Chloride Is Essential for Capacitation and for the Capacitation-associated Increase in Tyrosine Phosphorylation

Chloride Is Essential for Capacitation and for theCapacitation-associated Increase inTyrosine Phosphorylation*

Received for publication, June 16, 2008, and in revised form, September 19, 2008 Published, JBC Papers in Press, October 27, 2008, DOI 10.1074/jbc.M804586200

Eva V. Wertheimer‡, Ana M. Salicioni‡, Weimin Liu‡, Claudia L. Trevino§, Julio Chavez§,Enrique O. Hernandez-Gonzalez¶, Alberto Darszon§, and Pablo E. Visconti‡1

From the ‡Department of Veterinary and Animal Science, Paige Laboratories, University of Massachusetts, Amherst,Massachusetts 01003, the §Departamento de Genetica del Desarrollo y Fisiología Molecular, Instituto deBiotecnologia-Universidad Nacional Autonoma de Mexico, 62250 Cuernavaca, Mexico, and the ¶Departamento de BiologíaCelular, Centro de Investigacion y de Estudios Avanzados del Instituto Politecnico Nacional, 07360 Mexico City, Mexico

After epididymal maturation, sperm capacitation, whichencompasses a complex series of molecular events, endows thesperm with the ability to fertilize an egg. This process can bemimicked in vitro in definedmedia, the composition of which isbasedon the electrolyte concentrationof the oviductal fluid. It iswell established that capacitation requires Na�, HCO3

�, Ca2�,and a cholesterol acceptor; however, little is known about thefunction of Cl� during this important process. To determinewhether Cl�, in addition to maintaining osmolarity, activelyparticipates in signaling pathways that regulate capacitation,Cl� was replaced by either methanesulfonate or gluconate twononpermeable anions. The absence of Cl� did not affect spermviability, but capacitation-associated processes such as theincrease in tyrosine phosphorylation, the increase in cAMP lev-els, hyperactivation, the zona pellucidae-induced acrosomereaction, and most importantly, fertilization were abolished orsignificantly reduced. Interestingly, the addition of cyclic AMPagonists to sperm incubated in Cl�-free medium rescued theincrease in tyrosine phosphorylation and hyperactivation sug-gesting that Cl� acts upstream of the cAMP/protein kinase Asignaling pathway. To investigate Cl� transport, sperm incu-bated in complete capacitationmediumwere exposed to a batteryof anion transport inhibitors. Among them, bumetanide and furo-semide, two blockers of Na�/K�/Cl� cotransporters (NKCC),inhibited all capacitation-associated events, suggesting that thesetransporters may mediate Cl� movements in sperm. Consistentwith these results, Western blots using anti-NKCC1 antibodiesshowed the presence of this cotransporter inmature sperm.

Before becoming fertilization-competent, mammalian spermmust undergo a series of maturational processes in the femalereproductive tract (1). The molecular, biochemical, and physi-ological changes that occur in sperm, whereas in the femaletract are collectively referred to as capacitation. These func-tional changes associated with capacitation are not one eventbut are a combination of sequential and concomitant processesinvolvingmodifications at themolecular level occurring both inthe head (i.e. preparation for the acrosome reaction) and the tail(i.e. motility changes such as hyperactivation). Molecularevents implicated in the initiation of capacitation can be mim-icked in vitro and have been partially defined. These includeremoval of cholesterol from the sperm plasma membrane;modifications in plasma membrane phospholipids; fluxes ofHCO3

� and other intracellular ions; increased protein tyrosinephosphorylation; and hyperpolarization of the sperm plasmamembrane potential (Em) in mouse and other species (forreview see Ref. 2).With respect to the changes in the plasma membrane Em in

mouse sperm, it is hypothesized that the capacitation-associ-ated hyperpolarization results from changes in the activity ofion-selective channels and transporters. Consistent with thishypothesis, our studies in sperm from this species have revealedthe presence of amiloride-sensitive epithelial Na� channels(ENaCs)2 and the cystic fibrosis transmembrane regulator(CFTR) (3, 4). Experiments in those reports suggest that closingof ENaCs, with the consequent reduction in Na� influx,induces the hyperpolarization of the sperm Em observed duringcapacitation. Down-regulation of ENaC activity appears to be aconsequence of either the activation of CFTR or the influx ofCl�. Independent of our work, the presence of CFTR in spermwas also reported by Xu et al. (5); this group hypothesized that* This work was supported, in whole or in part, by National Institutes of Health

Grants HD38082 and HD44044 (to P. E. V.) from the Eunice Kennedy ShriverNICHD. This work was also supported by Fogarty International ResearchCollaboration Award Grant RO3 TW006121 (to P. E. V. and A. D.) and byfunds from the Consejo Nacional de Ciencia y Tecnología (CONACyT 49113to A. D.), the Direccion General de Asuntos del Personal Academico-Uni-versidad Nacional Autonoma de Mexico (DGAPA IN225406 to A. D. andIN227806-3 to C. T.), and the Wellcome Trust. The costs of publication ofthis article were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom correspondence should be addressed: Dept. of Veterinary andAnimal Sciences, 208 Paige Laboratories, University of Massachusetts,Amherst, MA 01003. Tel.: 413-545-5565; Fax: 413-545-6326; E-mail:[email protected].

2 The abbreviations used are: ENaC, epithelial Na� channel; NKCC, Na�/K�/Cl� cotransporter; CFTR, cystic fibrosis transmembrane regulator; ZP, zonapellucida; Sp-cAMPS, Sp-diastereomer of adenosine 3�,5�-cyclic mono-phosphothiorate; IBMX, 3-isobutyl-1-methylxanthine; DPC, diphe-nylamine-2-carboxylate; inh-172, 5-[(4-carboxyphenyl)methylene]-2-thioxo-3-[(3-trifluoromethyl)phenyl-4-thiazolidinone; DIDS, 4,4�-diiso-thiocyanatostilbene-2,2�-disulfonic acid; SITS, disodium 4-acetamido-4�-isothiocyanato-stilben-2,2�-disulfonate; DMSO, dimethyl sulfoxide; BSA,bovine serum albumin; PBS, phosphate-buffered saline; IVF, in vitro fertili-zation; PY, phosphotyrosine; CASA, computer-assisted semen analysis;ClC, Cl�-selective ion channel; KCC, K�/Cl� cotransporter; sAC, soluble ad-enylyl cyclase; PKA, protein kinase A.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 51, pp. 35539 –35550, December 19, 2008© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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in addition to its role as aCl� transporter, CFTR also transportsHCO3

�. As CFTR is primarily a Cl� channel and other aniontransporters are likely to be involved in capacitation (4), thepresent work focuses on the role of Cl� on sperm function.

Cl� can be transported through various systems across theplasma membrane including different types of Cl� channelsand a series of specialized carriers. Intracellular Cl� levels aredetermined by the relative contributions of all Cl� transporterspresent in the plasmamembrane of a given cell type. Cl� chan-nels are a subset of ion channels selective to Cl� and oftenpermeable to other small monovalent anions. Four structuralCl� channel families have been identified to date: 1) Cl�-selec-tive ion channels (ClCs); 2) CFTR channels; 3) the �-aminobu-tyric acid- and glycine-gated neurotransmitter receptors; and4) Ca2�-activated Cl� channels. In addition, functional datasuggest that other not yet identified Cl� channels may exist.TheCl� carrier proteins include: 1) the electroneutral and elec-trogenic Cl�/HCO3

� exchanger families (6) and 2) the electro-neutral cation-Cl� cotransporter family. All of these are sec-ondary active transporters, which means that the translocationof one ion is coupled to the translocation of another ion ineither the opposite direction (antiporter) or the same direction(cotransporter or symporter). The flow of the ion translocatedup the electrochemical gradient is coupled to the flow of a sec-ond ion down its electrochemical gradient, and thus the energyrequired does not come directly from ATP (7).In the present work we have analyzed whether Cl� ions reg-

ulate different events in the capacitation process. The centralobservations of this work are that: 1) Cl� is necessary for thecapacitation-associated increase in cAMP and tyrosine phos-phorylation; 2) sperm incubated in a medium without Cl� donot acquire either responsiveness to zona pellucidae (ZP) forthe induction of acrosome reactionnor hyperactivatedmotility;3) fertilization is inhibited if sperm are incubated under capac-itating medium without Cl�; 4) in the absence of Cl�, cAMP-permeable analogs can induce both the increase in tyrosinephosphorylation as well as hyperactivated motility; 5) bumet-anide and furosemide, two Na�/K�/Cl� cotransporter(NKCC) inhibitors, block capacitation and the capacitation-associated processes; and 6) at least one member of the NKCCfamily is present in mature sperm.

EXPERIMENTAL PROCEDURES

Materials—Chemicals were obtained from the followingsources. Bovine serum albumin (BSA, fatty acid-free), sodiummethanesulfonate, sodium gluconate, potassium gluconate,dibutyryl cyclic AMP, Sp-cAMPS, 8-bromo-cAMP, 3-isobutyl-1-methylxanthine (IBMX), carbonyl cyanide m-chlorophenyl-hydrazone, valinomycin, chlorotoxin, diphenylamine-2-car-boxylate (DPC), 5-[(4-carboxyphenyl)methylene]-2-thioxo-3-[(3-trifluoromethyl)phenyl-4-thiazolidinone (inh-172), bumeta-nide, furosemide,9-(hydroxymethyl)anthracene ,R(�)-butylinda-zone, (�)-bicuculline, 5-nitro-2-(3-phenylpropylamino)benzoicacid, indanyloxyacetic acid 94, hydrochlorothiazide, picrotoxin,DIDS, and SITS were purchased from Sigma. The fluorescentdyes 3,3-dipropylthiadicarbocyanine iodide (DiSC3-(5)) andHoestch 33342 were obtained from Invitrogen. Niflumic acidwas fromCalbiochem. Cyclic AMP analogs and inhibitors were

prepared fresh the day of the experiment in eitherMilli-Qwateror DMSO depending on solubility.Anti-phosphotyrosine (PY) monoclonal antibody (clone

4G10) was purchased from Upstate Biotechnology (LakePlacid, NY). In addition, for immunodetection of NKCC and�-tubulin, an anti-NKCC1 monoclonal antibody (clone T4)developed by Lytle et al. (8) and an anti-�-tubulin monoclonalantibody (Clone E7) developed by Chu and Klymkowsky (9)were obtained from the Developmental Studies HybridomaBank developed under the auspices of the National Institutes ofHealth, NICHD, and maintained by The University of IowaDepartment of Biological Sciences, Iowa City, IA. For negativecontrol donkey anti-mouse affinity-purified IgGwas purchasedfrom Jackson ImmunoResearch Laboratories (West Grove,PA).Mouse Sperm Preparation—Cauda epididymal mouse sperm

were collected from CD1 retired male breeders (Charles RiverLaboratories, Wilmington, MA) and sacrificed in accordancewith the Institutional Animal Care and Use Committee guide-lines.Minced cauda epididymis from each animal was placed in500 �l of a modified Krebs-Ringer medium (Whitten’s HEPES-buffered medium) (10). This medium does not support capac-itation unless supplemented with 5mg/ml BSA (fatty acid-free)and 15 mM NaHCO3. After 10 min, the sperm suspension waswashed by adding 1 ml of noncapacitating medium and poste-rior centrifugation at 800 � g for 5 min at room temperature.Sperm were then resuspended to a final concentration of 2 �107 cells/ml and diluted 10 times in the appropriate mediumdepending on the experiment performed. In experimentswhere capacitation was investigated, 5 mg/ml BSA and 15 mMNaHCO3 were added, and spermwere incubated at 37 °C for atleast 1 h. To study the role of Cl� in capacitation, NaCl and KClin the media were replaced either by sodium gluconate andpotassium gluconate or by sodium methanesulfonate andKOH. In all cases pHwasmaintained at 7.2.When differentCl�concentrations were assessed, the total NaCl plus sodium glu-conate (or sodium methanesulfonate) was maintained at 100mM. For the experiments in K�-free media, KCl and KH2PO4were replaced by NaCl and NaH2PO4, respectively. To test theeffect of the different inhibitors in capacitation, they were pre-incubatedwith sperm for 15min preceding the beginning of thecapacitating incubation. For the in vitro fertilization (IVF)assays, sperm were obtained and incubated for capacitation inWhitten’smediumwithoutHEPES containing 22mMNaHCO3and 15 mg/ml BSA and equilibrated in a humidified atmo-sphere of 5% CO2 in air (11).Sperm Membrane Purification—The preparation of sperm

fractions was carried out as described previously (12). Briefly,sperm (20� 107 cells) were homogenized using 10 strokes witha Teflon Dounce homogenizer in TE buffer (50 mM Tris-HCl,pH 7.5, 1 mM EDTA) supplemented with protease inhibitors(protease inhibitor mixture (Roche Applied Science) as indi-cated by themanufacturer plus 0.4 mM leupeptin, 0.4 mM apro-tinin, 0.1 mM pepstatin, 0.3 M benzamidine, and 0.32 mg/mlcalpain I and II inhibitor). After homogenization, the samplewas sonicated three times for 15 s on ice at intervals of 1 min.Cell debris was pelleted (1000 � g for 10 min at 4 °C), and thesupernatant was centrifuged at 10,000 � g for 10 min at 4 °C.

Chloride Is Essential for Capacitation

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Again, the resultant pellet was saved, and the supernatant thenwas centrifuged at 100,000 � g for 1 h at 4 °C. The final pellet,which contained the membrane fraction, was resuspended insample buffer and used for SDS-PAGE and immunoblotting.SDS-PAGE and Immunoblotting—After incubation under

different experimental conditions, sperm were spun down,washed in 1 ml of phosphate-buffered saline (PBS), resus-pended in Laemmli sample buffer (13) without�-mercaptoeth-anol, and boiled for 5 min. After centrifugation, the superna-tants were saved, and �-mercaptoethanol was added to a finalconcentration of 5%. The samples were boiled for 5 min andsubjected to SDS-PAGE using 8–10% mini-gels; proteinextracts equivalent to 1–2 � 106 sperm were loaded per lane.Each gel contained dual prestained molecular weight standard(Bio-Rad). Electrophoretic transfer of proteins to Immobilon(Bio-Rad) and immunodetection of tyrosine-phosphorylatedproteins were carried out using PY monoclonal antibodies asdescribed previously (14). For loading controls, membraneswere stripped, and an anti-�-tubulin monoclonal antibody wasused at a 5.2 ng/ml. For immunodetection of the NKCC, ananti-NKCC monoclonal antibody was used at a concentrationof 2.3 �g/ml. Immunoblots were developed with the appropri-ate secondary antibody conjugated to horseradish peroxidase(Jackson ImmunoResearch Laboratories) and ECL chemilumi-nescence reagents.Kidneys from CD1male retired breeders were collected, and

proteins were extracted using radioimmune precipitation assaybuffer (10 mM Tris, pH 7.2, 150 mMNaCl, 0.1% SDS, 1% TritonX-100, 1% deoxycholate, 5 mM EDTA, and protease and phos-phatase inhibitors). Protein concentration was assayed usingthe BCA kit from Pierce. In each lane 50 �g of total protein wasloaded in an SDS-8% polyacrylamide gel. Proteins were trans-ferred onto polyvinylidene difluoridemembranes, andWesternblotting was performed using anti-NKCC1 antibodies.Sperm Motility Analysis—Samples of sperm were incubated

in Whitten’s medium without HEPES at 37 °C and 5% CO2 for1 h. After incubation, the sperm suspension was loaded on a20-�m chamber slide (Leja slide, Spectrum Technologies) andplaced on amicroscope stage heated to 37 °C. Spermmovementwas examined using the CEROS computer-assisted semenanalysis (CASA) system (Hamilton Throne Research, Beverly,MA). The parameters used were: frames acquired � 30, framerate � 60 Hz, minimum cell size � 4 pixels, low average pathvelocity cutoff � 5 mm/s, static head size � 0.2–2.99, statichead intensity � 0.26–1.31, and static head elongation �0–100. Sperm with hyperactivated motility, defined as motilitywith high amplitude thrashing patterns and short distance oftravel, were sorted using the criteria established by Bray et al.(15): 1) curvilinear velocity (VCL, velocity calculated from thesum of track-point to track-point velocity) � 180 �m/s; 2)amplitude of lateral head displacement (ALH, mean width ofthe sperm head oscillation as the cell swims), � 9.5 �m; 3)linearity (LIN, departure of the cell from a straight line) � 38%.Acrosome Reaction Assay—Capacitation was measured indi-

rectly by determining the zona pellucidae-induced acrosomereaction based on the premise that only capacitated sperm willundergo exocytosis. Zona pellucidae were prepared fromhomogenized ovaries of virgin female 22-day-old outbred CD1

mice (Charles River Laboratories) as described (16, 17) and sol-ubilized by the procedures outlined previously (18). The per-centage of acrosome reaction was measured using CoomassieBlue G-250 staining as described by Visconti et al. (19). Briefly,following a 45-min incubation at 37 °C under the conditionsmentioned for each experiment, 5 zona pellucida equiva-lents/�l were added. After an additional 30min of incubation at37 °C, a fixative solution consisting of 5% final concentration offormaldehyde in PBS was added to each tube. Following fixa-tion, 10-�l aliquots of sperm suspension were spread onto glassslides and air-dried. The slides were then stained with 0.22%Coomassie Blue G-250 in 50% methanol and 10% glacial aceticacid for 3–5 min, gently rinsed with deionized H2O, air-dried,and mounted with 50% (v/v) glycerol in phosphate-bufferedsaline. To calculate the percentage of acrosome reaction, atleast 100 sperm were assayed per experimental condition forthe presence or absence of the characteristic dark blue acroso-mal crescent. The percentage of acrosome-reacted spermato-zoawas calculated for each experimental condition dividing thenumber of acrosome-reacted spermatozoa by the total numberof spermatozoa scored (sum of acrosome-reacted and non-ac-rosome-reacted) and multiplying this ratio by 100.Mouse Eggs Collection and IVF Assays—Egg collection was

performed as described previously (20). Briefly, metaphase II-arrested eggswere collected from6–8-week-old superovulatedCD1 female mice (Charles River Laboratories) at 13 h afterhuman chorionic gonadotropin (Sigma) injection. Cumuluscells were removed by brief incubation (�5 min) in Whitten’sHEPES-buffered medium with 7 mM NaHCO3, 5 mg/ml BSA,and 0.02% type IV-S hyaluronidase (Sigma). After cumulus cellremoval, eggs were placed in a drop of Whitten’s medium con-taining 22mMNaHCO3 and 15mg/ml BSA and then allowed torecover for 30 min in an incubator with 5% CO2 at 37 °C (11).Fertilization drops (200�l each) containing 10–20 eggs were

inseminated with sperm (final concentration of 2.5 � 106cells/ml) that had been incubated for 1 h under capacitating condi-tions. After 4 h of insemination, eggs were washed throughthree drops of Whitten’s medium containing 22 mM NaHCO3and 15 mg/ml BSA using a thin bore pipette to detach anyloosely attached sperm. After another 3 h of incubation, eggswere fixed with 3.7% paraformaldehyde/PBS for 15 min,washed, and stainedwithHoestch 33342 (Sigma; 10�g/ml finalconcentration) in PBS for 10 min at room temperature. Toassess fertilization the three following criteria were considered:1) the formation of the male and female pronuclei, 2) the emis-sion of the second polar body, and 3) the presence of the spermtail. The percentage of fertilization was calculated for eachfertilization drop by dividing the number of fertilized eggs bythe total number of eggs in that drop (sum of fertilized andnonfertilized eggs) and multiplying this ratio by 100.RNA Isolation and Reverse Transcription-PCR—Total RNA

was prepared from isolated mouse pachytene, round and elon-gated spermatids (60) using TRIzol reagent (Sigma) accordingto the manufacturer’s instructions. cDNA was synthesizedfrom total RNA samples with random hexamer-primed reversetranscription (Superscript II RNase H-reverse transcriptase;Invitrogen). cDNA was then subjected to PCR amplificationusing TaqDNA polymerase (Invitrogen). The NKCC1 primers




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were designed using the mouse-reported nucleotide sequencefor these genes (mouse NKCC GenBankTM accession numberNM_009194.2). Primer sequences for mouse NKCC1 were asfollows: forward, 5�-CCT GCT TTA CTT CAT C-3�; reverse,5�- GTC AAA CCT CCA TCA-3�. The absence of genomiccontamination in the RNA samples was confirmedwith reversetranscription negative controls (no reverse transcriptase) foreach experiment. Amplified products were analyzed by DNAsequencing in order to confirm their identity.Indirect Immunofluorescence—Sperm obtained by the swim-

upmethod inWhitten’sHEPES-bufferedmediumwerewashedonce, resuspended in PBS at a concentration of 1–2 � 105sperm/ml, and seeded on 8-well glass slides. After air-drying,sperm were fixed with 3.7% paraformaldehyde in PBS for 15min at room temperature, washed with PBS (four washes eachfor 5min), and permeabilizedwith 0.5%TritonX-100 for 5min.Following permeabilization, sperm were treated with 10% BSAin PBS for 1 h at room temperature and then incubated eitherwith the respective primary antibody (1:50–1:250) diluted inPBS containing 1% BSA or with the same concentration of thecorresponding affinity-purified IgG. Incubations were thencarried out at 4 °C overnight. After incubation, sperm werewashed thoroughly with PBS and incubated with the corre-sponding Alexa 555-conjugated secondary antibody (1:200)diluted in PBS containing 1% BSA for 1 h at room temperature;these solutions also contained Alexa 488-conjugated peanutagglutinin (1:100) for staining acrosomes. Incubation with thesecondary antibody was followed by four washes in PBS,mounted using SlowFade Light reagents (Molecular Probes,Eugene, OR), and observation by epifluorescence microscopyusing a TE300 Eclipse microscope (magnification �60)(Nikon).Differential interference contrast imageswere taken inparallel and served as control for sperm morphology. Negativecontrols using secondary antibody alone were also used tocheck for antibody specificity (not shown).cAMP Measurements—Sperm (5 � 106 sperm/ml) were

incubated for 1 h in noncapacitating (without BSA andNaHCO3) or capacitating medium (with BSA and NaHCO3) inthe presence or absence of Cl� or bumetanide (1mM). All treat-ments were supplemented with IBMX (0.1mM). Afterward, thesperm were centrifuged for 2 min at 800 � g, the pellet wasresuspended in 200 �l of HCl (0.1 mM), vortexed twice for 2 s,and incubated for 20 min at room temperature. Next, spermwere centrifuged at 5000� g for 5min, and the supernatantwasused to measure cAMP levels by using the BIOMOL format Acyclic AMP “PLUS” EIA kit (BIOMOL International, PlymouthMeeting, PA). A standard curve was run for each assay, and theunknown cAMP concentrations were obtained utilizing aweighted four-parameter logistic curve fitting (as recom-mended by manufacturer) with the aid of GraphPad software.Statistical Analysis—The data are expressed as the means �

S.E. The IVF experimental results were normalized to the con-trol values, which were considered as 100%. In order to assumenormal distribution, percentages were converted to ratios andall data subjected to the arcsine square root transformation(21). Statistical analysis was performed with the aid of Graph-Pad software using the parametric t test for simple comparisonsand either theTukey test following one-way analysis of variance

or the Bonferroni post-tests after two-way analysis of variancefor multiple comparisons.

RESULTS

Cl� Is Necessary for Capacitation—Cl� is involved in theregulation of the capacitation-associated hyperpolarization ofthe sperm Em (4). However, it has not been established whetherCl� acts upstream, downstream, or independently of other sig-naling pathways involved in capacitation. To analyze the role ofCl� in capacitation, media with different Cl� concentrationswere prepared by replacing this anion with gluconate asdescribed under “Experimental Procedures.” Initially, thecapacitation-associated increase in tyrosine phosphorylationwas analyzed by Western blot using anti-PY antibodies. Asshown previously (18), in the absence of BSA andHCO3

�, eitherwith or without Cl�, there is no increase in tyrosine phospho-rylation (Fig. 1A). When BSA and HCO3

� were present, theincrease in tyrosine phosphorylation was observed only in thepresence of Cl� (Fig. 1A). Moreover, the increase in tyrosinephosphorylation was dependent on the Cl� concentration(Fig. 1B). Because hexokinase (116 kDa) is a protein that isconstitutively phosphorylated in tyrosine residues (18), it iscommonly used as a loading control for Western blots withanti-PY antibodies. To confirm this understanding, as anadditional loading control each membrane was stripped andreproved with an anti-tubulin antibody (described under“Experimental Procedures”).Capacitation is linked to sperm flagellum hyperactivation

and to sperm preparation for agonist-induced acrosome reac-tion. To investigate the effect of Cl� on hyperactivatedmotility,CASA of sperm incubated in capacitating media with or with-out Cl� was carried out. Although the percentage of motilesperm did not differ in either medium (with or without Cl�),the percentage of hyperactivated cells was significantly reducedin the absence of Cl� (Fig. 1C). To assure that the absence ofCl� did not affect sperm viability, sperm membrane integritywas tested using propidium iodide, a nonpermeable dye com-monly used to assay this parameter. No difference in the per-centage of viable spermwas observed between sperm incubatedin media with and without Cl� (data not shown).Second, to test whether the presence of Cl� during capacita-

tion is needed for the acrosome reaction, mouse sperm wereincubated in media with or without Cl�, and the ZP-inducedacrosome reaction was determined. The ZP-induced acrosomereaction was significantly reduced when Cl� was not present(Fig. 1D). Because Cl� has been reported to be involved in theactual process of acrosome reaction (22–24), to ensure thatthe observed effect is on capacitation, sperm were incubated inthe absence of Cl� for 1 h. At that time the Cl�-free mediumwas supplemented with 100mMCl�, and ZP was added. Underthese conditions, the ZP-induced acrosome reaction was stillsignificantly inhibited (data not shown), indicating that theeffect observed was related to capacitation.Finally, only capacitated sperm are able to fertilize eggs in

vitro; therefore, the need for Cl� was tested on in vitro fertili-zation assays. Sperm incubated in capacitating media with dif-ferent concentrations of Cl� were co-incubated with eggs asdescribed under “Experimental Procedures,” and fertilization




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was evaluated at the pronuclei stage. In the absence of Cl� thepercentage of fertilized eggs was zero (Fig. 1E). To rule out apossible role of Cl� in the direct interaction between gametes,sperm capacitated in media containing Cl� were used to fertil-ize eggs in a medium without Cl�. Under these conditions thepercentage of fertilized eggs in media without Cl� was not sig-nificantly different from the control (Fig. 1F). Interestingly, theminimumCl� concentration needed for the increase in proteintyrosine phosphorylationwas between 50 and 75mM, similar tothe Cl� concentration needed for the ZP-induced acrosomereaction and for the IVF assays (Fig. 1B,D, and E).Whenmeth-anesulfonatewas used instead of gluconate to replaceCl� in theincubation media, the same set of capacitation-associatedparameters was analyzed, and in all cases similar results wereobtained (data not shown).Previously we showed that Na�/HCO3

� cotransporters arepresent in sperm (25). In particular, that study demonstratedthat HCO3

� additions induce membrane potential hyperpolar-ization in non-capacitated mouse sperm. Because HCO3

� is

involved in the regulation of solubleadenylyl cyclase (sAC), we analyzedwhether this initial HCO3

� trans-port was affected in the absence ofCl�. Similar to findings in controlmedia, the addition of HCO3

� tosperm in the absence of Cl� eliciteda membrane potential hyperpolar-ization (data not shown), suggestingthat HCO3

� transport through theNa�/HCO3

� cotransporter is inde-pendent of the presence of Cl� inthe incubation medium.Cyclic AMP Is Downstream of the

Effect of Cl� in Capacitation—It hasbeen reported that protein tyrosinephosphorylation in mammaliansperm is downstream of the cAMPpathway (26–29). To assesswhether the requirement for Cl� ismediated by cAMP, sperm wereincubated in media with or withoutCl� and in the presence or absenceof cAMP agonists (dibutyryl cAMP,SP-cAMPS, or 8-bromo-cAMP)and the phosphodiesterase inhibitorIBMX as described in the legend forFig. 2. Under these conditions thethree cAMP analogs were able toincrease tyrosine phosphorylationin Cl�-free medium (Fig. 2A), sug-gesting that cAMP is involved in theCl� requirement for this phospho-rylation event.CASA revealed that in the pres-

ence of dibutyryl cAMP and IBMX,the percentage of hyperactivatedsperm incubated in Cl� -freemedium is similar to the control

with Cl� (Fig. 2B), suggesting that the role of Cl� in hyperacti-vated motility is also mediated by cAMP. In contrast, whenIBMXanddibutyryl cAMPwere used in IVF experiments, thesecompounds were not able to increase the percentage of fertil-ized eggs when the experiment was conducted in the absence ofCl� (Fig. 2C). Because IBMX is dissolved in DMSO, 0.1%DMSO was present under all conditions tested to control fordeleterious solvent effect on IVF. The lack of action of cAMPagonists in this case suggests that in addition to its role in theincrease in tyrosine phosphorylation and hyperactivation,likely mediated by a cAMP pathway, Cl� might have additionalcAMP-independent functions in the capacitation process.Consistent with findings by Branham et al. (30), cAMP-perme-able analogs induced the acrosome reaction under all condi-tions tested, even in the absence of ZP (data not shown). There-fore, no conclusions were obtained in these experiments.Effect of Cl�Transport Inhibitors on Capacitation-associated

Processes—The observation that Cl� was required for theincrease in tyrosine phosphorylation, hyperactivation, acro-

FIGURE 1. Cl� is necessary for capacitation-associated events and for fertilization. A, capacitation-associ-ated tyrosine phosphorylation is inhibited in Cl�-free medium. Mouse sperm were incubated for 60 min inmedia that do not support (�BSA, �HCO3

�) (NC) or support capacitation (�BSA, �HCO3�) (C) in the presence or

absence of Cl� (replaced by gluconate). Subsequently, aliquots from each condition were processed for West-ern blotting with anti-PY antibodies (upper panel). The increase in tyrosine phosphorylated proteins wasobserved only when sperm were capacitated in the presence of Cl�. The stripped membranes were reblottedwith an anti-�-tubulin antibody for loading control (lower panel). B, the increase in tyrosine phosphorylation isdependent on Cl� concentration. Sperm were incubated in Whitten’s media containing BSA, HCO3

�, and dif-ferent Cl� concentrations. Sperm proteins were analyzed by Western blots with anti-PY antibodies (upperpanel). After being stripped, membranes were reblotted with an anti-�-tubulin antibody for loading control(lower panel). C, the percentage of hyperactivated sperm is reduced in the absence of Cl�. After capacitation inthe presence or the absence of Cl�, motility parameters were analyzed by CASA. The percentage of motile cells(upper panel) and the percentage of hyperactivation (lower panel) were compared between treatments asexplained under “Experimental Procedures.” D, Cl� is needed for the induction of acrosome reaction. Spermwere incubated with different Cl� concentrations in capacitation media, and then solubilized ZP was addedand incubated for induction of acrosome reaction. After fixation and staining with Coomassie Blue, thestatus of the acrosome was assessed in at least 100 sperm/treatment. E, IVF is inhibited in the absence ofCl�. Sperm incubated in capacitating media with different Cl� concentrations were used in IVF assays. Theinsemination drops contained 10 –20 eggs and had the same Cl� concentration as the capacitation media.F, the absence of Cl� during the co-incubation does not affect the interaction between gametes. Eggswere co-incubated in the absence of Cl� with sperm capacitated in control media. The percentage offertilization in the control was 75 � 8. Each experiment was repeated at least three times. Hyperactivationexperiments: *, p � 0.05 versus control (100 mM Cl�) using two-tailed Student’s t test. Acrosome reaction:*, p � 0.05 comparing basal with ZP-induced acrosome reaction for each Cl� concentration using Bonfer-roni post tests. IVF experiments: Tukey’s multiple comparison test was performed. The means of groupsthat have different letters differ significantly (p � 0.05).




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some reaction, and IVF suggests that Cl� transport in spermplays an important role in capacitation. Moreover, thishypothesis is consistent with previous results indicating thatduring capacitation there is an increase in intracellular Cl�concentration (4). Despite these data, howCl� enters the spermis not known. To investigate the nature of the transporters medi-atingCl� fluxes during capacitation, the increase in tyrosinephos-phorylation was used as an end point to test a battery of knowninhibitors. Previous observations (4) indicated that the CFTRantagonistsDPCand inh-172 arenot able to inhibit the increase intyrosine phosphorylation.Herewe show the same lack of effect oncapacitation-dependent tyrosine phosphorylation, hyperactiva-tion, and motility even at concentrations higher than the oneseffective in CFTR regulation (Fig. 3, A and B, and Table 1).Similarly, bicuculline and picrotoxin, �-aminobutyric acid-Areceptor/Cl� channel antagonists (31); chlorotoxin and niflumicacid, two Ca2�-dependent Cl� channel inhibitors (32, 33); and5-nitro-2-(3-phenylpropylamino)benzoic acid, 9-(hydroxym-ethyl)anthracene, indanyloxyacetic acid 94, and propionic acid,knownClC family blockers (34–36), were not able to inhibit thecapacitation-associated increase in tyrosine phosphorylation(Fig. 3C andTable 1). Although these results do not rule out thepresence of the respective Cl� transporters in sperm, they sug-gest that these transporters do not play a key role in the eventsleading to the increase in tyrosine phosphorylation. On theother hand, as expected (25), more general Cl�-transportinhibitors such as stilbene compounds (SITS, DIDS) were ableto block the increase in tyrosine phosphorylation as well assignificantly reduce sperm hyperactivation and IVF (Fig. 3,D–F, and Table 1). Although these results suggest that aniontransport participates in sperm capacitation, it is not possible toconclude which type of transporter is involved because thesecompounds can inhibit a range of molecules.We then tested whether the family of electroneutral Cl�

cotransporters participated in the capacitation-associatedevents. This later transporter family is composed of threesubfamilies with a total of seven members (37, 38): one Na�/Cl� cotransporter (NCC), which can be inhibited by diuret-ics such as hydrochlorothiazide; two NKCCs, inhibited bybumetanide and less potently by furosemide; and four Na�-independent K�/Cl� cotransporters (KCC), which can be

specifically blocked by R(�)-bu-tylindazone (39). No effects wereobserved on tyrosine phosphoryl-ation (Fig. 3C and Table 1) witheither hydrochlorothiazide orR(�)-butylindazone. Notably, bu-metanide and furosemide had aconcentration-dependent inhibi-tory effect on the increase in pro-tein tyrosine phosphorylation(Fig. 4A; Table 1). In addition,although bumetanide had noeffect on the percentage of motilecells (Fig. 4B, left), it significantlyreduced the percentage of hyper-activated sperm (Fig. 4B, right)and inhibited ZP-induced acro-

some reaction and in vitro fertilization (Fig. 4, C and D).Bumetanide is an NKCC inhibitor; however, its effect on

the increase in protein tyrosine phosphorylation, hyperacti-vation, and IVF was observed at higher concentrations ofthese compounds than the ones known to inhibit NKCCtransporters in other cell types (40). On the other hand, theZP-induced acrosome reaction was inhibited with lowerconcentrations saturating at 10 �M, a concentration com-patible with NKCC inhibition (Fig. 4C). To test whether theeffect of low bumetanide concentrations was related tocapacitation or directly affecting the ZP-induced acrosomereaction, the inhibitor was either added during capacitationand removed by centrifugation before addition of ZP oradded at the same time as the ZP. Under both conditions, theacrosome reaction was inhibited (Fig. 4C). Hence, in addi-tion to the effect of bumetanide and furosemide in the pro-cesses that make the sperm able to respond to ZP induction,the actual acrosome reaction process is affected by the inhib-itors. Similar results were obtained with furosemide (datanot shown).The role of NKCC in capacitation was further analyzed by

eliminating external K� from capacitation media, a condi-tion that would stop NKCC function. Although the lack ofK� had no effect on the increase in tyrosine phosphorylation(Fig. 5A) and sperm hyperactivation (Fig. 5B), sperm incu-bated in K�-free medium were unable to acrosome-react inthe presence of ZP (Fig. 5C). Consistent with these results,the absence of K� also inhibited IVF (data not shown). Theseobservations provide additional evidence that NKCC isinvolved in the regulation of the acrosome reaction. Theanalysis of Cl� transporter inhibitors in different capacita-tion-associated parameters is summarized in Table 1. All ofthe inhibitors were tried at a wide range of concentrations.However, some of them were deleterious to the cells whenused at high concentrations, and hence these concentrationswere excluded from the table.As in thecaseof sperm incubated inCl�-freemedium, theaddi-

tion of cAMP agonists to sperm capacitated in the presence of 1mM bumetanide completely overcame the inhibition of theincrease in protein tyrosine phosphorylation (Fig. 6A), suggestingthat the signal transduction machinery is functional under these

FIGURE 2. Cyclic AMP is downstream of the effect of Cl� on capacitation. Sperm were incubated in mediawithout Cl� in the absence or presence of IBMX (0.2 mM) and different cAMP analogs: dibutyryl cyclic AMP (db;5 mM), sp-cAMPS (sp; 1 mM), or 8-bromo-cAMP (8Br; 0.5 mM). The increase in tyrosine phosphorylation (A) andthe percentage of hyperactivation (B) were evaluated. In IVF assays (C), gamete co-incubation was conductedin drops with the same composition as the capacitation media. The percentage of fertilization in the controlwas 85 � 6. Each experiment was repeated at least three times. For hyperactivation and IVF experiments,Tukey’s multiple comparison tests were performed. The means of groups that have different letters differsignificantly (p � 0.01) (B and C).




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conditions. Moreover in the presence of bumetanide, permeablecAMP analogs increased both the percentage of hyperactivation(Fig. 6B) and the percentage of fertilization in IVF assays (Fig. 6C).This result, together with the ability of cAMP-permeable analogsto induce capacitation-associated events in the absence of Cl�,suggests that Cl� transport is upstream of a cAMP pathway. Tofurther investigate this hypothesis cAMPaccumulationwasmeas-

ured. When capacitation was carriedout in thepresenceofCl�, therewas a2.5-fold increase on the cAMP levelscompared with sperm that were keptunder noncapacitating conditions(without BSA and HCO3

�). On theother hand, when sperm wereincubated in capacitation mediaeither in the absence of Cl� or inthe presence of bumetanide, nosignificant increases in the cAMPlevels were observed (Fig. 7A). Inall of these measurements, thecAMP concentration falls into thelinear part of the cAMP standardcurve (Fig. 7B).NKCC1 Is Present in Mouse

Sperm—Although pharmacologi-cal approaches are limited by thespecificity of the inhibitors, theyrestrict the possible transportercandidates. In this case, the inhibi-tion of the ZP-induced acrosomereaction in the absence of K� andby low concentrations of bumet-anide suggests that NKCC trans-porters might be present in mousesperm. In particular, NKCC1 ispresent in pachytene and roundspermatids and is involved in sper-matogenesis (41) (Fig. 8A). Takingthese results into consideration, toanalyze the presence of the proteinin mature sperm, an anti-NKCC1monoclonal antibody was used.Western blots indicate the presenceof the predicted 145-kDa molecularmass protein (8) in total spermextracts as well as in the kidney pos-itive control (Fig. 8B). Consistentwith its predicted localization,NKCC1 was present in spermplasma membrane purified frac-tions. In both kidney and sperm, theanti-NKCC1 antibody recognizedadditional lower molecular weightproteins. Interestingly, these lowerbands are also present in the spermplasma membrane fraction andcould represent degradation prod-ucts, an alternative form of NKCC1,

or additional spermproteins not related toNKCC. Immunoflu-orescence experiments using this antibody show staining in dif-ferent sperm compartments including the principal piece, themid-piece, and the anterior head (Fig. 8C). Notably, staining inthe head was not observed when PNA staining was negative,suggesting thatNKCC is present in the plasmamembrane over-laying the acrosome.

FIGURE 3. Effect of Cl� channel inhibitors in capacitation. A and B, CFTR inhibitors do not inhibit capacita-tion-associated increase in tyrosine phosphorylation, motility, or hyperactivation. Sperm were incubated incomplete capacitating media in the presence of two different CFTR inhibitors: inh-172 (250 �M) or DPC (1 mM),and the increase in tyrosine phosphorylation (A, upper panel) and percentage of motile sperm and hyperacti-vation (B) were analyzed. C, upper panel, Cl� channel inhibitors with no effect on tyrosine phosphorylation.Sperm were incubated under noncapacitating (NC) or capacitating (C) conditions in the presence of differentanionic channel blockers: picrotoxin (1 mM), bicuculline (1 mM), indanyloxyacetic acid 94 (IAA-94; 0.2 mM),propionic acid (1 mM), 9-(hydroxymethyl)anthracene (AC-9; 0.25 mM), chlorotoxin (0.01 mM), thiazide (1 mM),R(�)-butylindazone (DIO-A; 0.2 mM), 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 0.2 mM); and niflu-mic acid (NFA; 0.5 mM). Then, Western blotting was conducted using anti-PY antibodies. D, upper panel, DIDSand SITS block capacitation-associated increase in tyrosine phosphorylation. Sperm were incubated undernoncapacitating or capacitating conditions in the presence or absence of DIDS or SITS (disulfonic stilbenederivatives), two broad anionic channel blockers. Next, Western blotting was conducted using anti-PY anti-bodies. E and F, DIDS and SITS block hyperactivation and IVF. Sperm incubated in capacitating media in thepresence or absence of DIDS or SITS (1 mM) were used to assay for hyperactivation (E) and IVF (F). The inhibitorswere present also during the gametes co-incubation. The percentage of fertilization in the control was 78 � 13.All membranes were stripped and reblotted with an anti-�-tubulin antibody for loading control (A, C, and D,lower panels). Each experiment was repeated at least three times. For motility, hyperactivation and IVF exper-iments, two-tailed Student’s t test (B) or Tukey’s multiple comparison test (E and F) were performed. The meansof groups that have different letters differ significantly (p � 0.01) (E and F).




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DISCUSSION

Mammalian sperm are not able to fertilize eggs immedi-ately after ejaculation. In 1951, Chang (42) and Austin (43)demonstrated independently that sperm acquire fertiliza-tion capacity only after residing in the female tract for a finiteperiod of time in a process known as sperm capacitation.Taking these initial investigations into account, sperm

capacitation became defined usingfertilization as the end-point.However, a variety of evidencesuggests that the functionalchanges occurring in sperm dur-ing capacitation are not one eventbut a combination of sequentialand concomitant processes. Someof them are: 1) sperm hyperactiva-tion, 2) preparation to undergo anagonist-induced acrosome reac-tion (e.g. zona pellucidae or pro-gesterone), and 3) acquisition ofthe ability to fertilize the egg.Additionally, at the molecularlevel, during the last 10 years, aseries of studies has establishedthat some signaling pathways areactivated during sperm capacita-tion. In particular, capacitationhas been associated with a cAMP/PKA-dependent increase in pro-tein tyrosine phosphorylation (18,26). This pathway appears to bedownstream of a series of eventsthat occur at the level of the spermplasma membrane. Synthesis ofcAMP is mediated by an atypicalsoluble adenylyl cyclase known as

sAC (44, 45). This enzyme was first reported by Okamura etal. (46) as a HCO3

� -stimulated cyclase and is essential for theregulation of multiple aspects of sperm capacitation includ-ing activation of motility (47), the cAMP/PKA-dependentincrease in protein tyrosine phosphorylation (18, 26, 48), andhyperactivation (49, 50). In contrast to all the evidenceindicating a role for HCO3

� function in the regulation of

FIGURE 4. Effect of bumetanide on capacitation-associated events. A and B, bumetanide, an NKCC inhibitor,blocks the increase in tyrosine phosphorylation and hyperactivation with no effect on the percentage onsperm motility. Sperm were incubated under noncapacitating (NC) or capacitating (C) conditions in the pres-ence of increasing bumetanide concentrations. Subsequently, Western blotting with anti-PY antibodies(A, upper panel) and CASA (B) were carried out. Membranes were stripped and reblotted with an anti-�-tubulinantibody for loading control (A, lower panel). C, bumetanide inhibits the ZP-induced acrosome reaction. Bumet-anide was added to sperm during capacitation or at the same time as the ZP. Subsequently, ZP-inducedacrosome reaction was evaluated. D, bumetanide inhibits IVF. Eggs were inseminated with sperm capacitatedin the presence of different concentrations of bumetanide. The inhibitor was also added to each co-incubationdrop at the same concentration as during capacitation. The percentage of fertilization in the control was 80 �9. Each experiment was repeated at least three times. For hyperactivation experiments (B), *, p � 0.01 versuscontrol (0 mM bumetanide plus DMSO) using two-tailed Student’s t test; for acrosome reaction (C), *, p � 0.05comparing basal with ZP-induced acrosome reaction for each bumetanide concentration and using Bonferronipost tests; for IVF experiments, Tukey’s multiple comparison test was performed. The means of groups thathave different letters differ significantly (p � 0.05) (D).

TABLE 1Effect of inhibitors on capacitation associated events and fertilizationThe effect of different Cl� channel inhibitors was tested first on capacitation-associated tyrosine phosphorylation. Those inhibitors that affected the increase in phosphorylationwere controlled for effects on motility (as a parameter of viability) and further for hyperactivation and fertilization. GABA-A, � -aminobutyric acid-A; NPPB, 5-nitro-2-(3-phenylpropylamino)benzoic acid; IAA-94, indanyloxyacetic acid 94; AC-9, 9-(hydroxymethyl)anthracene; —, not determined. VRAC, volume-regulated anion channel.

Inhibitor Concentration Effect on tyrosinephosphorylation

Effect onmotility

Effect onhyperactivation Effect on IVF

DPC (CFTR inhibitor) 1 mM None None None NoneBumetanide (NKCC inhibitor) 1–100 �M None — — —

250 �M–1 mM Inhibition None Inhibition InhibitionFurosemide (NKCC inhibitor) 100–250 �M None — — —

250 �M–1 mM Inhibition None Inhibition InhibitionThiazide (NCC inhibitor) 250 �M–1 mM None — — NoneR(�)-Butylindazone (KCC inhibitor) 200 �M None — — —DIDS (chloride channel blocker) 500 �M–1 mM Inhibition None Inhibition InhibitionSITS (chloride channel blocker) 1 mM Inhibition None Inhibition InhibitionChlorotoxin (calcium-activated Cl� channel inhibitor,

small conductance Cl� channel inhibitor)1 �M–10 �M None — — —

Niflumic acid (calcium-activated Cl-channelsinhibitor and VRAC inhibitor)

200–750 �M None — — —

Picrotoxin (GABA-A receptor/Cl�channel antagonist)

200 �M–1 mM None — — —

Bicuculline (GABA-A receptor/Cl�channel antagonist)

10 �M–1 mM — — — —

NPPB (CLC channels inhibitor) 200 �M None None None —IAA-94 (CLC channels inhibitor) 200 �M None — — —Propionic acid (CLC channels inhibitor) 1 mM None — — —AC-9 (CLC channels inhibitor) 100–250 �M None None None —




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these signaling pathways, much less is known about the roleof Cl�, the predominant anion in the capacitation medium.Previously, we had shown that Cl� is needed for the mem-

brane potential changes that accompany capacitation (4). Wehave hypothesized that CFTRmediates the effect of Cl� on thecapacitation-associated hyperpolarization downstream ofcAMP through PKA activation of this Cl� channel. However,

CFTR inhibition does not altereither tyrosine phosphorylation orsperm hyperactivation, two pro-cesses that are also downstream of acAMP pathway. In contrast to theseresults, sperm incubated in Cl�-freemedium did not hyperactivate anddid not undergo the capacitation-associated increase in tyrosinephosphorylation. Altogether, theseexperiments suggest that in addi-tion to CFTR, other Cl� transportsystems are present in sperm. Toinvestigate this hypothesis, a batteryof Cl� transport inhibitors wastested in capacitation assays. Asexpected, general Cl� transportblockers such as stilbenesmimickedthe absence of Cl�; however, morespecific inhibitors were not able toreduce either the increase in tyro-sine phosphorylation or hyperacti-vation. DIDS was significantly moreeffective than SITS in inhibitingIVF, but it had equal inhibitorypower when other sperm parame-ters such as the increase in tyrosinephosphorylation and hyperactiva-tion were assayed. These com-pounds are known to reactcovalently with lysine and cysteineresidues (51); however, the ability ofDIDS to bind covalently to theseresidues ismuch greater than that ofSITS (52). In contrastwithmeasure-ments of tyrosine phosphorylationand hyperactivation, longer incuba-tion times are needed for IVF.Under these conditions covalentbinding might be essential for theinhibitory properties of the stilbenecompounds.Bumetanide and furosemide, two

NKCC inhibitors, reduced spermcapacitation parameters to levelssimilar to those observed in theabsence of Cl�. However, the con-centration necessary for the inhibi-tion of tyrosine phosphorylationand hyperactivation was higherthan that reported to be effective in

inhibiting NKCC (40, 53, 54). Moreover, even though the pres-ence of Cl� andNa� (25) is essential for the increase in tyrosinephosphorylation, K�, another ion needed for the function ofNKCCs, was not required for these processes. These resultssuggest that high concentrations of bumetanide might have atarget different from NKCC, that is essential for the onset ofhyperactivation and the increase in tyrosine phosphorylation.

FIGURE 5. Analysis of sperm capacitation in the absence of K�. A and B, the lack of K� during capacitationdoes not affect either the increase in tyrosine phosphorylation or sperm hyperactivation. Sperm were incu-bated under noncapacitating (NC) or capacitating (C) medium with or without K�, and the increase in tyrosinephosphorylation (A, upper panel), the percentage of motility (B, upper panel), and hyperactivation (B, lowerpanel) were evaluated. Membranes were stripped and reblotted with an anti-�-tubulin antibody for loadingcontrol (A, lower panel). C, K� ions are necessary for the ZP-induced acrosome reaction to occur. Sperm wereincubated in control or K�-free capacitating media, and ZP-induced acrosome reaction was assayed. Eachexperiment was repeated at least three times. For hyperactivation (B), *, p � 0.01 versus control, using two-tailed Student’s t test. For acrosome reaction (C), *, p � 0.01 comparing basal with ZP-induced acrosomereaction, using Bonferroni post tests.

FIGURE 6. Cyclic AMP agonists rescue the inhibition of capacitation by bumetanide. Capacitation wascarried out in the presence or absence of bumetanide (1 mM), IBMX (0.2 mM), and dibutyryl cyclic AMP (db cAMP;5 mM). Then, protein tyrosine phosphorylation (A), sperm hyperactivation (B), and fertilization (C) were evalu-ated. The percentage of fertilization in the control was 78 � 7. Each experiment was repeated at least threetimes. For hyperactivation and IVF experiments, Tukey’s multiple comparison tests were performed. Themeans of groups that have different letters (a and b) differ significantly (p � 0.01) (B and C).

FIGURE 7. Capacitation-associated increase in cAMP levels are prevented in the absence of Cl� or in thepresence of bumetanide. Capacitation was carried out in the presence or absence of Cl� or bumetanide (1mM). Then, sperm were centrifuged and resuspended in HCl (0.1 mM) for 20 min. After that, sperm werecentrifuged again and the supernatant was used to measure cAMP levels (A). A standard curve was run for eachassay. A typical standard curve is depicted (B). Tukey’s multiple comparison tests were performed. The meansof groups that have different letters (a and b) differ significantly (p � 0.05) (A).




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In contrast to these results, the ZP-induced acrosome reactionwas dependent on the presence of Cl�, K� (this study) andNa�

(25) andwas inhibited at amuch lower concentration of bumet-anide and furosemide, suggesting that NKCCmight have a rolein the capacitation-dependent preparation of sperm for theacrosome reaction or directly in the acrosome reaction processitself. Similarly, DPC and inh-172, two CFTR antagonists usedin this work, were not able to inhibit either hyperactivation orthe increase in tyrosine phosphorylation, yet they block theZP-induced acrosome reaction (4). On the other hand, in sAC

null mutants, the inhibition of the increase in tyrosine phos-phorylation and hyperactivation did not block the ability of thesperm to acrosome react in the presence of ZP (55). Altogether,these experiments suggest that different aspects of capacitationare regulated by different molecular pathways.The presence of NKCCwas studied usingWestern blot anal-

yses in both total sperm extracts and purified membrane prep-arations. It is worth noting that in addition to the 145-kDa bandthat corresponds to the reportedNKCC1 (8), a lowermolecularmass band was detected in sperm. Interestingly, two NKCC1transcripts were reported in specific testis cell populations (41,56). Besides the normal NKCC1 transcript (6.5 kb) present insomatic tissues, an alternate transcript of 4.2 kb was detected inpachytene spermatocytes and round spermatids. Furthermore,null mutants of this protein were shown to have defects in sper-matogenesis and are infertile (41). It remains to be investigatedwhether the shorter transcript codes for the lower molecularweight protein recognized by anti-NKCC1. Alternatively, it isnot possible to discard the possibility that the anti-NKCC anti-body recognized additional sperm proteins; therefore, theimmunofluorescence staining may not correspond completelyto NKCC.Asmentioned previously, bumetanide inhibited hyperactiva-

tion, the increase in tyrosine phosphorylation, and fertilizationat higher concentrations than those needed to block NKCC1 inother systems. It is likely that higher concentrations of thisinhibitor target molecule(s) different from NKCC1. Studies inhuman erythrocytes (39) have shown that most of the classicalCl� transport inhibitors, including furosemide and bumet-anide, can inhibit anion exchangers and, to a lesser extent,KCCs when used at high concentrations. Taking into accountthat thiazide, the specific KCC inhibitor, did not affect tyrosinephosphorylation, it would be more likely that an anionexchanger was affected by bumetanide (and furosemide) whenused at high concentration. An alternative hypothesis is thathigh concentrations of bumetanide are needed to inhibit anadditional sperm-specific NKCC and that this protein has lessaffinity to the inhibitor than the classical NKCC found insomatic tissues. Consistent with this hypothesis, changes inbumetanide binding affinity have been reported in several chi-meras of human colonic NKCC1 (57). In addition, although K�

is needed for a functional NKCC in most cases, in a few it hasbeen reported that it can transport only Na� and Cl� (58). Thefinding of a lowermolecular weight NKCC1 in sperm, as well asthe aforementioned smaller NKCC1 transcripts, supports thispossibility. Further isolation and identification of the 95-kDaband will be necessary to verify this hypothesis. In contrast tohyperactivation and the increase in tyrosine phosphorylation,the classical NKCC1 appears to have a role in other aspects ofcapacitation, such as the preparation of the sperm for the ZP-induced acrosome reaction. This conclusion is based on therequirement of external Cl�, K�, and Na� for capacitation andthe exocytotic reaction, as well as on the lower bumetanideconcentrations needed to inhibit these processes.Although it is not yet possible to define the complete set of

Cl� transporters regulating Cl� homeostasis in sperm, thisstudy provides strong evidence that this anion is required forcapacitation.Moreover, the results presented here indicate that

FIGURE 8. NKCC is present in mouse sperm. A, NKCC mRNA is present inspermatogenic cells. Reverse transcription-PCR of spermatogenic cells wascarried out using oligonucleotides for NKCC1 and cDNA of pachytene, roundcells, and elongated spermatids (�); negative controls (�) without reversetranscriptase are also shown for each PCR. B, NKCC1 is present in maturemouse sperm. Total kidney and sperm extracts were analyzed by Westernblotting with a monoclonal anti-NKCC1 antibody (left). In addition, spermmembrane purifications were carried out as described under “ExperimentalProcedures” and SDS extracts from 100,000 � g pellet were analyzed usinganti-NKCC antibodies (right). C, NKCC1 localizes to different compartments insperm including anterior head, mid and principal piece. For immunolocaliza-tion assays, mouse sperm were air-dried, fixed, permeabilized, and probedwith anti-NKCC1 antibodies. Peanut agglutinin (PNA) was used to follow theacrosomal status. An NKCC1-specific signal in the head is not observed whenpeanut agglutinin is negative (arrows). Each experiment was repeated at leastthree times.




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cAMP-permeable analogs are able to overcome the absence ofCl� as well as bumetanide inhibition of capacitation-associatedparameters. This result, coupled with the finding that accumu-lation of cAMP in sperm incubated under capacitating condi-tions does not occur in the absence of Cl� or in the presence ofbumetanide, suggests that the observed increase inCl� concen-tration during capacitation (4) is upstream of cAMP signalingpathways. Although less likely, another possibility is that Cl�and cAMP are part of parallel and coincident signaling path-ways involved in sperm capacitation and that addition of exog-enous permeable cAMP analogs can compensate for the loss ofCl�-mediated signaling.

Our working hypothesis (Fig. 9) is that inward translocationof Cl�, to whichNKCC1 is likely to contribute, is coupled to theactivity of a Cl�/HCO3

� antiporter. Inward fluxes of HCO3�

through this exchanger would stimulate sAC and increase thecAMP levels. Recently, Nolan et al. (59) generated mice thatlack the sperm-specific PKA catalytic subunit C�2. These miceare infertile despite normal mating behavior, and their spermpresent defects in motility and capacitation-associated eventssuch as the increase in tyrosine phosphorylation. Also, cAMPlevels in C�2 knock-out mice are up-regulated, suggesting thatPKAphosphorylation exerts a negative feedback on cAMPpro-duction. Consistent with these findings, incubation of wild typesperm with the PKA inhibitor H89 increases the basal levels ofcAMP (59). Because cAMP is needed for later events associatedwith capacitation, it is possible to hypothesize that the negativefeedbackmechanismon cAMP levels exerted by PKAphospho-rylation is released at some point during the capacitation proc-ess. Because Cl� is required for subsequent cAMP-dependentprocesses, we postulate that the release of the negative feedbackin cAMP synthesis is dependent on the activation of other reg-

ulatory pathways downstream of cholesterol release and Cl�transport. Further investigation should test whether two wavesof cAMP synthesis by sAC are needed in the capacitation proc-ess: 1) an acute Cl�-independent increase in cAMP levelsinvolving sAC activation by HCO3

� and mediated through aNa�/HCO3

� cotransporter as postulated previously (25) (Fig.9A); and 2) a sustained increase in cAMP concentration medi-ated by an inward flux of Cl� coupled to the activation of aCl�/HCO3

� antiporter (Fig. 9B).

Acknowledgments—We thank Matias Okawa for helpful discussionand critical reading of the manuscript and Laura Brassard and Dr.Randolph Lisle for proofreading. We also acknowledge technicalassistance from theAnimalCare and Sequencing facilities at Institutode the Biotecnologia-Universidad Nacional Autonoma de Mexico.

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FIGURE 9. Model for the involvement of Cl� during capacitation. A, initialevents during capacitation. At first, an acute Cl�-independent increase incAMP levels involving sAC activation by HCO3

� and mediated through a Na�/HCO3

� cotransporter occurs allowing the consequent activation of PKA. Sub-sequently, PKA phosphorylation exerts a negative feedback on cAMP levels.B, later events during sperm capacitation. Because of the need of cAMP forlater events, it is possible to hypothesize that the negative feedback mecha-nism on the cAMP levels exerted by PKA phosphorylation is released at somepoint during the capacitation process. Our working hypothesis is that inwardtranslocation of Cl� (through NKCC and other Cl� transporters) is coupled tothe activity of a Cl�/HCO3

� antiporter. Inward fluxes of HCO3� through this

exchanger would stimulate sAC and raise the cAMP levels necessary for theposterior increase in tyrosine phosphorylation. Also, NKCC-dependent Cl�

currents would be necessary to prepare sperm for ZP-induced acrosome reac-tion. In addition, Cl�-dependent unknown mechanisms occurring in spermare essential for fertilization to occur.




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Enrique O. Hernández-González, Alberto Darszon and Pablo E. ViscontiEva V. Wertheimer, Ana M. Salicioni, Weimin Liu, Claudia L. Trevino, Julio Chavez,

in Tyrosine PhosphorylationChloride Is Essential for Capacitation and for the Capacitation-associated Increase

doi: 10.1074/jbc.M804586200 originally published online October 27, 20082008, 283:35539-35550.J. Biol. Chem.

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Chloride Is Essential for Capacitation and for the Capacitation-associated Increase in Tyrosine Phosphorylation