The Role of Soluble Adenylyl Cyclase in the Regulation of Flagellar Motility in Ascidian Sperm

Abstract

Flagellar motility in sperm is activated and regulated by factors related to the eggs at fertilization. In the ascidian Ciona intestinalis, a sulfated steroid called the SAAF (sperm activating and attracting factor) induces both sperm motility activation and chemotaxis. Cyclic AMP (cAMP) is one of the most important intracellular factors in the sperm signaling pathway. Adenylyl cyclase (AC) is the key enzyme that synthesizes cAMP at the onset of the signaling pathway in all cellular functions. We previously reported that both transmembrane AC (tmAC) and soluble AC (sAC) play important roles in sperm motility in Ciona. The tmAC plays a major role in the SAAF-induced activation of sperm motility. On the other hand, sAC is involved in the regulation of flagellar beat frequency and the Ca²⁺-dependent chemotactic movement of sperm. In this study, we focused on the role of sAC in the regulation of flagellar motility in Ciona sperm chemotaxis. The immunochemical analysis revealed that several isoforms of sAC protein were expressed in Ciona sperm, as reported in mammals and sea urchins. We demonstrated that sAC inhibition caused strong and transient asymmetrization during the chemotactic turn, and then sperm failed to turn toward the SAAF. In addition, real-time Ca²⁺ imaging in sperm flagella revealed that sAC inhibition induced an excessive and prolonged Ca²⁺ influx to flagella. These results indicate that sAC plays a key role in sperm chemotaxis by regulating the clearance of [Ca²⁺]_i and by modulating Ca²⁺-dependent flagellar waveform conversion.

Keywords: calcium; cilia; protein kinase; sperm motility.

Figure 1

(A) Schematic representation of the Ci-sAC. The bold line shows the region used for the antigen to raise a polyclonal antibody. (B) Western blots of whole sperm protein with the antibodies against the two regions of Ci-sAC. CBB-stained pattern of whole sperm proteins (left) and corresponding immunoblots (right) are shown. Original images of (B) can be found in Figure S1. (C,D) Immunolocalization of Ci-sAC in sperm with the antibodies against the two protein regions. The differential interference contrast (DIC) image, Ci-sAC (magenta), alpha-tubulin, acetylated (green) and the merged image are shown. Scale bar: 20 µm.

Figure 2

Effects of a sAC inhibitor KH-7 on the flagellar motility in Ciona sperm during chemotactic turn. Data that are shown comprise flagellar bending patterns (A,D), changes in asymmetric index (B,E) and flagellar curvature (C,F) during the chemotactic turn of sperm treated with 0.5% DMSO (control, A–C) and sperm treated with 10 μM KH-7 (D–F). (A,D) Flagellar bending patterns. Data from 100 (A) and 162 (D) waveforms are overwritten at 5 msec intervals. Dots and arrows indicate head position and direction of movement, respectively. (B,E) Changes in asymmetric index. Asymmetric index was calculated as the ratio of maximal curvatures of both bends (P-bend_Max/R-bend_Max). A raw value from each waveform (gray dot) and a smoothened value (red line) obtained using the Savitzky–Golay method (9 points) are shown above. (C,F) Flagellar curvature is plotted against the distance from the base of flagellum. Data from 20 waveforms from a–c (B) or d–f (E) are separately overwritten in (C) or (F), respectively.

Figure 3

Comparison of the maximal asymmetric index, duration of asymmetric wave formation and number of asymmetries during one turn between Ciona sperm treated with 0.5% DMSO (control) and 10 μM KH-7 (KH7). These parameters for wave asymmetry were estimated from the comparison between the P- and R-bend. Asymmetric index > 1.5 is defined as an asymmetric wave. Distribution of values is plotted in a box plot. *** Significant at p < 0.001, * p < 0.05 (Student’s t-test) in comparison to the control.

Figure 4

Effects of a sAC inhibitor KH-7 on [Ca²⁺]_i dynamics during sperm chemotaxis. (A,B) Trajectories of the sperm head (A) and changes in [Ca²⁺]_i signals yielded by the sperm tail (B) in Ciona sperm treated with 0.5% DMSO (control; DMSO) and 10 μM KH-7 (KH7) around the tip of a capillary containing 1 μM SAAF. In image (A), the origin of the coordinates indicates the capillary tip. The arrows indicate the swimming direction of the sperm. The dots show the head position with the color representing the average intensity of [Ca²⁺]_i signals in pseudo colors. The color scale represents the lookup table (LUT) for fluorescence signals. (C) Comparisons of [Ca²⁺]_i dynamics between Ciona sperm treated with 0.5% DMSO (control; DMSO) and 10 μM KH-7 (KH7). Data represent the mean and maximum of [Ca²⁺]_i, duration of [Ca²⁺]_i increase, time to the peak of [Ca²⁺]_i and the increasing and decreasing rate of [Ca²⁺]_I in the sperm around the tip of a capillary containing 1 μM SAAF. [Ca²⁺]_i is expressed as F/F0, which is the value of the fluorescent intensity from the whole flagella (F) divided by the average intensity of basal [Ca²⁺]_i before SAAF stimulation of the whole flagella (F0). Distribution of values is plotted in a box plot. *** Significant at p < 0.001, ** p < 0.01 (Student’s t-test) in comparison to the control.

Figure 5

Roles of sAC in the turn movement of Ciona sperm. The turn movement toward the egg consists of a weakly asymmetric wave (steady phase) to a highly asymmetric wave (turn phase), followed by a symmetric wave (straight swimming phase). 1. Steady state. While the SAAFs are bound to the plasma membrane Ca²⁺-ATPase (PMCA), which pump Ca²⁺ out from the cytoplasm. [Ca²⁺]_i is kept at a constant balance with the calcium efflux and a calcium channel, such as CatSper. 2. Turn state. When the SAAF dissociates from the PMCA, the Ca²⁺ efflux caused by the PMCA ceases and the calcium influx becomes dominant, inducing the increase in [Ca²⁺]_i. The increase in [Ca²⁺]_i induces an asymmetric wave through calaxin. Ca²⁺ simultaneously activates sAC and promotes cAMP synthesis. cAMP induces hyperpolarization by activating the tetrameric, cyclic nucleotide-gated, K+-selective channel (tetraKCNG). cAMP and hyperpolarization activate the hyperpolarization-activated and cyclic nucleotide-gated channel (HCN). Hyperpolarization also promotes the calcium efflux from the Na⁺/Ca²⁺ exchanger (NCX). 3. Straight swimming phase. In addition to the sAC-mediated Ca²⁺, the SAAF recruits PMCA again, resulting in a decrease in [Ca²⁺]_i and the formation of symmetric waves. cAMP also induces phosphorylation and dephosphorylation of axonemal proteins via the cAMP-dependent protein kinase (PKA), which causes the change in sperm’s flagellar waveform.