Solzin J et al. (2004), Revisiting the role of H+ in chemotactic signal...

ECB-ART-39102

J Gen Physiol 2004 Aug 01;1242:115-24. doi: 10.1085/jgp.200409030.

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Revisiting the role of H+ in chemotactic signaling of sperm.

Solzin J , Helbig A , Van Q , Brown JE , Hildebrand E , Weyand I , Kaupp UB .

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Chemotaxis of sperm is an important step toward fertilization. During chemotaxis, sperm change their swimming behavior in a gradient of the chemoattractant that is released by the eggs, and finally sperm accumulate near the eggs. A well established model to study chemotaxis is the sea urchin Arbacia punctulata. Resact, the chemoattractant of Arbacia, is a peptide that binds to a receptor guanylyl cyclase. The signaling pathway underlying chemotaxis is still poorly understood. Stimulation of sperm with resact induces a variety of cellular events, including a rise in intracellular pH (pHi) and an influx of Ca2+; the Ca2+ entry is essential for the chemotactic behavior. Previous studies proposed that the influx of Ca2+ is initiated by the rise in pHi. According to this proposal, a cGMP-induced hyperpolarization activates a voltage-dependent Na+/H+ exchanger that expels H+ from the cell. Because some aspects of the proposed signaling pathway are inconsistent with recent results (Kaupp, U.B., J. Solzin, J.E. Brown, A. Helbig, V. Hagen, M. Beyermann, E. Hildebrand, and I. Weyand. 2003. Nat. Cell Biol. 5:109-117), we reexamined the role of protons in chemotaxis of sperm using kinetic measurements of the changes in pHi and intracellular Ca2+ concentration. We show that for physiological concentrations of resact (<25 pM), the influx of Ca2+ precedes the rise in pHi. Moreover, buffering of pHi completely abolishes the resact-induced pHi signal, but leaves the Ca2+ signal and the chemotactic motor response unaffected. We conclude that an elevation of pHi is required neither to open Ca(2+)-permeable channels nor to control the chemotactic behavior. Intracellular release of cGMP from a caged compound does not cause an increase in pHi, indicating that the rise in pHi is induced by cellular events unrelated to cGMP itself, but probably triggered by the consumption and subsequent replenishment of GTP. These results show that the resact-induced rise in pHi is not an obligatory step in sperm chemotactic signaling. A rise in pHi is also not required for peptide-induced Ca2+ entry into sperm of the sea urchin Strongylocentrotus purpuratus. Speract, a peptide of S. purpuratus may act as a chemoattractant as well or may serve functions other than chemotaxis.

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Species referenced: Echinodermata
Genes referenced: hcn3 LOC100887844 LOC115919910 LOC576642 LOC576733 LOC583082 LOC590297

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	Figure 1. . Model of the cellular pathway involved in chemotactic signaling of sperm (from Darszon et al., 1999, 2001). Top, molecular components; bottom, sequence of cellular events. Binding of peptides to a receptor guanylyl cyclase (GC) increases the intracellular cGMP concentration. The sperm hyperpolarizes by opening cGMP-regulated K+ channels. Two transporters, a Na+/H+ and a Na+/Ca2+ exchanger, export protons and Ca2+, respectively, in response to the hyperpolarization; accordingly pHi rises and [Ca2+]i falls. The rise in pHi activates an adenylyl cyclase. The increase in cAMP concentration causes an influx of Ca2+. In principle, cAMP can stimulate the Ca2+ entry by two different mechanisms. First, the hyperpolarization and cAMP activate HCN channels (SPIH; Gauss et al., 1998); the ensuing depolarization of the cell activates voltage-dependent Ca2+ channels. Second, cAMP could activate a Ca2+ channel directly.
	Figure 2. . Changes in intracellular pH and [Ca2+] in sperm from A. punctulata induced by resact. (A) Changes in pHi detected by ΔF518 of BCECF. Sperm were stimulated at t = 0 with resact concentrations ranging from 0.125 pM to 2.5 nM; each trace represents the average of four recordings. (B) Resact-induced (25 pM) changes in pHi from four different sperm samples collected at different times during the season. All signals were normalized to the maximum. (C) Comparison of the kinetics of normalized Ca2+ and pHi signals after stimulation with 6.25 pM resact. (D) Dependence of the delay of the Ca2+ responses (gray trace) and the pHi responses (black trace) on the resact concentration. The delay was defined by the intercept between the regression line of the slope of the response rise and the time axis. Vertical bars represent the standard deviation from at least three experiments. The data of the Ca2+ responses are from Kaupp et al. (2003). The pH data were collected at the end of the season. (E) Effect of imidazole on pHi of sperm. The traces represent three different preincubation/mixing conditions. Sperm were preincubated for 5 min in ASW and then mixed with ASW/10 mM imidazole (ASW, +I); as a control, sperm were preincubated in ASW and then mixed with ASW (ASW, ASW); or sperm were preincubated in ASW/10 mM imidazole and then mixed with ASW (+I, ASW). F518 values at t = 0 have been set to zero. (F) Effect of imidazole on resact-induced changes in pHi. Sperm were preincubated for 5 min in ASW without imidazole (gray traces) or in ASW/10 mM imidazole (black traces). In the absence of imidazole, stimulation with 25 pM resact (−I, +R) induced an intracellular alkalinization. However, in the presence of imidazole, 25 pM resact (+I, +R) induced an acidification rather than an alkalinization. This acidification is due to the twofold dilution of extracellular imidazole by the mixing. As a consequence, imidazole escapes the cell, each molecule leaving a proton behind. Mixing of imidazole-incubated sperm with ASW without resact (+I, −R) resulted also in an acidification. Mixing of sperm with ASW (−I, −R) (control) did not change pHi. (G) Effect of imidazole on the resact-induced increase in [Ca2+]i. Sperm were preincubated in ASW without imidazole (gray traces) or in ASW/10 mM imidazole (black traces). 25 pM resact induced an increase in [Ca2+]i both in the absence (−I, +R) as well as in the presence of imidazole (+I, +R). Unstimulated sperm did not show an increase in [Ca2+]i upon mixing with ASW, neither in the presence (+I, −R) nor in the absence of imidazole (−I, −R).
	Figure 3. . Effect of cyclic nucleotides on pHi in A. punctulata sperm. (A) pHi responses induced by resact (25 pM) or by UV irradiation (arrow) of sperm loaded with caged cGMP (30 μM). (B) pHi responses induced by resact (25 pM) or by cAMP released from caged cAMP (30 μM).
	Figure 4. . Dependence of the pHi signal and cGMP synthesis on extracellular Ca2+ in A. punctulata sperm. (A) pHi signals at normal and low [Ca2+]o. Sperm were preincubated for 5 min in ASW and then mixed with 25 pM resact in EGTA-ASW. The final [Ca2+]o after mixing was <10−6 M. Resact induced a small and slow change in pHi (low Ca2+, +R). In the controls, sperm were either mixed with 25 pM resact in ASW (Ca2+, +R), or with ASW alone (Ca2+, −R). (B) Relative changes in cGMP concentration upon stimulation of sperm with different concentrations of resact. White bars represent the change in [cGMP] at normal extracellular [Ca2+] and black bars at low extracellular [Ca2+] (<10−6 M). Mean values ± SD are given for a stimulation period of 200 ms when the cGMP response was maximal at normal extracellular [Ca2+] (Kaupp et al., 2003). Data represent the means of at least three experiments (1 nM resact, 2.5 nM resact, n = 7; 25 nM resact, n = 4; 250 nM resact, n = 3). In each experiment triplicate measurements were done.
	Figure 5. . Behavior of A. punctulata sperm upon stimulation by resact released from caged resact. (A) Swimming trajectories of single sperm cells in a resact gradient produced by a flash of UV light directed to the central area of the visual field. The concentration of caged resact was 1 μM. Gray traces before and black traces after the UV flash. Intervals between consecutive dots, 80 ms. Bar, 100 μm. (B) Accumulation of sperm in the area of released resact in the presence of imidazole (10 mM). Distribution of sperm before photolysis (left) and 26 s after a 400-ms UV flash (right). Bar, 100 μm.
	Figure 6. . Changes in intracellular pH and [Ca2+] in S. purpuratus sperm induced by speract. (A) Changes in [Ca2+]i detected by ΔF518 of Fluo-4. Sperm were stimulated at t = 0 with speract concentrations ranging from 2.5 pM to 2.5 nM; each trace represents the average of three recordings. (B) Changes in pHi detected by ΔF518 of BCECF. Speract concentrations and number of signals for averaging as in panel A. (C) Comparison of the kinetics of normalized Ca2+ and pHi signals after stimulation with 6.25 pM speract. (D) Dependence of the delay of the Ca2+ responses (gray trace) and the pHi responses (black trace) on the speract concentration. Details as in Figure 2 D. (E) Effect of imidazole on speract-induced increase in [Ca2+]i. Sperm were preincubated in ASW without imidazole (gray traces) or in ASW/10 mM imidazole (black traces). 25 pM speract induced an increase in [Ca2+]i both in the absence (−I, +S) and in the presence of imidazole (+I, +S). Unstimulated sperm did not show an increase in [Ca2+]i upon mixing with ASW, neither in the presence (+I, −S) nor in the absence of imidazole (−I, −S). (F) Effect of imidazole on speract-induced changes in pHi. Sperm were preincubated in ASW without imidazole (gray traces) or in ASW/20 mM imidazole (black traces). In the absence of imidazole, stimulation with 25 pM speract (−I, +S) induced an intracellular alkalinization. However, in the presence of imidazole, 25 pM speract (+I, +S) induced an acidification rather than an alkalinization. Mixing of imidazole-incubated sperm with ASW, but without speract (+I, −S) resulted also in an acidification. Mixing of sperm with ASW (−I, −S) (control) did not substantially change pHi. The peptide-induced pHi responses of both S. purpuratus and A. punctulata sperm (Fig. 2 F) are inhibited in the presence of imidazole.
	Figure 7. . Effect of cyclic nucleotides on [Ca2+]i and pHi in S. purpuratus sperm. (A) Ca2+ response induced by UV irradiation (arrow) of sperm loaded with caged cGMP or caged cAMP (incubation with 30 μM each). (B) pHi response induced by UV irradiation (arrow) of sperm loaded with caged cGMP or caged cAMP (same concentration as in A).

References [+] :

Babcock, Early persistent activation of sperm K+ channels by the egg peptide speract. 1992, Pubmed, Echinobase