Real-time analysis of the role of Ca(2+) in flagellar movement and motility in single sea urchin sperm

Abstract

Eggs of many marine and mammalian species attract sperm by releasing chemoattractants that modify the bending properties of flagella to redirect sperm paths toward the egg. This process, called chemotaxis, is dependent on extracellular Ca(2+). We used stroboscopic fluorescence imaging to measure intracellular Ca(2+) concentration ([Ca(2+)]i) in the flagella of swimming sea urchin sperm. Uncaging of cyclic GMP induced Ca(2+) entry via at least two distinct pathways, and we identified a nimodipine-sensitive pathway, compartmentalized in the flagella, as a key regulator of flagellar bending and directed motility changes. We found that, contrary to current models, the degree of flagellar bending does not vary in proportion to the overall [Ca(2+)]i. Instead we propose a new model whereby flagella bending is increased by Ca(2+) flux through the nimodipine-sensitive pathway, and is unaffected by [Ca(2+)]i increases through alternative pathways.

Figure 1.

Uncaging cGMP increases Ca^{2 +} in swimming sperm. (A and B) Fluorescence images of caged cGMP- and fluo-4–loaded sperm before and after UV flash in (A) ASW and (B) ASW containing 30 μM nimodipine. Interval between images = 23 ms. Bars, 20 μm. Color bar shows fluorescence intensity after background subtraction. (C–H) Normalized increases in fluo-4 fluorescence (F/F₀) in flagellum (black) and head (gray) of individual swimming sperm after uncaging of cGMP. Arrow with line denotes UV flash. (C and D) ASW, (E and F) ASW with 30 μM nimodipine, (G) ASW with 50 mM K⁺, (H) zero Ca²⁺ ASW. Video 1 shows animated sequence of images in A and B (Video 1 available at http://www.jcb.org/cgi/content/full/jcb.200411001/DC1).

Figure 2.

Uncaging cGMP induces characteristic motility changes in single sperm. (a) Trajectory of an individual caged cGMP-loaded sperm in ASW before (red) and after (black) a UV flash. Points along trajectory trace are 23 ms apart. Position of the sperm at the moment of UV flash is marked by a white circle. Bar, 60 μm. Curved arrow shows sperm swimming direction. Graph below shows curvature of sperm trajectory over time. Arrow and solid line indicate UV flash. Dashed line = 0.05 radians/μm. (b–e) Sperm trajectories during a series of UV flashes (b) ASW, (c) ASW containing 30 μM nimodipine, (d) high K⁺ ASW, (e) zero Ca²⁺ ASW.

Figure 3.

Momentary increases in flagellar asymmetry accompany the fast, transient increase in flagellar [Ca^{2 +} ]i and are inhibited by nimodipine. (a) Top panel shows position of the flagellum from an untreated single sperm in relation to a normalized head axis. Red trace is from first visible image of the flagellum after initial, or first, UV flash. Black traces are the position of the same flagellum in the subsequent three images. Blue traces are the position of the flagellum in final five images presented. Position of flagellum recorded every 23 ms. Bar, 10 μm. Dashed line indicates long axis of head. Bottom panel shows [Ca²⁺]i increase in flagellum shown above. Points are color-coded to corresponding traces. Arrow with line denotes UV flash. Dashed line indicates F/F₀ value of 1. (b) Position of flagella from untreated cGMP-loaded sperm after a secondary UV flash. Color coding of flagellum, scale, and interval between flagellum traces as in panel a. Bottom panel shows [Ca²⁺]i increase in flagellum shown above. Color coding, UV flash as in panel a. Dashed line shows an F/F₀ value of 3. (c) Sperm in ASW containing 30 μM nimodipine. Color coding of flagellum, scale, and interval between flagellum traces as in panel a. Bottom panel shows [Ca²⁺]i increase in flagellum shown above. Color coding, UV flash, and dashed line as in panel a.

Figure 4.

Uncaging of cGMP induces a directed relocation of sperm toward a hypothetical stimulus source. (a) Schematic depiction of the calculation of the distance and angle data in the polar plots. The flash axis (black line) is defined by the center of the sperm circular trajectory before the UV flash (red circle) and the position of the sperm at which it receives a UV flash (green circle). The center of the sperm circular trajectory after the UV flash (black circle) is then described by two variables: its displacement from the center of circling before uncaging of cGMP (the length of the arrow) and its angular displacement from the flash axis. The origins of the polar graphs are the center of the sperm circular trajectory before the UV flash, with the flash axis normalized as the 0° axis for each individual UV flash. Curved arrow shows sperm swimming direction. (b) Relocation of sperm after uncaging of cGMP in sperm in ASW. Concentric circles at 10-μm intervals. Open circles = relocation of the center of the circular swimming path after first UV flash; closed circles = relocation of the center of the circular swimming path up to a maximum of four subsequent UV flashes per sperm (i.e., applied after first UV flash). (c) Sperm in ASW containing 30 μM nimodipine. (d) Sperm in high K⁺ ASW. (e) Sperm in zero Ca²⁺ ASW.