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.

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).

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.

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.

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.

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.

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.

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.

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