The CatSper channel: a polymodal chemosensor in human sperm

Abstract

The sperm-specific CatSper channel controls the intracellular Ca(2+) concentration ([Ca(2+)](i)) and, thereby, the swimming behaviour of sperm. In humans, CatSper is directly activated by progesterone and prostaglandins-female factors that stimulate Ca(2+) influx. Other factors including neurotransmitters, chemokines, and odorants also affect sperm function by changing [Ca(2+)](i). Several ligands, notably odorants, have been proposed to control Ca(2+) entry and motility via G protein-coupled receptors (GPCRs) and cAMP-signalling pathways. Here, we show that odorants directly activate CatSper without involving GPCRs and cAMP. Moreover, membrane-permeable analogues of cyclic nucleotides that have been frequently used to study cAMP-mediated Ca(2+) signalling also activate CatSper directly via an extracellular site. Thus, CatSper or associated protein(s) harbour promiscuous binding sites that can host various ligands. These results contest current concepts of Ca(2+) signalling by GPCR and cAMP in mammalian sperm: ligands thought to activate metabotropic pathways, in fact, act via a common ionotropic mechanism. We propose that the CatSper channel complex serves as a polymodal sensor for multiple chemical cues that assist sperm during their voyage across the female genital tract.

Figure 1

Odorant-induced Ca²⁺ signals in human sperm. (A) Ca²⁺ signals induced by bourgeonal. ΔF/F₀ (%) indicates the percent change in fluorescence (ΔF) with respect to the mean basal fluorescence (F₀) before application of bourgeonal. (B) Dose–response relationship of the signal amplitudes from (A) (K_1/2=65 μM). (C) Bourgeonal-induced Ca²⁺ signals in the presence of the CatSper channel inhibitors NNC 55-0396 and mibefradil. (D) Ca²⁺ signals induced by bourgeonal in non-capacitated sperm. (E) Ca²⁺ signals induced by bourgeonal and simultaneous application of bourgeonal and undecanal. (F) Ca²⁺ signals induced by undecanal. (G) Dose–response relationship of the signal amplitudes from (F) (K_1/2=30 μM). (H) Undecanal-induced Ca²⁺ signals in the presence of the CatSper channel inhibitors NNC 55-0396 and mibefradil.

Figure 2

Electrophysiological characterization of whole-cell CatSper currents from human sperm. Currents were recorded at pH_i 7.3 in the absence of intracellular divalent ions. The membrane voltage was stepped from −80 to +80 mV in steps of 10 mV from a holding potential of 0 mV. (A) Currents in extracellular solution containing Mg²⁺ and Ca²⁺ (HS) and monovalent CatSper currents in divalent-free Na⁺-based bath solution (NaDVF); perfusion with 80 μM bourgeonal potentiated monovalent currents (NaDVF+Bg). CatSper currents and currents evoked by bourgeonal were completely blocked by 30 μM mibefradil (NaDVF+Bg+Mib). (B) Current–voltage relationship from (A). (C) Currents in solution containing Ca²⁺ and Mg²⁺ (HS) and CatSper currents in divalent-free solution (NaDVF). Perfusion with 50 μM undecanal potentiated monovalent currents (NaDVF+Un). Currents were completely blocked by 30 μM mibefradil (NaDVF+Un+Mib). (D) Current–voltage relationship from (C).

Figure 3

Bourgeonal does not activate a cAMP-signalling pathway. (A) Changes in total cAMP concentration in human sperm bathed in high (25 mM) bicarbonate (HCO₃⁻) evoked by (in mM): 50 HCO₃⁻, 0.5 IBMX; (in μM) 50 bourgeonal, and 50 adenosine (5 measurements from 2 donors). The red lines indicate the 95% confidence intervals (CIs). Overlapping CIs indicate that there is no significant difference between conditions. (B) cAMP changes in sperm bathed in low (4 mM) bicarbonate evoked by (in mM): 50 HCO₃⁻, 0.5 IBMX; (in μM) 50 bourgeonal,50 adenosine, 30 chloro-adenosine, 500 SQ22536, 100 MDL12330A; (in nM) 100 FPP, 10 angiotensin II, 1.5 calcitonin (5–133 measurements from 4 to 16 donors). Data comprise data from Strünker et al (2011) combined with data from additional experiments.

Figure 4

Pharmacology of inhibitors for transmembrane adenylyl cyclases (tmACs) and phospholipase C (PLC). (A) Ca²⁺ signals induced by bourgeonal in the presence of the tmAC inhibitor SQ22536. (B) Mean amplitude of bourgeonal-induced Ca²⁺ signals in the absence and presence of 200 μM SQ22536 (four experiments). (C–E) Ca²⁺ signals induced by bourgeonal (C), progesterone (D) and ammonium chloride (NH₄Cl) (E) in the presence of the tmAC inhibitor MDL12330A. (F) Relative inhibition of the signal amplitudes by 100 μM MDL12330A (⩾4 experiments). (G) Whole-cell membrane currents in extracellular solution containing Ca²⁺ and Mg²⁺ (HS). Monovalent CatSper currents in divalent-free Na⁺-based bath solution before (NaDVF) and after intracellular alkalization by ammonium chloride (10 mM) (NaDVF+NH₄Cl) in the absence and presence of MDL12330A (100 μM). MDL12330A completely inhibited basal and alkaline-activated monovalent CatSper currents. Currents were recorded at pH_i 7.3 in the absence of intracellular divalent ions. (H) Ca²⁺ signals induced by the PLC inhibitor U73122.

Figure 5

Activation of CatSper by odorants does not involve a metabotropic pathway. (A) Bourgeonal-induced Ca²⁺ signals recorded in a stopped-flow apparatus. (B) Ca²⁺ signals from (A) shown on an extended time scale. Intracellular Ca²⁺ concentration rose within time resolution of the stopped-flow apparatus (36.6 ms). (C) Whole-cell membrane currents in extracellular solution containing Ca²⁺ and Mg²⁺ (HS). Monovalent CatSper currents in divalent-free Na⁺-based bath solution before (NaDVF) and after perfusion with 50 μM bourgeonal (NaDVF+Bg). Currents were recorded at pH_i 7.3 in the absence of intracellular divalent ions. The pipette solution contained GDPβS (250 μM) to preclude activation of G proteins.

Figure 6

Ca²⁺ signals evoked by 8-Bromo analogues of cyclic nucleotides. Signals induced by 8-Br-cGMP (A) or 8-Br-cAMP (B). (C) Dose–response relationship for 8-Br-cGMP based on the signal amplitudes from (A) (K_1/2=6.5 mM). (D) 8-Br-cGMP-induced Ca²⁺ signals in the presence of the CatSper inhibitors NNC 55-0396 or mibefradil. (E) 8-Br-cAMP-induced Ca²⁺ signals in the presence of NNC 55-0396 or mibefradil. (F) Relative inhibition of the signal amplitudes by NNC 55-0396 and mibefradil. Inhibition of signal amplitudes >100% indicates that 8-Br-cGMP evoked a decrease in [Ca²⁺]_i in the presence of the inhibitors.

Figure 7

Cyclic nucleotides and ATP do not stimulate Ca²⁺ influx. (A) Time course of [Ca²⁺]_i after stimulation with cAMP and cGMP. (B) Signal amplitudes evoked by cAMP, cGMP, and ammonium chloride (NH₄Cl) (four experiments). (C) Time course of [Ca²⁺]_i after stimulation with ATP. (D) Signal amplitudes induced by ATP and NH₄Cl (four experiments). (E) Whole-cell membrane currents in extracellular solution containing Ca²⁺ and Mg²⁺ (HS) before and after perfusion with ATP (100 μM). ATP did not stimulate membrane currents. Monovalent CatSper currents in divalent-free Na⁺-based bath solution (NaDVF) served as a positive control. Currents were recorded at pH_i 7.3 in the absence of intracellular divalent ions.

Figure 8

Whole-cell CatSper currents are potentiated by extracellular 8-Br-cGMP. Currents were recorded at pH_i 7.3 in the absence of intracellular divalent ions. Voltage was stepped from −80 to +80 mV in steps of 10 mV from a holding potential of 0 mV. (A) Currents in extracellular solution containing Ca²⁺ and Mg²⁺ (HS) and monovalent CatSper currents in divalent-free Na⁺-based bath solution (NaDVF). Extracellular 8-Br-cGMP (5 mM) potentiated monovalent currents (NaDVF+8-Br-cGMP). Currents were completely blocked by 30 μM mibefradil (NaDVF+8-Br-cGMP + Mib). (B) Current–voltage relationship from (A). (C) Currents recorded as described in (A), with 5 mM cGMP included in the pipette solution. (D) Current–voltage relationship from (C). (E) Currents recorded as described in (A), with 5 mM 8-Br-cGMP included in the pipette solution. (F) Current–voltage relationship from (E). (G) Mean current amplitudes at −60 mV (4–10 experiments) recorded in Ca²⁺ and Mg²⁺ containing solution (HS), in divalent-free solution (NaDVF), after perfusion with 5 mM 8-Br-cGMP (NaDVF+8-Br-cGMP), and after perfusion with 5 mM 8-Br-cGMP and 30 μM mibefradil (NaDVF+8-Br-cGMP+Mib). The currents were recorded either in the absence or in the presence of 5 mM cGMP or 8-Br-cGMP in the pipette solution. Extracellular but not intracellular application of cyclic nucleotides potentiated CatSper currents.

Figure 9

Normalized dose–response relationships for various compounds that activate CatSper. Each data point represents the mean of n⩾3.