The CatSper channel controls chemosensation in sea urchin sperm

Abstract

Sperm guidance is controlled by chemical and physical cues. In many species, Ca(2+) bursts in the flagellum govern navigation to the egg. In Arbacia punctulata, a model system of sperm chemotaxis, a cGMP signaling pathway controls these Ca(2+) bursts. The underlying Ca(2+) channel and its mechanisms of activation are unknown. Here, we identify CatSper Ca(2+) channels in the flagellum of A. punctulata sperm. We show that CatSper mediates the chemoattractant-evoked Ca(2+) influx and controls chemotactic steering; a concomitant alkalization serves as a highly cooperative mechanism that enables CatSper to transduce periodic voltage changes into Ca(2+) bursts. Our results reveal intriguing phylogenetic commonalities but also variations between marine invertebrates and mammals regarding the function and control of CatSper. The variations probably reflect functional and mechanistic adaptations that evolved during the transition from external to internal fertilization.

Keywords: Ca2+ signaling; CatSper; chemotaxis; sperm.

Figure 1

Features of ApCatSper 1–4, β, γ, and δ and localization of CatSper in A. punctulata sperm

1. Predicted membrane topology and predicted molecular weight of ApCatSper 1–4 cloned from A. punctulata testis, and of ApCatSper β, δ, and γ. A gene encoding ApCatSper δ was identified in the A. punctulata genome; transcripts encoding ApCatSper β and ApCatSper γ were identified in the A. punctulata transcriptome. S1 to S6, transmembrane segments; +, positively charged amino acids in S4; gray cylinder, coiled-coil domain.

2. Upper panel, alignment of pore regions of ApCatSper 1–4. Amino acids of the Ca²⁺ selectivity-filter motif are highlighted. Lower panel, alignment of S4 voltage-sensor segments of ApCatSper 1–4 and of D. melanogaster ShakerB K_v channel. Positively charged amino acids are highlighted. Numbers indicate start and end position of amino acids of the selected region.

3. Western blots of total protein of CHO cells transfected with ApCatSper 2 or 3, non-transfected control cells (-), and A. punctulata sperm (Sp). The Western blots were probed with anti-HA, anti-ApCatSper 2, or anti-ApCatSper 3 antibodies. Arrows indicate bands representing ApCatSper 2 and 3.

4. Immunocytochemical analysis of sperm stained with anti-ApCatSper 3, anti-GC, or anti-CNGK antibodies; superposition of images obtained by fluorescence and bright-field microscopy; scale bar = 10 μm. The DNA was stained with DAPI (blue).

5. Western blot analysis of co-immunoprecipitation (IP) of A. punctulata sperm proteins. The input (I), flow through (FT), washes (W1-5), and the eluate (E) of the IP using the anti-ApCatSper 2 antibody were probed with the anti-ApCatSper 3 antibody (upper panel) and vice versa (lower panel). ApCatSper 3 and ApCatSper 2 were co-immunoprecipitated with the anti-ApCatSper 2 (upper panel) and anti-ApCatSper 3 antibody (lower panel), respectively.

6. Analysis by mass spectrometry of immunoprecipitated proteins. ApCatSper 1-4, β, γ, and δ were identified in the immunoprecipitates obtained with both the anti-ApCatSper 2 and anti-ApCatSper 3 antibodies. The number of tryptic peptides identified and the respective sequence coverage are given.

Source data are available online for this figure.

Figure 2

CatSper inhibitors abolish alkaline-evoked Ca²⁺ influx in A. punctulata sperm

1. Alkaline-evoked Ca²⁺ signals in sperm mixed with NH₄Cl; sperm were loaded with the Ca²⁺ indicator Fluo-4. ΔF/F (%) indicates the change in Fluo-4 fluorescence (ΔF) with respect to the basal fluorescence (F, mean of the first 3–5 data points).

2. Ca²⁺ signals evoked by mixing of sperm simultaneously with NH₄Cl (30 mM) and the CatSper inhibitor MDL12330A.

3. Dose–response relation of inhibition of the Ca²⁺ signals shown in panel (B), K_i = 20 μM, amplitudes were determined at t = 4 s.

Figure 3

Determination of the threshold pH_i for alkaline-evoked Ca²⁺ influx

1. Changes in pH_i evoked by mixing with pH_i-clamp solutions (see explanation in the text); sperm were loaded with the pH_i indicator BCECF. ΔR/R (%) indicates the change in the BCECF fluorescence emission ratio (ΔR = F₄₉₄/F₅₄₀) with respect to the basal ratio (R, mean of the first 3–5 data points).

2. Steady-state change (at t = 14 s) of BCECF fluorescence for the pH_i signals shown in (A). The intercept of the fitted straight line with the x-axis yields the resting pH_i; the slope of the straight line yields the ΔR/R (%) × ΔpH⁻¹. Inset: calibrated changes in pH_i evoked by various pH_i-clamp solutions.

3. Ca²⁺ signals evoked by mixing of sperm with pH_i-clamp solutions.

4. Dose–response relation for the Ca²⁺ signals shown in (C).

5. Calibrated pH_i increase (red) and respective Ca²⁺ response (black) evoked by mixing of sperm with a pH_i 7.6-clamp solution; depicted are, on an extended time scale, the first 500 ms of the respective pH_i increase and Ca²⁺ signal shown in (B, inset) and (C), respectively. The threshold pH_i for CatSper activation was deduced from the latency of the Ca²⁺ signal.

6. Threshold pH_i and latency of Ca²⁺ signals evoked by various pH_i-clamp solutions (mean ± SD; n ≥ 3).

Figure 4

The threshold pH_i for alkaline-evoked Ca²⁺ influx is controlled by V_m

1. Depolarization-evoked Ca²⁺ signals in sperm mixed with ASW containing high KCl concentrations.

2. Ca²⁺ signals evoked by mixing of sperm with 80 mM KCl and the CatSper inhibitor MDL12330A.

3. Ca²⁺ signals evoked by mixing of sperm with 160 mM KCl and MDL12330A.

4. Dose–response relation for the Ca²⁺ signals shown in (B, C) at t = 1–2 s.

5. Threshold pH_i for Ca²⁺ signals evoked by pH_i-clamp solutions in sperm bathed in ASW containing low (3 mM), high (191 mM), and normal (9 mM) KCl (mean ± SD; n ≥ 3); data for 9 mM KCl are from Fig3F.

6. Resting pH_i and resting V_m in sperm bathed in ASW containing low (3 mM), high (191 mM), and normal (9 mM) KCl (black) (mean ± SD; n ≥ 3). Mean threshold pH_i for CatSper activation at different membrane potentials (red); mean threshold pH_i was derived from data shown in (E).

Figure 5

CatSper inhibitors abolish chemoattractant- and cGMP-induced Ca²⁺ influx

1. Ca²⁺ signals in sperm evoked by photorelease (at t = 0) of resact from caged resact in the presence of the CatSper inhibitor MDL12330A.

2. Ca²⁺ signals evoked by intracellular photorelease (at t = 0) of cGMP in sperm loaded with caged cGMP in the presence of the CatSper inhibitor MDL12330A.

3. Normalized dose–response relation for inhibition of the resact- and cGMP-induced Ca²⁺ signals shown in (A, B) (K_i = 6.2 and 4.3 μM, respectively).

4. Normalized dose-response relation for inhibition of the resact- and cGMP-induced Ca²⁺ signals shown in Supplementary Fig S4 by the CatSper inhibitor mibefradil (K_i = 7.7 and 20.9 μM, respectively).

Figure 6

The chemoattractant-induced Ca²⁺ influx is orchestrated by changes in pH_i and V_m

1. Calibrated V_m (left) and pH_i (right) changes and Ca²⁺ signals (middle) evoked by resact.

2. Normalized V_m, pH_i, and Ca²⁺ signals evoked by 10 pM resact; the first 600 ms after mixing are shown. The hyperpolarization precedes the pH_i increase, whereas the pH_i increase precedes the Ca²⁺ increase.

3. Latency of the V_m, the pH_i, and the Ca²⁺ signals evoked by various resact concentrations (mean ± SD; n = 3).

4. Normalized pH_i and Ca²⁺ signals evoked by photorelease of cGMP in sperm loaded with caged cGMP.

5. Calibrated V_m and Ca²⁺ signals evoked by 1 nM resact; the threshold voltage (V_thr) for the Ca²⁺ influx was deduced from the latency of the Ca²⁺ signal.

6. Calibrated pH_i and Ca²⁺ signals evoked by 1 nM resact; the threshold pH_i (pH_thr) at which the Ca²⁺ influx commences was deduced from the latency of the Ca²⁺ signal.

7. Linear relationship between pH_thr and V_thr for activation of Ca²⁺ influx by various resact concentrations (data derived from Supplementary Fig S6; mean ± SD; n ≥ 3).

Figure 7

CatSper inhibitors abolish chemotaxis of sperm in an chemoattractant gradient

1. Dark-field microscopy images of a sperm suspension before (top) and after (bottom) photorelease of a resact gradient (middle) in the absence (control; left) or presence of the CatSper inhibitor MDL12330A (10 μM; right). MDL12330A abolishes resact-induced sperm accumulation.

2. Relative change of the sperm dispersion in the field of view evoked by photorelease of resact (t = 0, flash) in the absence (control; blue) or presence of MDL12330A (red); a decrease in dispersion indicates sperm accumulation in the irradiated area (mean ± SD; n = 4).

Figure 8

Role of CatSper in chemosensory signaling pathways of sea urchin and mammalian sperm

1. Signaling pathways in sperm of A. punctulata (upper panel) and humans (lower panel). Questionmarks above dashed lines in the lower panel indicate that the mechanism of how ΔpH_i is produced and the hyperpolarization by Slo3 has not been firmly established. Solid lines indicate that the action of ΔpH_i on CatSper and of ΔpH_i and Ca²⁺ on Slo3 activity has been firmly established.

2. Model of the pH_i-induced change in the voltage dependence and V_thr of CatSper; the resact-induced hyperpolarization (black arrow) evokes a rapid alkalization that shifts the voltage dependence of CatSper activation to more negative values (blue arrow) and, thereby, primes CatSper to open during the subsequent depolarization (red arrow).

3. Changes in pH_i evoked by repetitive release (arrows) of cGMP from caged cGMP. The first UV flash was delivered at t = 0.