release of cGMP elicited Ca2+ spikes in the flagellum; thus, the flagellum became intermittently visible. In the four segments of the trajectory, where a spike-like increase of [Ca2+]i occurred (Figure 2B, upper part of panels 1–4), [Ca2+]i began to rise before the curvature increased and before the sperm turned (Figure 2B, lower part of panels 1–4). This result demonstrates that sperm adopt a new swimming direction after a rise of [Ca2+]i. The Ca2+ changes in the flagellum appeared as a train of asymmetrical spikes with the following properties. The delay for the first Ca2+ spike was 0.23±0.06 s (n=8), which agrees with the delay of Ca2+ responses measured in sperm suspensions (Kaupp et al, 2003). The time-to-peak of a Ca2+ spike (ca. 60–120 ms) was shorter than the decay time (Figure 2B, lower part of panels 1–4, and Figure 3, upper panel), and the half-width was 0.4±0.1 s (n=8). The short duration of spikes demonstrates that Ca2+ is removed from the cell by a powerful transport system, probably a flagellar Na+/Ca2+−K+ exchanger (Su and Vacquier, 2002). The cGMP-induced Ca2+ spikes were largely similar in amplitude and ceased abruptly (Figure 3, upper panel; see Supplementary Tables S1 and S2); the mean duration of the oscillatory period was 6.6±2.6 s (n=8), which matches the mean duration of the period between the flash and before sperm resume a circular swimming trajectory (4.9±1.4 s, n=7). During the time period between the spikes, [Ca2+]i drops below the detection limit. Because the curvature in-between spikes drops well below the value of unstimulated sperm, it is conceivable that [Ca2+]i also drops below the resting value. At low [Ca2+]e (⩽10−7 M), Ca2+ spikes were abolished. Figure 3.Ca2+ fluctuations in the flagellum (upper) and head (lower) after the release of cGMP (arrow); same cell as shown in Figure 2. Fluorescence intensity is given as photons per flagellum or head. Download figure Download PowerPoint In some cells, the pattern of the cGMP-induced Ca2+ elevations was more complex. For example, a Ca2+ spike was missing in a sequel of regularly spaced spikes, spikes were overlapping, or spikes were different in size (Supplementary Figure S1). Even in those examples, the curvature faithfully reflected the changes in [Ca2+]i, and the number of Ca2+ spikes, whether small or large, correlated with the number of peaks in the curvature (Supplementary Tables S1 and S2). These figures also illustrate that, while [Ca2+]i was still above its resting level, the curvature fell to values that were significantly lower than those in unstimulated sperm, that is, the trajectory became straighter. This observation suggests that the kinetics of the relative changes in [Ca2+]i rather than the absolute [Ca2+]i controls the curvature. We propose that cGMP elicits two distinct processes that define a motor response unit: a Ca2+-controlled increase of the asymmetry of the flagellar beat ('turn' or 'bend') and an opponent process that causes straight swimming. Although the straightening of the trajectory after a turn seems to be related to the decline of [Ca2+]i from its peak value, additional factors may be involved in the control of the trajectory (Figure 1C). The Ca2+ spikes in the flagellum came along with Ca2+ changes in the head. However, the relative changes in the head were up to 100-fold smaller than those in the flagellum (Figure 3, compare upper and lower panels). Moreover, [Ca2+]i in the head fluctuated without obvious changes in the trajectory; therefore, changes in [Ca2+]i in the head are unlikely to control the swimming behavior. The onset of the Ca2+ rise of the flagellum preceded the rise of the head by one or two frames, that is, 60–120 ms. Occasionally, Ca2+ fluctuations in the head occurred without corresponding changes in the flagellum. In contrast to the flagellum, the Ca2+ dynamics in the head was characterized by two components, a steady increase of [Ca2+]i superimposed by small Ca2+ fluctuations (Figure 3, lower panel). Steady and oscillatory components of changes in [Ca2+]i have been observed in the head of immobilized sperm; however, changes in the head were larger than those in the flagellum (Wood et al, 2003). Stimulus function and Ca2+ dynamics We recorded the trajectories and changes in [Ca2+]i while sperm were swimming in a resact gradient (Figure 4; see also Supplementary movie S3). Sperm were suspended in caged resact (Kaupp et al, 2003); a gradient was established by irradiation with UV light that was not homogeneously distributed. The profile of the gradient was derived from the spatial distribution of the flash energy (Figure 4A). After the gradient had been established, Ca2+ spikes appeared and sperm began moving up-gradient (Figure 4B). The pattern of Ca2+ spikes of sperm that swim in a gradient was more complex than that stimulated by cGMP (Figure 4C). In particular, the initial burst of larger Ca2+ spikes was followed by smaller Ca2+ fluctuations. The spikes became smaller when sperm approached the source and when the gradient became shallower. We attribute this difference to the continuous stimulation and the initiation of adaptation. Figure 4.Ca2+ dynamics and trajectories of Arbacia sperm cells swimming in a resact gradient. (A) Contour lines of concentrations of the resact gradient established by a flash of UV light. (B) Swimming trajectories of five sperm cells in the resact gradient. Black traces represent trajectories before and colored traces after photolysis of caged resact (150 nM) (red dots). Resact concentration is depicted as a topographical map of the gradient shown in panel A. Adjacent lines represent steps of 2.5 of the relative resact concentration. Scale bar, 50 μm. (C) Changes in [Ca2+]i (fluorescence, blue) and curvature (red) during chemotaxis of sperm nos. 4 and 5 in the resact gradient. The trajectory analysis of sperm nos. 1–3 is shown in Supplementary Figure S2. The numbers of the panels refer to the sperm number in panel B. Note the different scaling of the ordinates in the panels. Lower panel: Trajectory of sperm No. 5 during the boxed periods a–c. Numbered arrows indicate the turns during the curvature peaks 1 and 2. Download figure Download PowerPoint Soon after the onset of stimulation, changes in [Ca2+]i, whether small or large, were closely correlated with changes in the curvature (Figure 4C). At a later stage, when sperm had returned to an almost circular trajectory, [Ca2+]i continued to spike in most cells, albeit with smaller amplitude; the corresponding changes in curvature became smaller and almost indistinguishable from the noise level. This behavior is particularly pronounced in sperm nos. 4 and 5 (Figure 4C; see also Supplementary Figure S2 for sperm nos. 1–3). The small changes in curvature probably reflect minute adjustments in the diameter and position of the circles. The reduction of the Ca2+ spike activity and the minute adjustments probably reflect adaptive processes in the sustained presence of the attractant. Such adaptive mechanisms may also explain why some sperm display changes in curvature without a Ca2+ spike and vice versa. We suspect that a Ca2+ threshold must be reached to elicit a turn and that this threshold is variable. Due to the loop-like trajectory, sperm swimming in a gradient encounter a more or less periodic change of resact concentration ('stimulus function'). Are the stimulus function and the Ca2+ spikes correlated? To answer this question, we performed a crosscorrelation analysis, which reveals the temporal relationship between two time series, for example, the changes in resact concentration and Ca2+ spikes. Figure 5A shows the stimulus function and the Ca2+ responses along the sperm trajectory in a gradient. The stimulus function first increases instantaneously followed by a periodic increase due to the epicycloid movement of sperm toward the source. It is plausible that our experimental paradigm recapitulates features of the time-dependent pattern of resact concentrations encountered by sperm in their natural habitat, that is, a combination of a sudden increase of resact due to convections and currents in the sea water and a chemical gradient. The step-like stimulation of sperm with resact (homogeneous irradiation, no gradient) also elicits a train of Ca2+ spikes, similar to those elicited by cGMP (Supplementary Table S3). We examined the possibility that the first few Ca2+ spikes produced by the step-like increase of resact compromise the analysis. However, the result was largely independent of whether we included or excluded the initial Ca2+ spikes (data not shown). Such an outcome is expected if the Ca2+ spike frequency and angular frequency of swimming are similar. Future work needs to address this possibility in more detail. The analysis reveals that stimulus function and Ca2+ spikes are synchronized (Figure 5B). The phase shift between the crosscorrelation and the autocorrelation of the stimulus function was 280±92 ms (n=19) or 156±43°. The delay of the Ca2+ increase and, consequently, of the motor responses seems to be a crucial element for the adjustment of the trajectory. Figure 5.Stimulus function and Ca2+ response of Arbacia sperm navigating in a resact gradient. (A) Time course of the stimulus function (green) and changes in Ca2+ fluorescence (blue). The resact gradient (shown in Figure 4A) was established by a flash of UV light (arrow) resulting in a step in the stimulus function. As a result of the repetitive loop-like movement toward the center of the gradient, the sperm cell is repetitively exposed to increasing and decreasing resact concentrations producing a periodic stimulus function. The Ca2+ spikes follow the periodic stimulus function with a delay. (B) Autocorrelation of the stimulus function (green) and crosscorrelation with the train of Ca2+ spikes (blue); same experiment as in panel A. Download figure Download PowerPoint Attraction of Asterias sperm by asterosap We have chosen asterosap, a peptide from the starfish A. amurensis, to re-examine the relative importance of cAMP and cGMP for Ca2+ entry, because asterosap binds to a receptor GC and activates only a cGMP-signaling pathway (Matsumoto et al, 2003). However, the function of asterosap is not clear. At high concentrations (1 μM), it serves as a cofactor for acrosomal exocytosis (Nishigaki et al, 1996); at picomolar concentrations, asterosap stimulates Ca2+ entry similar to that observed in sea urchin sperm when stimulated by resact (Kaupp et al, 2003; Matsumoto et al, 2003; Solzin et al, 2004). To test the hypothesis that asterosap serves as a chemoattractant, we recorded the swimming behavior of sperm near a source of asterosap. Like sea urchin sperm, unstimulated A. amurensis sperm swim in circles parallel to the surface of the chamber. The circle diameter was 81.3±14.8 μm (n=15). After a minute ejection (0.1–0.3 μl) from a capillary containing 1 μM asterosap, sperm were attracted to the opening of the capillary (Supplementary movie S4). In most experiments, sperm accumulated in front of the tip within ∼20 s (Figure 6A). The average number of sperm in a given area near the capillary mouth was 6.8±4.0 (n=16) before the ejection and 14.5±6.9 thereafter, that is, a 2.1-fold increase of sperm density. Cells far from the asterosap source kept their original swimming trajectory for some time, probably because the gradient had to build up by diffusion. Sperm reoriented their swimming trajectory toward the capillary in epicycloid-like movements (Figure 6B), generated by alternating periods of higher and lower curvature of the trajectory (see below; Figure 7). Thus, the behavior of sperm from the sea urchin Arbacia and the starfish Asterias was similar in all aspects, including swimming in very narrow circles (not shown) in the presence of asterosap and IBMX, an inhibitor of phosphodiesterases (Kaupp et al, 2003). These results show that asterosap is a chemotactically active peptide. Figure 6.Swimming behavior of Asterias sperm upon stimulation with asterosap. (A) Accumulation of sperm in front of the capillary filled with asterosap. Distribution of sperm before (left) and 17 s after the ejection of 1 μM asterosap (right) is shown. Number of sperm in the dotted square is as follows: n=12, before; n=33, after ejection. The black dashed line represents opening of the capillary. (B) Swimming trajectories of four single sperm cells before (left) the ejection of asterosap and thereafter (right). The interval between consecutive dots is 80 ms. Scale bars, 100 μm. Inset: Trajectory of a single sperm (red). Download figure Download PowerPoint Figure 7.Swimming behavior of Asterias sperm upon stimulation with cyclic nucleotides and asterosap. (A) Swimming trajectory of a single sperm cell upon stimulation with DEACM-cGMP. Scale bar, 100 μm. Inset: Magnification of the area marked by dashed lines. Blue traces, before; green traces, after UV flash; red dot, UV flash. The interval between consecutive dots is 80 ms. (B) Curvature of the trajectory shown in panel A. Arrow, UV flash; n