Human spermatozoa possess a calcium-dependent chloride channel that may participate in the acrosomal reaction

Abstract

Motility, maturation and the acrosome reaction (AR) are fundamental functions of mammalian spermatozoa. While travelling through the female reproductive tract, spermatozoa must mature through a process named capacitation, so that they can reach the egg and undergo the AR, an exocytotic event necessary to fertilize the egg. Though Cl⁻ is important for sperm capacitation and for the AR, not much is known about the molecular identity of the Cl⁻ transporters involved in these processes.We implemented a modified perforated patch-clamp strategy to obtain whole cell recordings sealing on the head of mature human spermatozoa.Our whole cell recordings revealed the presence of a Ca²⁺-dependent Cl⁻ current. The biophysical characteristics of this current and its sensitivity to niflumic acid (NFA) and 4,4-diisothiocyano-2,2-stilbene disulphonic acid (DIDIS) are consistent with those displayed by the Ca²⁺-dependent Cl⁻ channel from the anoctamin family (TMEM16). Whole cell patch clamp recordings in the cytoplasmic droplet of human spermatozoa corroborated the presence of these currents, which were sensitive to NFA and to a small molecule TMEM16A inhibitor (TMEM16Ainh, an aminophenylthiazole). Importantly, the human sperm AR induced by a recombinant human glycoprotein from the zona pellucida, rhZP3, displayed a similar sensitivity to NFA, DIDS and TMEM16Ainh as the sperm Ca²⁺-dependent Cl⁻ currents. Our findings indicate the presence of Ca²⁺-dependent Cl⁻ currents in human spermatozoa, that TMEM16A may contribute to these currents and also that sperm Ca²⁺-dependent Cl⁻ currents may participate in the rhZP3-induced AR.

Figure 1. Whole-cell patch-clamp recordings from mature human spermatozoa

A, fluorescence image sequences of a spermatozoon after achieving the whole-cell configuration in the perforated-patch mode. Fluorescein (1 μm) diffused from the pipette throughout the interior of the spermatozoon in ∼15 s. The pipette fluorescence is not seen because the approach of the pipette is perpendicular to the sperm head (see supplemental Fig. S1). B, whole-cell currents elicited by voltage steps from a holding potential of 0 mV (inset: step protocol) in physiological salt solution conditions (see Methods) without and with NFA (10 μm). C, current–voltage relationships (I–V curve) of results as in B in the absence and presence of 10 μm NFA, showing inward and outward rectification at hyperpolarized and depolarized membrane potentials. Note that the macroscopic current family and I–V plot show a NFA-sensitive current component. For C, data represent means ± SEM with n = 4.

Figure 2. The sperm macroscopic currents depend on the concentration of extracellular Cl⁻

A, family of macroscopic currents obtained from a spermatozoon exposed to high and low extracellular Cl⁻ concentrations ([Cl⁻]_e) using TEA-Cl solutions (see Methods). Currents were elicited by voltage steps, indicted in the voltage step protocol, from a holding potential of 0 mV. Note that lowering of [Cl⁻]_e from 147 to 12 mm strongly decreased outward currents. B, the I–V plot clearly shows the [Cl⁻]_e dependence of the current and the reversal potential (E_rev). Reducing [Cl⁻]_e shifts E_rev to the right, as expected for a Cl⁻ selective channel. C, The relation between the measured E_rev of the current and the calculated equilibrium potential for Cl⁻ is very close. The dashed line is the fitting expected for an ideal Cl⁻ channel. In all conditions [Ca²⁺]_i = 250 nm. For C, data represent means ± SEM with n = 5.

Figure 3. Behaviour of the Cl⁻ currents at different [Ca²⁺]_i

A, representative whole-cell currents recorded from one of four human spermatozoa dialysed with pipette solutions containing (from top to bottom) 1, 25, 250 and 1000 nm free Ca²⁺ with TEA-Cl standard solution in the bath. Currents were obtained from a holding potential of 0 mV with the voltage step protocol shown in the top panel. Note the strong [Ca²⁺]_i dependence of the macroscopic current; voltage is not sufficient to activate the current in [Ca²⁺]_i = 1 nm, and 25 nm activates only 4% of the current with respect to 1000 nm, the maximal concentration used. B, I–V plot of the currents obtained in A. C and D, illustrate concentration–response curves of the effect of different [Ca²⁺]_i on the macroscopic inward and outward Cl⁻ currents obtained from four independent experiments sealing on the sperm head and three on the cytoplasmic droplet (see Methods). Currents were normalized against the current obtained at 1000 nm[Ca²⁺]_i recorded at –120 mV (C) and +100 mV (D). The continuous lines represent the data fitted to a Hill equation with the following parameters: K_d = 236 nm and n_H = 1.3 at –120 mV and K_d = 163 nm and n_H = 1.9 at +100 mV. Data represent means ± SEM with n = 7.

Figure 4. Inhibition of the Ca²⁺-dependent Cl⁻ currents by NFA

A, typical family of whole-cell currents recorded from human spermatozoa under three different conditions: control, 3 and 11 μm of NFA. Currents were obtained from holding potential of 0 mV with the indicated voltage step protocol (top panel) in the presence of external TEA-Cl standard solution and 250 nm free Ca²⁺ in the pipette solution. B, I–V plots of the results obtained within the indicated range of NFA concentrations (A displays only three conditions). The current blocked was close to 100% with 35 μm NFA and after 10 min of washout ∼25% of the control current was recovered. C, NFA dose-dependently blocks the Ca²⁺-dependent Cl⁻ currents. The continuous line is the fit of the dose–response curve to the Hill equation which estimates an apparent IC₅₀ = 8.8 ± 1.7 μm. For C, data represent means ± SEM with n = 5–7.

Figure 5. Inhibition of the Ca²⁺-dependent Cl⁻ currents by DIDS

A, control whole-cell currents recorded from a human spermatozoon and blockage caused by 20 μm DIDS. Currents were obtained from a holding potential of 0 mV with the indicated voltage-step protocol (top panel) and recorded in the presence of external TEA-Cl standard solution and 250 nm free Ca²⁺ in the pipette solution. DIDS (20 μm) blocked the macroscopic currents by ∼55%. B, I–V relationships from five different spermatozoa without (control) or with 20 μm DIDS, which significantly inhibits the current (∼60% at 100 mV). C, DIDS dose-dependently blocks the Ca²⁺-dependent Cl⁻ currents. The continuous line is the fit of the dose–response data to the Hill equation which estimates an apparent IC₅₀ = 16 ± 1.4 μm. For B and C, data represent means ± SEM with n = 5–7.

Figure 6. Human spermatozoa patched at the cytoplasmic droplet display progesterone activated CatSper currents and CaCC currents inhibited by NFA and TMEM16A_inh

A, a representative monovalent whole-cell I_CatSper recorded from a human spermatozoon in HS solution (baseline trace, black) and in divalent-free solution (DVF) in the absence (dark grey), and presence (grey) of 500 nm of progesterone (n = 4). B, whole-cell CaCC currents recorded from a human spermatozoon dialysed with pipette solution containing 250 nm free Ca²⁺ and exposed to a bath TEA-Cl standard solution. Currents were obtained at a holding potential of 0 mV with the indicated voltage step protocol (top panel) in the absence or presence of 10 μm NFA. Note that 10 μm NFA inhibited nearly 50% of the current indicating that it is a similar current to that observed in Fig. 4. C, family of CaCC currents recorded from another spermatozoon before and after exposure to 10 μm TMEM16A_inh. D, current–voltage relationship (I–V) of currents normalized with respect to maximal current in control conditions measurements at the end of each voltage pulse of recordings as in B and C. E, dose dependent blockade of CaCC currents by TMEM16A_inh. Current amplitudes were measured at +100 mV by averaging seven to nine original current traces and normalizing with respect to the maximal blocked fraction. For E and D data represent the mean ± SEM with n = 6.

Figure 7. The rhZP3-induced AR in human spermatozoa is inhibited by CaCC/ TMEM16A_inh

Motile human spermatozoa were obtained by the swim-up technique and capacitated during 5 h. Sperm populations were preincubated for 15 min with different concentrations of NFA, DIDS and TMEM16A_inh. The AR was induced with rhZP3 (10 ng μl⁻¹). Cells were fixed with cold methanol and the acrosomal status was evaluated after staining sperm with FITC-PSA. Acrosomal reaction was expressed as an index (ARI, see Methods) and was used to estimate the percentage of AR inhibition (see Methods for details). NFA (A) and DIDS (B), two inhibitors of CaCCs, inhibited 90% of the AR. TMEM16A_inh (C) blocked approximately 80%, indicating that TMEM16A channels may have an important contribution in the AR. For all data, error bars represent ± SEM with n = 4–6. Statistical comparisons according to Student's unpaired t test indicate: *P < 0.05; **P < 0.01; ***P < 0.001 versus spermatozoa incubated with 0.1% DMSO + 10 ng μl⁻¹ of rhZP3.