J Physiol 590.11 (2012) pp 2659–2675 2659

The

Jou

rnal

of

Phys

iolo

gy

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

Gerardo Orta1, Gonzalo Ferreira2, Omar Jose1, Claudia L. Trevino1, Carmen Beltran1 and Alberto Darszon1

1Departamento de Genetica del Desarrollo y Fisiologıa Molecular, Instituto de Biotecnologıa, Universidad Nacional Autonoma de Mexico (UNAM),Cuernavaca, Morelos, C.P. 62210, Mexico2Departamento de Biofısica, Facultad de Medicina, Universidad de la Republica, Montevideo 11800, Uruguay

Key points

• Ion channels participate in crucial sperm functions such as motility, capacitation and theacrosome reaction.

• Chloride, the main anion in physiological solutions, is deeply involved in sperm physiology.• We implemented a modified perforated patch-clamp strategy to obtain whole cell recordings

sealing on the head of mature human spermatozoa to investigate their ion channels.• This work presents the first evidence for the presence of calcium-dependent chloride channels

(CaCCs) in human spermatozoa; they could be constituted by TMEM16.• The CaCCs play an important role in the physiology of human spermatozoa and participate in

the acrosome reaction.

Abstract Motility, maturation and the acrosome reaction (AR) are fundamental functions ofmammalian spermatozoa. While travelling through the female reproductive tract, spermatozoamust mature through a process named capacitation, so that they can reach the egg and undergothe AR, an exocytotic event necessary to fertilize the egg. Though Cl− is important for spermcapacitation and for the AR, not much is known about the molecular identity of the Cl− trans-porters involved in these processes. We implemented a modified perforated patch-clamp strategyto obtain whole cell recordings sealing on the head of mature human spermatozoa. Our whole cellrecordings revealed the presence of a Ca2+-dependent Cl− current. The biophysical characteristicsof this current and its sensitivity to niflumic acid (NFA) and 4,4′-diisothiocyano-2,2′-stilbenedisulphonic acid (DIDIS) are consistent with those displayed by the Ca2+-dependent Cl− channelfrom the anoctamin family (TMEM16). Whole cell patch clamp recordings in the cytoplasmicdroplet of human spermatozoa corroborated the presence of these currents, which were sensitiveto NFA and to a small molecule TMEM16A inhibitor (TMEM16Ainh, an aminophenylthiazole).Importantly, the human sperm AR induced by a recombinant human glycoprotein from thezona pellucida, rhZP3, displayed a similar sensitivity to NFA, DIDS and TMEM16Ainh as thesperm 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 thatsperm Ca2+-dependent Cl− currents may participate in the rhZP3-induced AR.

(Received 10 November 2011; accepted after revision 31 March 2012; first published online 2 April 2012)Corresponding author A. Darszon: Departamento de Genetica del Desarrollo y Fisiologıa Molecular, Instituto deBiotecnologıa, UNAM, Avenida Universidad No. 2001 Col. Chamilpa, CP 62210, Cuernavaca, Mor., Mexico.Email: darszon@ibt.unam.mx

Abbreviations AR, acrosome reaction; CaCC, calcium-dependent chloride channel; cAMP, adenosine 3′,5′-cyclicmonophosphate; DIDS, 4,4′-diisothiocyano-2,2′-stilbene disulphonic acid; NFA, niflumic acid; rhZP3, recombinanthuman glycoprotein from the zona pellucida number 3; TMEM16A, transmembrane protein 16A, also calledanoctamin-1; TMEM16Ainh, a small molecule TMEM16A inhibitor, ZP, zona pellucida.

C© 2012 The Authors. The Journal of Physiology C© 2012 The Physiological Society DOI: 10.1113/jphysiol.2011.224485

) by guest on March 26, 2014jp.physoc.orgDownloaded from J Physiol (

2660 G. Orta and others J Physiol 590.11

Introduction

From their germinal niche till they reach and fertilizethe egg, mammalian spermatozoa must travel a longand winding road. Upon ejaculation and during theirtransit through the female reproductive tract, spermatozoaacquire progressive motility and undergo molecular,biochemical and physiological changes referred to ascapacitation that enable them to reach and fertilize the egg(Bailey, 2010). To accomplish fertilization, spermatozoamust carry out the acrosome reaction (AR) (reviewedin Darszon et al. 2011). This exocytotic reaction enablesspermatozoa to penetrate the ZP matrix and fuse with theegg plasma membrane, generating a zygote. Though formany years it has been believed that the zona pellucida(ZP), a glycoproteinaceous matrix that surrounds themammalian oocyte, is the physiological inducer of the AR,how and where this reaction occurs has been re-examinedrecently (Ganguly et al. 2010; Inoue et al. 2011; Jinet al. 2011). The human ZP matrix is composed offour glycoproteins designated as ZP1 to ZP4; ZP3 isbelieved to be the main AR inducer (Conner et al. 2005;Caballero-Campo et al. 2006; Litscher et al. 2009). TheAR is a calcium-dependent process and it is inhibitedby several ion channel blockers, evidencing their pre-dominant role in this process (Espinosa et al. 1998;Mayorga et al. 2007). It is well established that motility,capacitation and the AR require diverse ions (Ca2+,HCO3

−, Na+, K+ and Cl−) (Visconti et al. 1995; Salicioniet al. 2007; Darszon et al. 2011).

In mouse spermatozoa, the absence of external Cl−

does not affect sperm viability, but capacitation-associatedprocesses such as the increase in tyrosine phosphorylation,the increase in cAMP levels, hyperactivation, theZP-induced AR and finally fertilization are abolished orsignificantly reduced (Wertheimer et al. 2008; Chen et al.2009). Similar results have been found in human sperm(Yeung & Cooper, 2008). As in other cells, Cl− is theprincipal anion that among other important functionsis implicated in sperm volume regulation and protectionfrom osmotic stress (Furst et al. 2002; Yeung et al.2005; Cooper & Yeung, 2007). Mammalian spermatozoaconfront drastic osmotic changes along their journey tofind the egg (Chen et al. 2010); for example, the acrosomeswelling that occurs after binding to ZP leads to AR(Zanetti & Mayorga, 2009). Therefore, it is likely that Cl−

plays a relevant role in sperm physiology. However, notmuch is known about the proteins that transport it acrossthe membrane of this fundamental cell.

Many different cell types in which cell volume controland secretion are critical (i.e. epithelial cells in exocrineglands and trachea, airway, vascular smooth muscle cells,reproductive tract smooth muscle cells, oviduct andductus epididymis cells, and mouse spermatids) expressCa2+-dependent Cl− channels (CaCCs), exhibiting similar

biophysical, pharmacological and molecular features(Hartzell et al. 2005; Huang et al. 2009; Kunzelmannet al. 2011). Interestingly, niflumic acid (NFA) and4,4′-diisothiocyanostilbene-2,2′-disulphonic acid (DIDS),two CaCC blockers, inhibit the ZP-induced mousespermatozoa AR in a similar dose-dependent manneras that with which they block CaCCs, indicating theirinvolvement in this exocytotic event (Espinosa et al.1998).

The long journey of spermatozoa is accompanied bydynamic changes in the concentration of intracellularCa2+ ([Ca2+]i) that trigger myriad signalling eventswhich could include the modulation of CaCCs, as wasdemonstrated in other cells (Arreola et al. 1996). Thoughcurrents mediated by CaCCs, initially documented inXenopus oocytes (Miledi, 1982), have now been recordedin many cells types, only recently has the transmembraneprotein TMEM16A been identified as one of the majormolecular counterparts of the CaCCs (Caputo et al. 2008;Schroeder et al. 2008; Yang et al. 2008). The developmentof small molecule inhibitors to investigate the contributionof TMEM16A to CaCC conductance in human tissues, air-way, intestinal epithelial and salivary gland cells allows thepharmacological dissection of TMEM16A/CaCC function(Namkung et al. 2010).

In spite of the evidence suggesting a role for CaCCsin human sperm physiology, they have never beenrecorded directly in these cells. In this work, we useda modified perforated patch-clamp technique to obtainwhole cell recordings sealing on the head of mature humanspermatozoa. Our findings show that these sperm possessCaCCs with biophysical and pharmacological propertiesresembling those reported in other native cells (Hartzellet al. 2005) and in CaCC expression cloning experimentsin amphibian and mammalian systems (Caputo et al. 2008;Schroeder et al. 2008; Yang et al. 2008). We corroborate thisby showing that these currents can be recorded sealing inthe cytoplasmic droplet, as has been done for CatSper, Hvand Slo3 (Kirichok et al. 2006; Lishko et al. 2010; Santi et al.2010). Our results are also consistent with a significantcontribution of TMEM16A like channels to the CaCCcurrents obtained by whole cell recordings from headsof human spermatozoa. In addition, our observationssuggest that these channels are involved in the humansperm rhZP3-mediated AR.

Methods

Ethical approval

This study was approved by the Bioethics Committeeat the Biotechnology Institute from the National Auto-nomous University of Mexico. All semen donors gavewritten informed consent.

C© 2012 The Authors. The Journal of Physiology C© 2012 The Physiological Society

) by guest on March 26, 2014jp.physoc.orgDownloaded from J Physiol (

J Physiol 590.11 Calcium-dependent chloride channel in human sperm 2661

Cell preparation, sperm capacitation and acrosomereaction assays

Ejaculates were obtained from healthy donors bymasturbation after at least 48 h of sexual abstinence.Only semen samples that fulfilled the World HealthOrganization guidelines were selected for experiments(WHO, 2010). Highly motile sperm were recovered aftera swim-up separation for 1 h in Ham’s F-10 medium at37◦C in a humidified atmosphere of 5% CO2–95% air.For electrophysiological experiments the cells were storedin physiological salt solutions immediately after swim-upseparation until used. For AR determinations the swim-upprocedure was carried out supplementing the F-10medium with 5 mg ml−1 of BSA and 2 mM CaCl2, pH 7.4.Cell concentration was then adjusted to 107 sperm ml−1

with the capacitating Ham’s F-10 medium, and incubationwas continued for 5 h. After capacitation, sperm werepreincubated with CaCC inhibitors (NFA, DIDS orTMEM16Ainh, 15 min in all cases) and the AR wasinduced in capacitated cells incubated for 30 min with10 ng μl−1 of recombinant human ZP3 (rhZP3), purifiedas reported previously (Jose et al. 2010). Stainingwith fluorescein isothiocyanate-labelled Pisum sativumagglutinin (FITC-PSA) was performed for evaluation ofthe AR. Sperm were washed with phosphate-bufferedsaline (PBS) and aliquots of the cell suspensions weresmeared onto AR glass slides and then air dried. Topermeabilize the plasma and outer acrosomal membranes,the slides were fixed with cold methanol for 30 s. Sampleswere incubated with FITC-PSA (25 μg ml−1 in PBS, pH7.4) for 30 min at room temperature in a moisturechamber. The excess of FITC-PSA was washed out withdistilled water, slides were dried again and finally theAR assays were evaluated with a fluorescence microscope(Zeiss Axioskop, excitation filter 450–490 nm, emissionfilter 520 nm). The AR patterns (bright fluorescence:acrosome intact; no fluorescence or only fluorescenceof the equatorial segment: acrosome reacted) wereevaluated in at least 200 cells per condition. Negative(no stimulation) and positive (A-23187 10 μM) controlswere included in all experiments. For each experiment,AR indexes (ARIs) were calculated by subtracting thepercentage of spontaneously reacted spermatozoa inthe negative control (value of 1.8 ± 0.4%, n = 3), fromthe raw mean values for all experimental conditions,expressing the results as a percentage of the ARobserved in the positive controls (value of 29.3 ± 0.6%,n = 3).

Data processing was done using the KyPlot 2.0 program(KyensLab Inc, Tokyo, Japan). In all cases, differencesbetween raw experimental and control data were testedby unpaired Student’s t tests. Differences were consideredsignificant when P < 0.05.

Whole-cell recording

We have applied the perforated-cell patch-clamptechnique to record ionic currents in the wholecell mode in mature human spermatozoa plated onpoly-lysine-coated coverslips. The patch pipette wasapplied at a 90 deg angle to the sperm cell head plasmamembrane using a three-axis piezo nano-positioning withservo-control (PZ 62E, Physik Instrumente, Germany) asrepresented in the online supplemental material Fig. S1.We used a mixture of saponin/β-escin (100 μg ml−1/5 μM)in the pipette tip (∼1 μl) to induce controlled patchpermeabilization, and were able to record stablewhole-cell currents after patch perforation from dialysedspermatozoa. This strategy has been used to record ioncurrents from cardiac myocytes and neurons (Fan &Palade, 1998; Sarantopoulos et al. 2004). It consists inperforating cell attached patches to obtain whole cellrecords, using a mixture of detergents in the pipettetip. The detergents were loaded through the back of thepipette after filling it with solution. The success rate toperforate the patch and record stable whole cell ioniccurrents for an adequate amount of time was about 50%.To visually confirm electrical and diffusion access to allsperm compartments (head, middle and principal pieces;Fig. 1A), using the adapted perforated patch technique;we dialysed the cell with internal solution containingfluorescein (1 μM) and recorded the fluorescence in aninverted fluorescence microscope (Olympus IX71) witha CCD camera. Glass pipettes (1BBL, World PrecisionInstruments, Inc., Sarasota, FL, USA) were designedto have 5–7 M� pipette resistance, when filled withinternal solution. The junction potential between inter-nal and external solutions, measured with the offsetcorrection of the patch amplifier, was usually about4–6 mV. Membrane potential was always corrected forthe liquid junction potential (∼4 mV). The chlorideconcentration was maintained at least at 5 mM to insurethe proper operation of the Ag–AgCl electrodes. Changingthe chloride concentration did have an effect on theliquid junction potential as we had to correct thepipette potential differentially for each concentration.The seal resistance was typically 3–5 G�. After sealingand achieving the whole cell configuration, capacitivetransients were obtained and used to measure spermcapacitance and series resistance (Fig. S2) to monitor thesuitability of the recording conditions with this method.Such transients are shown in Fig. S2A for the onset andoffset. High temporal resolution (>20 kHz) recordingsallowed estimates of clamp quality and time constants.Such a record is shown in Fig. S2B. A double exponentialof the form I = Io + I1 × (1 − exp(−t/τ1)) + I2 ×(1 − exp(−t/τ2)) was fitted to the capacitive transientdecay. Typical values of the best fit parameters were −52,

C© 2012 The Authors. The Journal of Physiology C© 2012 The Physiological Society

) by guest on March 26, 2014jp.physoc.orgDownloaded from J Physiol (

2662 G. Orta and others J Physiol 590.11

Figure 1. Whole-cell patch-clamp recordings frommature human spermatozoaA, fluorescence image sequences of a spermatozoonafter achieving the whole-cell configuration in theperforated-patch mode. Fluorescein (1 μM) diffusedfrom the pipette throughout the interior of thespermatozoon in ∼15 s. The pipette fluorescence is notseen because the approach of the pipette isperpendicular to the sperm head (see supplementalFig. S1). B, whole-cell currents elicited by voltage stepsfrom a holding potential of 0 mV (inset: step protocol)in physiological salt solution conditions (see Methods)without and with NFA (10 μM). C, current–voltagerelationships (I–V curve) of results as in B in the absenceand presence of 10 μM NFA, showing inward andoutward rectification at hyperpolarized and depolarizedmembrane potentials. Note that the macroscopiccurrent family and I–V plot show a NFA-sensitivecurrent component. For C, data representmeans ± SEM with n = 4.

38 and 14 pA for Io, I1 and I2, respectively, whereasthe time constants were 23 and 625 μS for the fast andslow components, respectively. The amplitude of the fastcomponent was about 2–3 times greater than that of theslow one, suggesting that the fast component may berelated to clamp conditions in the head and surroundingregions through the neck, whereas the slow one may berelated to the clamp conditions in the tail of the sperm.Hence, although the whole cell procedure reports currentsfrom all over the sperm, the recording conditions are betterand faster in the head and surrounding areas than in thetail. The area vs. time, charge (Q), under the capacitivetransients, needed to estimate membrane capacitance(Cm), sperm area and series resistance is shown in Fig. S2.The average charge measured under these conditions was24 ± 9 fC (n = 5) and the mean membrane capacitance ofthe whole sperm cell membrane was 1.3 ± 0.1 pF (n = 96).This measurement was quite similar to that reported by thecapacitance compensation from the Axopatch 200B. Thehuman sperm area determined by electron micrographsusing a stereological analysis method is approximately106 μm2 (Curry et al. 1996). The capacitance values foundby us are consistent with this value, assuming the specificCm to be 1 μF cm−2. For 1.3 pF average capacitance,the estimated sperm area under these assumptions isapproximately 130 μm2. Recordings were started after atleast 4 min dialysis to allow equilibration of the cytosoliccontent with the pipette solution.

Whole-cell macroscopic currents were acquired at10 kHz using an Axopatch 200B amplifier (MolecularDevices, Sunnyvale, CA, USA) interfaced with a PCequipped with a DigiData 1300A (Molecular Devices)

and filtered at 1–2 kHz (4-pole Bessel filter). Unlessindicated otherwise in the figure, membrane potentialwas changed from −120 to 100 mV in 10 mV steps bydelivering square pulses of 1 s duration every 5 s froma holding potential of 0 mV, followed by a 700 ms pulseto –120 mV. This protocol inactivates voltage dependentCa2+ and K+ currents. The leak of the seal, calculatedapplying Ohm’s law to current values measured at themiddle of the capacitive transient, was very low underthese circumstances (∼10–20 G�), suggesting that thistechnique did not alter the basic properties of themembrane.

During the course of this study Lishko et al. (2010)applied classical whole-cell patch clamp techniques forhuman sperm sealing at the cytoplasmic droplet. Usingthis strategy we were able to record CatSper currentsstimulated by progesterone (Fig. 6A) and Ca2+-dependentCl− currents inhibited by NFA and TMEM16Ainh (Fig. 6Band C).

Pulse protocols, and data capture and analysis wereperformed with pCLAMP software (Molecular Devices),SigmaPlot 9.0 (Systat Software Inc., San Jose, CA, USA)and Origin7.5 (OriginLab Corp., Northampton, MA,USA).

Solutions

Total whole-cell ionic currents were initially recordedusing normal physiological salt solutions (HS) withthe following composition; external solution (in mM):135 NaCl, 5 KCl, 1.8 CaCl2, 1 MgSO4, 5 dextrose, 10lactic acid, 1 sodium pyruvate, 10 Hepes, pH 7.35;

C© 2012 The Authors. The Journal of Physiology C© 2012 The Physiological Society

) by guest on March 26, 2014jp.physoc.orgDownloaded from J Physiol (

J Physiol 590.11 Calcium-dependent chloride channel in human sperm 2663

Table 1. Composition of solutions with different Cl−concentrations (in mM)

[Cl−]bath TEA-Cl CaCl2 D-Mannitol Dextrose TES

147 144 1.5 25 — 2070 67 1.5 147 20 2545 42 1.5 182 25 3512 8 2 228 30 50

[Cl−]pipette Ca-gluconate CaCl2 EGTA-TEA TEA-F TES

30 11.2 15 36.2 5 50

and internal solution (in mM): 10 NaCl, 140 KCl, 1MgSO4, 10 EGTA, 5 CaCl2, 5 ATP-Mg, 10 Hepes, pH7.25. The standard external solution for recording Cl−

currents had the following composition (in mM): 135TEA-Cl, 0.5 CaCl2, 45 D-mannitol, 20 TES, pH 7.35and 330 mosmol kg−1. The solutions with differentCl− concentrations are shown in Table 1. The tonicityof the internal solutions was 40 mosmol kg−1 morehypotonic than the external solutions, to get rid ofswelling-activated Cl− channels possibly present in thesecells (Yeung et al. 2005). The Ca2+-buffered pipettesolutions containing free [Ca2+] near 0 (1 nM) and up to1 μM were prepared as in Arreola et al. (1996). EGTA andN-(2-hydroxyethyl)ethylenedinitrilo-N ,N ′,N ′-triaceticacid (HEDTA) were selected as high and low affinityCa2+ buffers (K d values of 95 nM and 3.5 μM,respectively), to prepare Ca2+ buffers in the nano-and micromolar range, respectively (Table 2). ThepH of the pipette solutions was set to 7.25 withN-tris(hydroxymethyl)methyl-2-aminoethanesulphonicacid (TES). High concentrations of Ca2+ and pHbuffers assured control of both Ca2+ and pH duringthe course of these experiments. To determine the freeCa2+ concentrations we used the Maxchelator (v.2.1.)software program (written by Chris Patton, HopkinsMarine Station, Stanford University). Monovalent cationswere substituted by tetraethylammonium (TEA) in boththe internal and external solutions to avoid contaminationof the Cl− currents by K+ channels such as Slo3 or cationiccurrents through non-selective channels (Navarro et al.2007; Martinez-Lopez et al. 2009; Santi et al. 2009).

For recording monovalent CatSper currents, seals wereformed in HS solution and pipettes were filled with (inmM): 135 caesium methanesulphonate (CsMeSO3), 5CsCl, 10 EGTA, 5 Na-ATP, 0.5 Na-GTP, 10 Hepes, pH 7.3adjusted with CsOH. Bath divalent-free solution (DVF)for recording monovalent CatSper currents contained thefollowing (in mM): 140 CsMeSO3, 1 EDTA, 20 Hepes, pH7.4 adjusted with CsOH. HS solution was used to recordthe baseline current which was then exchanged by DVFfor measuring monovalent CatSper currents.

Chemicals

Bovine serum albumin (BSA), CaCl2, A23187, Ham’sF-10 medium, fluorescein isothiocyanate–Pisum sativumagglutinin (FITC-PSA), poly-lysine, niflumic acid,DIDS, saponin, β-escin, ionomycin, dimethyl sulfoxide(DMSO) and other inorganic salts were acquired fromSigma-Aldrich Chemical Co. (St Louis, MO, USA),and methanol was from J. T. Baker (Phillipsburg, NJ,USA). Stock solutions in DMSO were prepared for eachcompound and aliquots were stored at −20◦C.

Statistical analysis

Numeric results are expressed as means ± standard errormean (SEM); n, mean number of individuals, tested andanalysed with Student’s t test. Two-tailed P values <0.05were considered statistically significant.

Results

Whole-cell ionic currents

To investigate the mature human spermatozoa plasmamembrane ionic currents, we applied the perforated-cellpatch-clamp technique (see Methods) by attaching apatch pipette to the sperm head plasma membrane.Figure 1A shows the image of a spermatozoon afterachieving the whole-cell configuration and dialysing withthe fluorescent dye fluorescein (1 μM) through the patchpipette. Electrical access to all sperm compartments wascorroborated by the fluorescence throughout the whole

Table 2. Composition of internal solutions with different Ca2+ concentrations (in mM)

[Ca2+] TEA-Cl CaCl2 Ca- gluconate EGTA-TEA HEDTA-TEA TEA-F TES D-Mannitol

<10−6 47 — — 20 — 5 50 7425 × 10−6 37 5.2 — 25.2 — 5 50 94.5100 × 10−6 5 21 — 41 — 5 50 10250 × 10−6 — 23.5 2.7 36.2 — 5 50 —10−3 29.9 8.6 — — 38.6 5 50 18

C© 2012 The Authors. The Journal of Physiology C© 2012 The Physiological Society

) by guest on March 26, 2014jp.physoc.orgDownloaded from J Physiol (

2664 G. Orta and others J Physiol 590.11

interior of the human spermatozoon. The average timenecessary to accomplish cell dialysis after achieving agiga-ohm (G�) seal between the patch pipette and theplasma membrane of the sperm head was ∼15 s. Figure 1Bshows whole-cell macroscopic currents obtained fromthe same spermatozoon using physiological salt solutions(HS). Under these recording conditions the macroscopiccurrents result from a mixture of cationic (K+, Ca2+ orNa+) and anionic (Cl−) conductances. As Cl− is essentialfor sperm physiology (pointed out in the Introduction)and its transport systems not well defined, we decidedto explore the Cl− contribution to the sperm whole-cellmacroscopic currents, obtained by this methodology.The application of NFA decreased the total currentrevealing the presence of a minor but significant andreproducible NFA-sensitive component possibly due toCl− channels. The current–voltage relationship showedan inward and an outward rectifying component inthe negative and positive membrane potential values;specifically, at negative (−120 mV) and positive (100 mV)voltages, 10 μM NFA reduced the conductance 40% and17%, respectively (Fig. 1C).

Isolation and characterization of the Cl− conductance

Since NFA is best known as a blocker of Ca2+ regulatedCl− channels, we examined the Cl− dependence of themacroscopic current recorded in human spermatozoausing a modified bath solution containing differentTEA-Cl concentrations (see Table 1 and Fig. 2A). Toexamine if the currents could include a voltage-activatedCa2+ channel (CaV) current, we applied a pre-pulse to–120 mV after holding the cells at 0 mV. Under theseconditions we never observed such Ca2+ currents. Whenthe bath solution contained high Cl− concentrations,robust currents were recorded and they decreased asthe external Cl− concentration was reduced in theexternal medium. Lowering extracellular Cl− from 147to 12 mM strongly reduced the outward currents; seerecords in Fig. 2A and I–V curve in Fig. 2B. Differentextracellular Cl− concentrations, at a given intracellularCl− concentration, elicited a shift in the reversal potential(Erev) (Fig. 2C). Between the extreme extracellular Cl−

concentrations (147 and 12 mM), we found a shift of49 ± 4 mV in the reversal potential (Erev). The expectedshift for an ideal Cl− selective channel is 64 mV. Such adifference may indicate that the underlying channel has asmall, but significant, permeability to gluconate, as it hasbeen reported for several CaCCs (Frings et al. 2000; Kimet al. 2003). In addition, as there is external Ca2+ in themedium, CatSper (Lishko & Kirichok, 2010) and TRP-likechannels could contribute to the currents.

Ca2+-dependent Cl− currents

To determine if the Cl− currents recorded in maturehuman spermatozoa were due to CaCCs, we varied theCa2+ concentrations of the pipette internal solutionsand applied voltage-step protocols to avoid other ioniccurrents as described in Fig. 3A. The macroscopiccurrents were negligible when [Ca2+]i was 1 nM andvery small with 25 nM [Ca2+]i. Increasing the [Ca2+]i

to 250 or 1000 nM induced the appearance of a robustcurrent with an instantaneous component followed bya fractional slower component (Fig. 3A). This is clearlyillustrated in the current–voltage (I–V ) relationship atdifferent [Ca2+]i concentrations (Fig. 3B) and especiallyat [Ca2+]i = 1000 nM where noticeable increments arerecorded of the inward current at –120 mV and theoutward-rectifying current at +100 mV. The currentsat [Ca2+]i = 1000 nM are about 18- to 25-fold those at[Ca2+]i = 25 nM. Figure 3C shows steady-state averagecurrents measured at −120 mV normalized against thecurrent induced by 1000 nM of [Ca2+]i obtained fromrecordings in both the head, as well as by the nowestablished protocol sealing at the cytoplasmic spermdroplet (Lishko et al. 2010, 2011). To provide a quantitativedescription of the Ca2+ dependence, we fitted the Hillequation to these data (continuous line) and obtainedan apparent K d of 236 nM and a Hill coefficient (nH)of 1.3. Figure 3D shows the relative current normalizedagainst the current induced by 1000 nM of [Ca2+]i at+100 mV along with the fit of the Hill equation. Theapparent K d at +100 mV was 163 nM and nH of 1.9.The current at this potential was a very steep function of[Ca2+]i as indicated by the nH value. These results implythat the Ca2+ sensitivity of Cl− channels is a function ofmembrane potential as it has been reported for CaCCsin parotid acinar cells (Arreola et al. 1996). The K d andnH values at negative and positive membrane potentialare similar (nano-molar range) to those reported in ratparotid acinar cells (K d = 300 nM and nH = 1.2 at –66 mV;K d = 61 nM and nH = 2.7 at +74 mV) (Arreola et al.1996). Consistently, they are also similar to those reportedin heterologously expressed CaCCs (EC50 = 400 nM at60 mV) (Yang et al. 2008).

To further characterize the channels we recorded, weanalysed the dependence of the gating parameters ofCaCC activation with increasing [Ca2+]i. Figure S3Ashows typical records of CaCC obtained with the samepulse protocol as described before and [Ca2+]i = 500 nM.The gating for activation of the outward currents waswell described by a double exponential (see continuousline superimposed to the current trace). In Fig. S3B, weplotted the activation constants of the CaCC currentsversus [Ca2+]i. These results show that elevating [Ca2+]i

levels increased not only the currents amplitude, as

C© 2012 The Authors. The Journal of Physiology C© 2012 The Physiological Society

) by guest on March 26, 2014jp.physoc.orgDownloaded from J Physiol (

J Physiol 590.11 Calcium-dependent chloride channel in human sperm 2665

described in Fig. 3, but also accelerated the activationkinetics.

Pharmacology of Ca2+-dependent Cl− currents

To examine the pharmacological properties of the putativeCaCC currents recorded in mature human spermatozoa,two well known blockers of this channel were tested. Wefound that both NFA and DIDS potently affected theCa2+-dependent Cl− currents in cells containing 250 nM

of [Ca2+]i and under Cl− current recording conditions.As anticipated, the blockage became more pronounced asthe NFA concentration increased (Fig. 4A). The blockingeffect at the maximal NFA concentration (35 μM) testedwas partially reversible upon washout. Figure 4B shows theI–V curves obtained after the application and washout ofNFA. Best fit parameters of a Hill equation describing

the blockage of CaCC versus NFA concentration yieldedan IC50 = 8.8 ± 2 μM and an nH of 1.2. These results aresimilar to those reported from pharmacological studies inrabbit vascular smooth muscle cells (IC50 = 3.6 μM; Hogget al. 1994), as well as in the heterologous expressionof TMEM16A (IC50 = 29 μM; Schroeder et al. 2008).Figure 4C was constructed with the steady-state currentvalues obtained at 100 mV.

DIDS added to the bath solution had a similar blockingeffect to NFA on the currents. Figure 5A shows typicalrecordings of a family of currents evoked by voltage stepsfrom −120 to +100 mV under control conditions andafter adding DIDS (20 μM). The current decreased moreevidently in the positive range of the membrane potential.At 100 mV DIDS (20 μM) blocked about ∼52% of thecontrol current. I–V plots of the controls and 20 μM

DIDS are shown in Fig. 5B. The DIDS dose–response

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 inthe 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 thereversal potential (Erev). Reducing [Cl−]e shifts Erev to the right, as expected for a Cl− selective channel. C, Therelation between the measured Erev 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, datarepresent means ± SEM with n = 5.

C© 2012 The Authors. The Journal of Physiology C© 2012 The Physiological Society

) by guest on March 26, 2014jp.physoc.orgDownloaded from J Physiol (

2666 G. Orta and others J Physiol 590.11

Figure 3. Behaviour of the Cl− currents at different [Ca2+]iA, representative whole-cell currents recorded from one of four human spermatozoa dialysed with pipette solutionscontaining (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 concentrationused. B, I–V plot of the currents obtained in A. C and D, illustrate concentration–response curves of the effectof different [Ca2+]i on the macroscopic inward and outward Cl− currents obtained from four independentexperiments sealing on the sperm head and three on the cytoplasmic droplet (see Methods). Currents werenormalized against the current obtained at 1000 nM [Ca2+]i recorded at –120 mV (C) and +100 mV (D). Thecontinuous lines represent the data fitted to a Hill equation with the following parameters: Kd = 236 nM andnH = 1.3 at –120 mV and Kd = 163 nM and nH = 1.9 at +100 mV. Data represent means ± SEM with n = 7.

C© 2012 The Authors. The Journal of Physiology C© 2012 The Physiological Society

) by guest on March 26, 2014jp.physoc.orgDownloaded from J Physiol (

J Physiol 590.11 Calcium-dependent chloride channel in human sperm 2667

curve is shown in Fig. 5C. The normalized blockagefraction of the outward currents was plotted against theDIDS concentration in a semi log scale. The best fit ofa Hill equation is the continuous line between the datapoints. Best fit parameters were IC50 = 16 ± 1.4 μM andnH = 1.4. These results are similar to those reported frompharmacological studies in rat vascular smooth musclecells (IC50 = 16.5 μM; Baron et al. 1991), as well as inthe heterologous expression of TMEM16A (IC50 = 24 μM;Schroeder et al. 2008). This pharmacological profilefurther suggests that the outward rectifying currentsrecorded from the head of mature human spermatozoain our preparation are Ca2+-dependent chloridechannels.

Whole-cell patch clamp recordings in the cytoplasmicdroplet without detergent reveal Ca2+-dependent Cl−

currents blocked by TMEM16Ainh, a small moleculeTMEM16A Cl− channel inhibitor

The use of detergent to obtain perforated whole cellpatch clamp recordings in human spermatozoa couldalter the observed Ca2+-dependent Cl− currents. To ruleout this possibility, similar experiments were carriedsealing in the cytoplasmic droplet and achieving thewhole-cell configuration as described by Kirichok et al.(2006) and Lishko et al. (2010). Monovalent ICatSper

currents stimulated by progesterone were recorded as apositive control using this methodology (Fig. 6A). This

Figure 4. Inhibition of the Ca2+-dependent Cl− currents by NFAA, 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 voltagestep protocol (top panel) in the presence of external TEA-Cl standard solution and 250 nM free Ca2+ in the pipettesolution. B, I–V plots of the results obtained within the indicated range of NFA concentrations (A displays onlythree conditions). The current blocked was close to 100% with 35 μM NFA and after 10 min of washout ∼25% ofthe control current was recovered. C, NFA dose-dependently blocks the Ca2+-dependent Cl− currents. Thecontinuous line is the fit of the dose–response curve to the Hill equation which estimates an apparentIC50 = 8.8 ± 1.7 μM. For C, data represent means ± SEM with n = 5–7.

C© 2012 The Authors. The Journal of Physiology C© 2012 The Physiological Society

) by guest on March 26, 2014jp.physoc.orgDownloaded from J Physiol (

2668 G. Orta and others J Physiol 590.11

later experiment revealed NFA sensitive Ca2+-dependentCl− currents displaying similar characteristics (Fig. 6B)to those described in the human sperm perforated patchrecordings with similar reversal potential (approximately–15 mV) and outward rectification to depolarizationvoltages. The shape of currents generated by the voltagesteps, the tail current after repolarizing to –120 mV andthe clear activation by 250 nM Ca2+ are the same as thoseobtained using the perforated patch. Figure 6B shows atypical family of CaCC currents with [Ca2+]i = 250 nM incontrol conditions and after application of NFA 10 μM,using the same voltage step protocol and solutionsdescribed earlier. NFA inhibited nearly 50% of CaCCcurrents, as shown previously in Fig. 4.

To investigate the contribution of TMEM16A to thehuman sperm CaCC currents, we used TMEM16Ainh,

a small molecule (aminophenylthiazole) TMEM16Ainhibitor (generously provided by Dr Verkman, Universityof California). Figure 6C shows CaCC currents obtainedbefore and after application of 10 μM TMEM16Ainh. Asimilar percentage of inhibition of the CaCC currentsat +100 mV was observed with 10 μM NFA (∼50%)and 10 μM TMEM16Ainh (∼60%) (Fig. 6B and C). TheI–V plots of the blockage of the CaCC currents by10 μM NFA and 10 μM TMEM16Ainh are illustrated inFig. 6D. The normalized blockage fraction of the outwardcurrents (100 mV) was plotted against the TMEM16Ainh

concentration in a semi log scale. The best fit to a Hillequation is the continuous line between the data points.Best fit parameters were IC50 = 482 nM and nH = 0.9(Fig. 6E). These findings indicate that a major componentof the CaCC currents is blocked by TMEM16Ainh

Figure 5. Inhibition of the Ca2+-dependent Cl− currents by DIDSA, control whole-cell currents recorded from a human spermatozoon and blockage caused by 20 μM DIDS. Currentswere obtained from a holding potential of 0 mV with the indicated voltage-step protocol (top panel) and recordedin 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 blocksthe Ca2+-dependent Cl− currents. The continuous line is the fit of the dose–response data to the Hill equationwhich estimates an apparent IC50 = 16 ± 1.4 μM. For B and C, data represent means ± SEM with n = 5–7.

C© 2012 The Authors. The Journal of Physiology C© 2012 The Physiological Society

) by guest on March 26, 2014jp.physoc.orgDownloaded from J Physiol (

J Physiol 590.11 Calcium-dependent chloride channel in human sperm 2669

(approximately 60%) and appears to be carried throughTMEM16A channels.

Acrosome reaction assays

Our previous work in mouse spermatozoa suggested theparticipation of Cl− channels in the induction of AR(Espinosa et al. 1998). Considering this we tested if theCaCC blockers could inhibit the rhZP3-induced AR inthe human spermatozoa. The results in Fig. 7 show thatin the absence of rhZP3 the control (no addition) andthe addition of DMSO (drugs vehicle) displayed a lowacrosome reaction index (ARI) (∼1%). This index relatesthe AR induced by A23187, the maximum considered as∼100%, to that induced by rhZP3 (8–10%). Applicationof rhZP3 elevated the ARI to ∼30%. Figure 7A and Billustrate the NFA and DIDS dose-dependent inhibitionof the acrosome reaction. The estimated IC50 for thesecompounds (3 and 1 μM, respectively) are similar to thosedetermined for the CaCCs. Figure 7C shows that theapplication of 10 and 20 μM TMEM16Ainh diminishedthe ARI to about 5–10% (AR was ∼6-fold less than thecontrol with rhZP3). These results indicate that CaCCs,and possibly TMEM16A channels, have an essential rolein the human sperm AR as anticipated from our resultsfrom mice spermatozoa.

Discussion

In the present study, we demonstrated that humanmature spermatozoa possess a typical Ca2+-dependentCl− current. We implemented a modified perforatedpatch-clamp strategy to obtain whole cell recordings onthe head of mature human spermatozoa by includingdetergents in the patch pipette (see Methods). This strategyrepresents an alternative to sealing on the cytoplasmicdroplet, which has already opened new avenues for electro-physiological research in mouse and human spermatozoa(Lishko et al. 2010, 2011). Our new approach offers, inprinciple, an alternative to whole cell recordings in specieswhere the sperm cytoplasmic droplet is lost at or afterejaculation (e.g. bull, mouse, domestic cat) (Axner &Linde Forsberg, 2007; Cooper, 2011), or in those casesin which mature spermatozoa do not possess a dropletat all (e.g. sea urchin, starfish, and fish) (Pudney, 1995;Arakaki et al. 1998; Smith & Ryan, 2010). Previous studiesof single channel activity in cell-attached patches fromthe head of human spermatozoa revealed the presenceof a Cl−-permeable channel in the equatorial region.However, its full characterization was precluded by thepresence of different channel types closely associated inthe same patch and the fact that channels were recorded incell attached conditions without knowing the membranepotential and without controlling the intracellular ionicconditions (Jimenez-Gonzalez et al. 2007).

Using the perforated patch-clamp strategy we recordedrobust whole-cell Cl− currents, activated by depolarizationand Ca2+ (K d = 163 nM), and blocked by NFA(IC50 = 8.8 μM) and DIDS (IC50 = 16 μM) in maturehuman spermatozoa (Figs. 2–5). Indirect evidence forother Cl− channels such as CFTR and the GABAA

receptor has been reported in mouse and humanspermatozoa (Meizel, 1997; Ritta et al. 1998; Gong et al.2001; Hernandez-Gonzalez et al. 2007). However, thebiophysical and pharmacological characteristics of theCl− currents described in the present work, plus their[Ca2+]i dependence, indicate that they are mediatedby CaCCs (Suzuki et al. 2006). The [Cl−]e, [Ca2+]i

and voltage-dependent patterns displayed by the humanspermatozoa Cl− currents reported in this paper are quitecharacteristic of CaCCs and in particular of the TMEM16Achannels heterologously expressed. As in the heterologousexpressed TMEM16A channels, these currents also showa high [Ca2+]i-dependent time constant of activation(Fig. S3). Besides the biophysical properties mentionedabove, the pharmacological profile of the spermatozoaCa2+-dependent Cl− currents closely follows that shownby native CaCCs and heterologously expressed TMEM16Achannels (IC50 of 16–250 μM for DIDS and 2–44 μM forNFA) (Hartzell et al. 2005; Caputo et al. 2008; Schroederet al. 2008; Yang et al. 2008). In general the IC50 valuesfor drugs and the calcium dependence obtained forCaCCs in native cells are lower than those calculated inheterologous expression systems. These observations mayindicate subtle but important differences between CaCCisoforms, lipid environments or the presence of specificauxiliary subunits capable of modifying the propertiesof the channels. It will be important to investigate thesepossibilities in the future.

We have corroborated that the Cl− currents recordedwith the perforated patch are reproduced in cyto-plasmic droplet recordings. Furthermore, we show thatthese later currents are inhibited by the small moleculeTMEM16A inhibitor, which is consistent with the proposalthat TMEM16A channels may significantly contributeto the CaCC currents in human mature spermatozoa(Fig. 6B–E).

Two families of proteins, the bestrophins andthe anoctamins (e.g. bestrophin 1 and TMEM16A,respectively), recapitulate the properties of CaCCs,but TMEM16A shows most of the biophysical andpharmacological properties that have been attributedto CaCC in various tissues. Additionally, bestrophin1 knockout mice show normal fertility and thriving(Kunzelmann et al. 2009). Though our results do notrule out a partial contribution of bestrophins or otherTMEM16 family members as part of the CaCC currents werecorded in the head and the cytoplasmic droplet of humanspermatozoa, their overall characteristics resemble mostlythose described for TMEM16A CaCC (Almaca et al. 2009).

C© 2012 The Authors. The Journal of Physiology C© 2012 The Physiological Society

) by guest on March 26, 2014jp.physoc.orgDownloaded from J Physiol (

2670 G. Orta and others J Physiol 590.11

Figure 6. Human spermatozoa patched at the cytoplasmic droplet display progesterone activatedCatSper currents and CaCC currents inhibited by NFA and TMEM16AinhA, a representative monovalent whole-cell ICatSper recorded from a human spermatozoon in HS solution (baselinetrace, black) and in divalent-free solution (DVF) in the absence (dark grey), and presence (grey) of 500 nM ofprogesterone (n = 4). B, whole-cell CaCC currents recorded from a human spermatozoon dialysed with pipette

C© 2012 The Authors. The Journal of Physiology C© 2012 The Physiological Society

) by guest on March 26, 2014jp.physoc.orgDownloaded from J Physiol (

J Physiol 590.11 Calcium-dependent chloride channel in human sperm 2671

Figure 7. The rhZP3-induced AR in human spermatozoa is inhibited by CaCC/ TMEM16AinhMotile human spermatozoa were obtained by the swim-up technique and capacitated during 5 h. Spermpopulations were preincubated for 15 min with different concentrations of NFA, DIDS and TMEM16Ainh. The ARwas induced with rhZP3 (10 ng μl−1). Cells were fixed with cold methanol and the acrosomal status was evaluatedafter staining sperm with FITC-PSA. Acrosomal reaction was expressed as an index (ARI, see Methods) and wasused to estimate the percentage of AR inhibition (see Methods for details). NFA (A) and DIDS (B), two inhibitorsof CaCCs, inhibited 90% of the AR. TMEM16Ainh (C) blocked approximately 80%, indicating that TMEM16Achannels 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 versusspermatozoa incubated with 0.1% DMSO + 10 ng μl−1 of rhZP3.

TMEM16A is expressed in different tissues such assmooth muscle cells of reproductive tracts like the oviductand ductus epididymis (Huang et al. 2009). Its highexpression in these reproductive tissues is consistent withour findings and raises the possibility that this channelcould be implicated in physiological processes such ascapacitation and the AR of the human spermatozoa,as we suggest here (Fig. 7). The TMEM16 family iscomposed of 10 members (A through K), also knownas anoctamins 1 through 10. Although only TMEM16Aand TMEM16B have been shown to express functionalCaCCs (Schroeder et al. 2008), other members of thefamily may also form Cl− channels (Galietta, 2009). Thesequence identity between the TMEM16 family membersis relatively high, in particular in the transmembraneregions (about 50–60%). Furthermore, it is not clear yetwhether the Cl− channel formed by TMEM16A is a mono-

mer or a hetero-oligomer formed by other proteins and/orother members of the TMEM16 family (Galietta, 2009).From the reports currently known and our results, wesuggest that the major component of the TMEM16 familypresent in the human spermatozoa head, is TMEM16A. Weattempted to identify this protein using a TMEM16A anti-body which in total protein extracts detected a faint bandof the reported molecular mass (117 kDa). This proteinwas enriched by immunoprecipitation yielding a singleband of the correct molecular mass. As this antibody didnot reliably work to immunolocalize the channel in spermand we did not have the peptide to compete, or access tothe null TMEM16A mouse to establish the specificity ofthe antibody, we consider these results preliminary.

It has been shown that NFA and DIDS block micespermatozoa Cl− channels. NFA was reported to inhibit aCa2+ induced hyperpolarization which is partially driven

solution containing 250 nM free Ca2+ and exposed to a bath TEA-Cl standard solution. Currents were obtainedat a holding potential of 0 mV with the indicated voltage step protocol (top panel) in the absence or presence of10 μM NFA. Note that 10 μM NFA inhibited nearly 50% of the current indicating that it is a similar current to thatobserved in Fig. 4. C, family of CaCC currents recorded from another spermatozoon before and after exposure to10 μM TMEM16Ainh. D, current–voltage relationship (I–V ) of currents normalized with respect to maximal currentin control conditions measurements at the end of each voltage pulse of recordings as in B and C. E, dose dependentblockade of CaCC currents by TMEM16Ainh. Current amplitudes were measured at +100 mV by averaging sevento nine original current traces and normalizing with respect to the maximal blocked fraction. For E and D datarepresent the mean ± SEM with n = 6.

C© 2012 The Authors. The Journal of Physiology C© 2012 The Physiological Society

) by guest on March 26, 2014jp.physoc.orgDownloaded from J Physiol (

2672 G. Orta and others J Physiol 590.11

by Cl− and the AR induced by solubilized ZP (Espinosa &Darszon, 1995; Espinosa et al. 1998). With this in mind, theeffect of these compounds plus TMEM16Ainh was tested onthe rhZP3-induced AR in human spermatozoa. Consistentwith our previous results in mice, both NFA and DIDSinhibited the rhZP3-induced AR in a dose-dependentmanner with IC50 values of 3 and 1 μM, respectively, whichresemble those found for the blocking of the CaCC spermcurrents (Fig. 7). In addition, the application of saturatingconcentrations of the TMEM16Ainh (10 and 20 μM)inhibited nearly 80% of the AR, indicating TMEM16Achannels may participate in the human spermatozoa AR.

In recent times a remarkable pharmacological overlaphas been revealed between Ca2+-dependent Cl− channelsand the large conductance, Ca2+-gated K+ channels (BKCa

or KCa1.1) (Greenwood & Leblanc, 2007; Sones et al.2009). A wide range of structurally disparate agentsconsidered to be Cl− channel blockers, such as NFA,anthracene-9-carboxylate and ethacrynic acid, enhanceKCa1.1 currents (Ottolia & Toro, 1994; Greenwood &Large, 1995; Toma et al. 1996). As evidence suggeststhe possible presence of BKCa in mammalian sperm(Wu et al. 1998; Rossato et al. 2001), the combinedeffects of NFA could enhance its inhibitory potencyon the AR. This reaction involves prominent spermhead membrane rearrangements triggered by [Ca2+]i

changes (∼25–1000 nM) leading to acrosome swelling anda regulatory volume decrease which may involve CaCCs.By blocking CaCCs, NFA, DIDS and TMEM16Ainh couldalter cell volume regulation, which might influence thedistance between the outer acrosomal membrane andthe plasma membrane, which is critical for acrosomeexocytosis (Rossato et al. 1996; Zanetti & Mayorga,2009).

Recently, Sones et al. (2010) showed that cholesteroldepletion increases the amplitude of Ca2+-dependentCl− currents from murine portal vein myocytes.The cholesterol removing agent methyl-β-cyclodextrin(M-βCD) affected the distribution of KCa1.1 andTMEM16A in lipid rafts. Cholesterol removal from thesperm plasma membrane during capacitation results inits reorganization, which is essential for the induction ofthe AR (Osheroff et al. 1999; Visconti et al. 1999; BouKhalil et al. 2006; Girouard et al. 2008). It is possible thatthese mechanisms could modulate the AR by regulatingCa2+-dependent Cl− channels together with other spermion channels such as K+, Ca2+ and TRP channels (De Blaset al. 2009; Jose et al. 2010; Santi et al. 2010).

In summary, our data demonstrate the presenceof robust CaCC currents in human spermatozoa.Their pharmacological and biophysical characteristicsare consistent with TMEM16A channels contributingimportantly to them. Our findings also indicate that CaCCcurrents, and possibly TMEM16A, actively participate inthe AR triggered by rhZP3 in human spermatozoa.

Future studies are necessary to determine the preciselocalization and the physiological mechanisms thatcontrol the activity of the TMEM16A channels and howthey modulate the AR.

References

Almaca J, Tian Y, Aldehni F, Ousingsawat J, Kongsuphol P,Rock JR, Harfe BD, Schreiber R & Kunzelmann K (2009).TMEM16 proteins produce volume-regulated chloridecurrents that are reduced in mice lacking TMEM16A. J BiolChem 284, 28571–28578.

Arakaki Y, Uehara T & Fagoonee I (1998). Comparative studiesof the genus Echinometra from Okinawa and Mauritius.Zoolog Sci 15, 159–168.

Arreola J, Melvin JE & Begenisich T (1996). Activation ofcalcium-dependent chloride channels in rat parotid acinarcells. J Gen Physiol 108, 35–47.

Axner E & Linde Forsberg C (2007). Sperm morphology in thedomestic cat, and its relation with fertility: a retrospectivestudy. Reprod Domest Anim 42, 282–291.

Bailey JL (2010). Factors regulating sperm capacitation. SystBiol Reprod Med 56, 334–348.

Baron A, Pacaud P, Loirand G, Mironneau C & Mironneau J(1991). Pharmacological block of Ca2+-activated Cl−current in rat vascular smooth muscle cells in short-termprimary culture. Pflugers Arch 419, 553–558.

Bou Khalil M, Chakrabandhu K, Xu H, Weerachatyanukul W,Buhr M, Berger T, Carmona E, Vuong N, Kumarathasan P,Wong PT, Carrier D & Tanphaichitr N (2006). Spermcapacitation induces an increase in lipid rafts having zonapellucida binding ability and containingsulfogalactosylglycerolipid. Dev Biol 290, 220–235.

Caballero-Campo P, Chirinos M, Fan XJ, Gonzalez-GonzalezME, Galicia-Chavarria M, Larrea F & Gerton GL (2006).Biological effects of recombinant human zona pellucidaproteins on sperm function. Biol Reprod 74, 760–768.

Caputo A, Caci E, Ferrera L, Pedemonte N, Barsanti C, SondoE, Pfeffer U, Ravazzolo R, Zegarra-Moran O & Galietta LJ(2008). TMEM16A, a membrane protein associated withcalcium-dependent chloride channel activity. Science 322,590–594.

Chen Q, Peng H, Lei L, Zhang Y, Kuang H, Cao Y, Shi QX, MaT & Duan E (2010). Aquaporin3 is a sperm water channelessential for postcopulatory sperm osmoadaptation andmigration. Cell Res 21, 922–933.

Chen WY, Xu WM, Chen ZH, Ni Y, Yuan YY, Zhou SC, ZhouWW, Tsang LL, Chung YW, Hoglund P, Chan HC & Shi QX(2009). Cl− is required for HCO3

− entry necessary for spermcapacitation in guinea pig: involvement of a Cl−/HCO3

−exchanger (SLC26A3) and CFTR. Biol Reprod 80, 115–123.

Conner SJ, Lefievre L, Hughes DC & Barratt CL (2005).Cracking the egg: increased complexity in the zona pellucida.Hum Reprod 20, 1148–1152.

Cooper TG (2011). The epididymis, cytoplasmic droplets andmale fertility. Asian J Androl 13, 130–138.

Cooper TG & Yeung CH (2007). Involvement of potassium andchloride channels and other transporters in volumeregulation by spermatozoa. Curr Pharm Des 13, 3222–3230.

C© 2012 The Authors. The Journal of Physiology C© 2012 The Physiological Society

) by guest on March 26, 2014jp.physoc.orgDownloaded from J Physiol (

J Physiol 590.11 Calcium-dependent chloride channel in human sperm 2673

Curry MR, Millar JD, Tamuli SM & Watson PF (1996). Surfacearea and volume measurements for ram and humanspermatozoa. Biol Reprod 55, 1325–1332.

Darszon A, Nishigaki T, Beltran C & Trevino CL (2011).Calcium channels in the development, maturation, andfunction of spermatozoa. Physiol Rev 91, 1305–1355.

De Blas GA, Darszon A, Ocampo AY, Serrano CJ, CastellanoLE, Hernandez-Gonzalez EO, Chirinos M, Larrea F, BeltranC & Trevino CL (2009). TRPM8, a versatile channel inhuman sperm. PloS One 4, e6095.

Espinosa F & Darszon A (1995). Mouse sperm membranepotential: changes induced by Ca2+. FEBS Lett 372, 119–125.

Espinosa F, de la Vega-Beltran JL, Lopez-Gonzalez I, DelgadoR, Labarca P & Darszon A (1998). Mouse spermpatch-clamp recordings reveal single Cl− channels sensitiveto niflumic acid, a blocker of the sperm acrosome reaction.FEBS Lett 426, 47–51.

Fan JS & Palade P (1998). Perforated patch recording withβ-escin. Pflugers Arch 436, 1021–1023.

Frings S, Reuter D & Kleene SJ (2000). NeuronalCa2+-activated Cl− channels – homing in on an elusivechannel species. Prog Neurobiol 60, 247–289.

Furst J, Gschwentner M, Ritter M, Botta G, Jakab M, Mayer M,Garavaglia L, Bazzini C, Rodighiero S, Meyer G, EichmullerS, Woll E & Paulmichl M (2002). Molecular and functionalaspects of anionic channels activated during regulatoryvolume decrease in mammalian cells. Pflugers Arch 444, 1–25.

Galietta LJ (2009). The TMEM16 protein family: a new class ofchloride channels? Biophys J 97, 3047–3053.

Ganguly A, Bukovsky A, Sharma RK, Bansal P, Bhandari B &Gupta SK (2010). In humans, zona pellucida glycoprotein-1binds to spermatozoa and induces acrosomal exocytosis.Hum Reprod 25, 1643–1656.

Girouard J, Frenette G & Sullivan R (2008). Seminal plasmaproteins regulate the association of lipids and proteins withindetergent-resistant membrane domains of bovinespermatozoa. Biol Reprod 78, 921–931.

Gong XD, Li JC, Cheung KH, Leung GP, Chew SB & Wong PY(2001). Expression of the cystic fibrosis transmembraneconductance regulator in rat spermatids: implication for thesite of action of antispermatogenic agents. Mol Hum Reprod7, 705–713.

Greenwood IA & Large WA (1995). Comparison of the effectsof fenamates on Ca-activated chloride and potassiumcurrents in rabbit portal vein smooth muscle cells. Br JPharmacol 116, 2939–2948.

Greenwood IA & Leblanc N (2007). Overlappingpharmacology of Ca2+-activated Cl− and K+ channels.Trends Pharmacol Sci 28, 1–5.

Hartzell C, Putzier I & Arreola J (2005). Calcium-activatedchloride channels. Annu Rev Physiol 67, 719–758.

Hernandez-Gonzalez EO, Trevino CL, Castellano LE, de laVega-Beltran JL, Ocampo AY, Wertheimer E, Visconti PE &Darszon A (2007). Involvement of cystic fibrosistransmembrane conductance regulator in mouse spermcapacitation. J Biol Chem 282, 24397–24406.

Hogg RC, Wang Q & Large WA (1994). Action of niflumic acidon evoked and spontaneous calcium-activated chloride andpotassium currents in smooth muscle cells from rabbitportal vein. Br J Pharmacol 112, 977–984.

Huang F, Rock JR, Harfe BD, Cheng T, Huang X, Jan YN & JanLY (2009). Studies on expression and function of theTMEM16A calcium-activated chloride channel. Proc NatlAcad Sci U S A 106, 21413–21418.

Inoue N, Satouh Y, Ikawa M, Okabe M & Yanagimachi R(2011). Acrosome-reacted mouse spermatozoa recoveredfrom the perivitelline space can fertilize other eggs. Proc NatlAcad Sci U S A 108, 20008–20011.

Jimenez-Gonzalez MC, Gu Y, Kirkman-Brown J, Barratt CL &Publicover S (2007). Patch-clamp ‘mapping’ of ion channelactivity in human sperm reveals regionalisation andco-localisation into mixed clusters. J Cell Physiol 213,801–808.

Jin M, Fujiwara E, Kakiuchi Y, Okabe M, Satouh Y, Baba SA,Chiba K & Hirohashi N (2011). Most fertilizing mousespermatozoa begin their acrosome reaction before contactwith the zona pellucida during in vitro fertilization. ProcNatl Acad Sci U S A 108, 4892–4896.

Jose O, Hernandez-Hernandez O, Chirinos M,Gonzalez-Gonzalez ME, Larrea F, Almanza A, Felix R,Darszon A & Trevino CL (2010). Recombinant humanZP3-induced sperm acrosome reaction: evidence for theinvolvement of T- and L-type voltage-gated calciumchannels. Biochem Biophys Res Commun 395, 530–534.

Kim SJ, Shin SY, Lee JE, Kim JH & Uhm DY (2003).Ca2+-activated Cl− channel currents in rat ventral prostateepithelial cells. Prostate 55, 118–127.

Kirichok Y, Navarro B & Clapham DE (2006). Whole-cellpatch-clamp measurements of spermatozoa reveal analkaline-activated Ca2+ channel. Nature 439, 737–740.

Kunzelmann K, Kongsuphol P, Aldehni F, Tian Y, OusingsawatJ, Warth R & Schreiber R (2009). Bestrophin andTMEM16-Ca2+ activated Cl− channels with differentfunctions. Cell Calcium 46, 233–241.

Kunzelmann K, Kongsuphol P, Chootip K, Toledo C, MartinsJR, Almaca J, Tian Y, Witzgall R, Ousingsawat J & SchreiberR (2011). Role of the Ca2+-activated Cl− channels bestrophinand anoctamin in epithelial cells. Biol Chem 392, 125–134.

Lishko PV, Botchkina IL, Fedorenko A & Kirichok Y (2010).Acid extrusion from human spermatozoa is mediated byflagellar voltage-gated proton channel. Cell 140, 327–337.

Lishko PV, Botchkina IL & Kirichok Y (2011). Progesteroneactivates the principal Ca2+ channel of human sperm.Nature 471, 387–391.

Lishko PV & Kirichok Y (2010). The role of Hv1 and CatSperchannels in sperm activation. J Physiol 588, 4667–4672.

Litscher ES, Williams Z & Wassarman PM (2009). Zonapellucida glycoprotein ZP3 and fertilization in mammals.Mol Reprod Dev 76, 933–941.

Martinez-Lopez P, Santi CM, Trevino CL, Ocampo-GutierrezAY, Acevedo JJ, Alisio A, Salkoff LB & Darszon A (2009).Mouse sperm K+ currents stimulated by pH and cAMPpossibly coded by Slo3 channels. Biochem Biophys ResCommun 381, 204–209.

Mayorga LS, Tomes CN & Belmonte SA (2007). Acrosomalexocytosis, a special type of regulated secretion. IUBMB Life59, 286–292.

Meizel S (1997). Amino acid neurotransmitterreceptor/chloride channels of mammalian sperm and theacrosome reaction. Biol Reprod 56, 569–574.

C© 2012 The Authors. The Journal of Physiology C© 2012 The Physiological Society

) by guest on March 26, 2014jp.physoc.orgDownloaded from J Physiol (

2674 G. Orta and others J Physiol 590.11

Miledi R (1982). A calcium-dependent transient outwardcurrent in Xenopus laevis oocytes. Proc R Soc Lond B Biol Sci215, 491–497.

Namkung W, Phuan PW & Verkman AS (2010). TMEM16Ainhibitors reveal TMEM16A as a minor component ofcalcium-activated chloride channel conductance in airwayand intestinal epithelial cells. J Biol Chem 286, 2365–2374.

Navarro B, Kirichok Y & Clapham DE (2007). KSper, apH-sensitive K+ current that controls sperm membranepotential. Proc Natl Acad Sci U S A 104, 7688–7692.

Osheroff JE, Visconti PE, Valenzuela JP, Travis AJ, Alvarez J &Kopf GS (1999). Regulation of human sperm capacitation bya cholesterol efflux-stimulated signal transduction pathwayleading to protein kinase A-mediated up-regulation ofprotein tyrosine phosphorylation. Mol Hum Reprod 5,1017–1026.

Ottolia M & Toro L (1994). Potentiation of large conductanceKCa channels by niflumic, flufenamic, and mefenamic acids.Biophys J 67, 2272–2279.

Pudney J (1995). Spermatogenesis in nonmammalianvertebrates. Microsc Res Tech 32, 459–497.

Ritta MN, Calamera JC & Bas DE (1998). Occurrence of GABAand GABA receptors in human spermatozoa. Mol HumReprod 4, 769–773.

Rossato M, Di Virgilio F, Rizzuto R, Galeazzi C & Foresta C(2001). Intracellular calcium store depletion and acrosomereaction in human spermatozoa: role of calcium and plasmamembrane potential. Mol Hum Reprod 7, 119–128.

Rossato M, Di Virgilio F & Foresta C (1996). Involvement ofosmosensitive calcium influx in human sperm activation.Mol Hum Reprod 2, 903–909.

Salicioni AM, Platt MD, Wertheimer EV, Arcelay E, Allaire A,Sosnik J & Visconti PE (2007). Signalling pathways involvedin sperm capacitation. Soc Reprod Fertil Suppl 65, 245–259.

Santi CM, Butler A, Kuhn J, Wei A & Salkoff L (2009). Bovineand mouse SLO3 K+ channels: evolutionary divergencepoints to an RCK1 region of critical function. J Biol Chem284, 21589–21598.

Santi CM, Martinez-Lopez P, de la Vega-Beltran JL, Butler A,Alisio A, Darszon A & Salkoff L (2010). The SLO3sperm-specific potassium channel plays a vital role in malefertility. FEBS Lett 584, 1041–1046.

Sarantopoulos C, McCallum JB, Kwok WM & Hogan Q (2004).β-Escin diminishes voltage-gated calcium current rundownin perforated patch-clamp recordings from rat primaryafferent neurons. J Neurosci Methods 139, 61–68.

Schroeder BC, Cheng T, Jan YN & Jan LY (2008). Expressioncloning of TMEM16A as a calcium-activated chloridechannel subunit. Cell 134, 1019–1029.

Smith CC & Ryan MJ (2010). Evolution of sperm quality butnot quantity in the internally fertilized fish Xiphophorusnigrensis. J Evol Biol 23, 1759–1771.

Sones WR, Davis AJ, Leblanc N & Greenwood IA (2010).Cholesterol depletion alters amplitude and pharmacology ofvascular calcium-activated chloride channels. Cardiovasc Res87, 476–484.

Sones WR, Leblanc N & Greenwood IA (2009). Inhibition ofvascular calcium-gated chloride currents by blockers ofKCa1.1, but not by modulators of KCa2.1 or KCa2.3channels. Br J Pharmacol 158, 521–531.

Suzuki M, Morita T & Iwamoto T (2006). Diversity of Cl−channels. Cell Mol Life Sci 63, 12–24.

Toma C, Greenwood IA, Helliwell RM & Large WA (1996).Activation of potassium currents by inhibitors ofcalcium-activated chloride conductance in rabbit portal veinsmooth muscle cells. Br J Pharmacol 118, 513–520.

Visconti PE, Bailey JL, Moore GD, Pan D, Olds-Clarke P &Kopf GS (1995). Capacitation of mouse spermatozoa. I.Correlation between the capacitation state and proteintyrosine phosphorylation. Development 121,1129–1137.

Visconti PE, Galantino-Homer H, Ning X, Moore GD,Valenzuela JP, Jorgez CJ, Alvarez JG & Kopf GS (1999).Cholesterol efflux-mediated signal transduction inmammalian sperm. β-Cyclodextrins initiate transmembranesignaling leading to an increase in protein tyrosinephosphorylation and capacitation. J Biol Chem 274,3235–3242.

Wertheimer EV, Salicioni AM, Liu W, Trevino CL, Chavez J,Hernandez-Gonzalez EO, Darszon A & Visconti PE (2008).Chloride Is essential for capacitation and for thecapacitation-associated increase in tyrosinephosphorylation. J Biol Chem 283, 35539–35550.

WHO (2010). WHO Laboratory Manual for the Examinationand Processing of Human Semen. WHO, Geneva.

Wu WL, So SC, Sun YP, Chung YW, Grima J, Wong PY, YanYC & Chan HC (1998). Functional expression of P2Ureceptors in rat spermatogenic cells: dual modulation of aCa2+-activated K+ channel. Biochem Biophys Res Commun248, 728–732.

Yang YD, Cho H, Koo JY, Tak MH, Cho Y, Shim WS, Park SP,Lee J, Lee B, Kim BM, Raouf R, Shin YK & Oh U (2008).TMEM16A confers receptor-activated calcium-dependentchloride conductance. Nature 455, 1210–1215.

Yeung CH, Barfield JP & Cooper TG (2005). Chloride channelsin physiological volume regulation of human spermatozoa.Biol Reprod 73, 1057–1063.

Yeung CH & Cooper TG (2008). Potassium channels involvedin human sperm volume regulation–quantitative studies atthe protein and mRNA levels. Mol Reprod Dev 75,659–668.

Zanetti N & Mayorga LS (2009). Acrosomal swelling andmembrane docking are required for hybrid vesicle formationduring the human sperm acrosome reaction. Biol Reprod 81,396–405.

Author contributions

The conception and design of the electrophysiologicalexperiments was done by G.O., G.F. and A.D. The acrosomereaction assays were performed by O.J., C.L.T. and A.D. Thecollection, analysis and interpretation of electrophysiologicaldata were performed by G.O., and G.F. The drafting of the articleand critical revision for important intellectual content were doneby G.O., G.F., A.D., O.J., C.L.T. and C.B. All authors providedfinal approval of the version to be published. All experimentswere carried out at the Universidad Nacional Autonoma deMexico.

C© 2012 The Authors. The Journal of Physiology C© 2012 The Physiological Society

) by guest on March 26, 2014jp.physoc.orgDownloaded from J Physiol (

J Physiol 590.11 Calcium-dependent chloride channel in human sperm 2675

Acknowledgements

We thank Orlando Trujillo Domınguez and Yoloxochitl SanchezGuevara for their technical assistance, Dr Alan S. Verkman for thegenerous donation of the TMEM16A inhibitor used in this study,Dr Enrique Balderas for composing supplementary Figure 1 andDr Froylan Gomez-Lagunas for valuable suggestions. This work

was supported by NIH Grants R01 HD038082-07A1 (to P.V.),CONACyT-Mexico, (49113 to A.D., 47011 and 99333 to C.T.),DGAPA/UNAM (IN211809 and IN225406 to A.D., IN204109and IN202212-3 to C.T. and IN217409 and IN204112–3 to C.B.).A.D. and G.F. are also grateful for international cooperation aidfrom CSIC (UdelaR, Uruguay) and from the SSRREE (Mexico).We have no conflict of interest or disclosures.

C© 2012 The Authors. The Journal of Physiology C© 2012 The Physiological Society

) by guest on March 26, 2014jp.physoc.orgDownloaded from J Physiol (