Chloride Is Essential for Capacitation and for the Capacitation-associated Increase in Tyrosine Phosphorylation
2008; Elsevier BV; Volume: 283; Issue: 51 Linguagem: Inglês
10.1074/jbc.m804586200
ISSN1083-351X
AutoresEva Wertheimer, Ana M. Salicioni, Weimin Liu, Claudia L. Treviño, Julio C. Chávez, Enrique O. Hernández‐González, Alberto Darszon, Pablo E. Visconti,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoAfter epididymal maturation, sperm capacitation, which encompasses a complex series of molecular events, endows the sperm with the ability to fertilize an egg. This process can be mimicked in vitro in defined media, the composition of which is based on the electrolyte concentration of the oviductal fluid. It is well established that capacitation requires Na+, HCO3-, Ca2+, and a cholesterol acceptor; however, little is known about the function of Cl– during this important process. To determine whether Cl–, in addition to maintaining osmolarity, actively participates in signaling pathways that regulate capacitation, Cl– was replaced by either methanesulfonate or gluconate two nonpermeable anions. The absence of Cl– did not affect sperm viability, but capacitation-associated processes such as the increase in tyrosine phosphorylation, the increase in cAMP levels, hyperactivation, the zona pellucidae-induced acrosome reaction, and most importantly, fertilization were abolished or significantly reduced. Interestingly, the addition of cyclic AMP agonists to sperm incubated in Cl–-free medium rescued the increase in tyrosine phosphorylation and hyperactivation suggesting that Cl– acts upstream of the cAMP/protein kinase A signaling pathway. To investigate Cl– transport, sperm incubated in complete capacitation medium were exposed to a battery of anion transport inhibitors. Among them, bumetanide and furosemide, two blockers of Na+/K+/Cl– cotransporters (NKCC), inhibited all capacitation-associated events, suggesting that these transporters may mediate Cl– movements in sperm. Consistent with these results, Western blots using anti-NKCC1 antibodies showed the presence of this cotransporter in mature sperm. After epididymal maturation, sperm capacitation, which encompasses a complex series of molecular events, endows the sperm with the ability to fertilize an egg. This process can be mimicked in vitro in defined media, the composition of which is based on the electrolyte concentration of the oviductal fluid. It is well established that capacitation requires Na+, HCO3-, Ca2+, and a cholesterol acceptor; however, little is known about the function of Cl– during this important process. To determine whether Cl–, in addition to maintaining osmolarity, actively participates in signaling pathways that regulate capacitation, Cl– was replaced by either methanesulfonate or gluconate two nonpermeable anions. The absence of Cl– did not affect sperm viability, but capacitation-associated processes such as the increase in tyrosine phosphorylation, the increase in cAMP levels, hyperactivation, the zona pellucidae-induced acrosome reaction, and most importantly, fertilization were abolished or significantly reduced. Interestingly, the addition of cyclic AMP agonists to sperm incubated in Cl–-free medium rescued the increase in tyrosine phosphorylation and hyperactivation suggesting that Cl– acts upstream of the cAMP/protein kinase A signaling pathway. To investigate Cl– transport, sperm incubated in complete capacitation medium were exposed to a battery of anion transport inhibitors. Among them, bumetanide and furosemide, two blockers of Na+/K+/Cl– cotransporters (NKCC), inhibited all capacitation-associated events, suggesting that these transporters may mediate Cl– movements in sperm. Consistent with these results, Western blots using anti-NKCC1 antibodies showed the presence of this cotransporter in mature sperm. Before becoming fertilization-competent, mammalian sperm must undergo a series of maturational processes in the female reproductive tract (1Yanagimachi R. Knobil E. Neill J.D. The Physiology of Reproduction (Knobil. eds. Raven Press, Ltd., New York1994: 187-317Google Scholar). The molecular, biochemical, and physiological changes that occur in sperm, whereas in the female tract are collectively referred to as capacitation. These functional changes associated with capacitation are not one event but are a combination of sequential and concomitant processes involving modifications at the molecular level occurring both in the head (i.e. preparation for the acrosome reaction) and the tail (i.e. motility changes such as hyperactivation). Molecular events implicated in the initiation of capacitation can be mimicked in vitro and have been partially defined. These include removal of cholesterol from the sperm plasma membrane; modifications in plasma membrane phospholipids; fluxes of HCO3- and other intracellular ions; increased protein tyrosine phosphorylation; and hyperpolarization of the sperm plasma membrane potential (Em) in mouse and other species (for review see Ref. 2Visconti P.E. Westbrook V.A. Chertihin O. Demarco I. Sleight S. Diekman A.B. J. Reprod. Immunol. 2002; 53: 133-150Crossref PubMed Scopus (289) Google Scholar). With respect to the changes in the plasma membrane Em in mouse sperm, it is hypothesized that the capacitation-associated hyperpolarization results from changes in the activity of ion-selective channels and transporters. Consistent with this hypothesis, our studies in sperm from this species have revealed the presence of amiloride-sensitive epithelial Na+ channels (ENaCs) 2The abbreviations used are:ENaCepithelial Na+ channelNKCCNa+/K+/Cl– cotransporterCFTRcystic fibrosis transmembrane regulatorZPzona pellucidaSp-cAMPSSp-diastereomer of adenosine 3′,5′-cyclic monophosphothiorateIBMX3-isobutyl-1-methylxanthineDPCdiphenylamine-2-carboxylateinh-1725-[(4-carboxyphenyl)methylene]-2-thioxo-3-[(3-trifluoromethyl)phenyl-4-thiazolidinoneDIDS4,4′-diisothiocyanatostilbene-2,2′-disulfonic acidSITSdisodium 4-acetamido-4′-isothiocyanato-stilben-2,2′-disulfonateDMSOdimethyl sulfoxideBSAbovine serum albuminPBSphosphate-buffered salineIVFin vitro fertilizationPYphosphotyrosineCASAcomputer-assisted semen analysisClCCl–-selective ion channelKCCK+/Cl– cotransportersACsoluble adenylyl cyclasePKAprotein kinase A and the cystic fibrosis transmembrane regulator (CFTR) (3Hernandez-Gonzalez E.O. Sosnik J. Edwards J. Acevedo J.J. Mendoza-Lujambio I. Lopez-Gonzalez I. Demarco I. Wertheimer E. Darszon A. Visconti P.E. J. Biol. Chem. 2006; 281: 5623-5633Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 4Hernandez-Gonzalez E.O. Trevino C.L. Castellano L.E. de la Vega-Beltran J.L. Ocampo A.Y. Wertheimer E. Visconti P.E. Darszon A. J. Biol. Chem. 2007; 282: 24397-24406Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Experiments in those reports suggest that closing of ENaCs, with the consequent reduction in Na+ influx, induces the hyperpolarization of the sperm Em observed during capacitation. Down-regulation of ENaC activity appears to be a consequence of either the activation of CFTR or the influx of Cl–. Independent of our work, the presence of CFTR in sperm was also reported by Xu et al. (5Xu W.M. Shi Q.X. Chen W.Y. Zhou C.X. Ni Y. Rowlands D.K. Yi Liu G. Zhu H. Ma Z.G. Wang X.F. Chen Z.H. Zhou S.C. Dong H.S. Zhang X.H. Chung Y.W. Yuan Y.Y. Yang W.X. Chan H.C. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 9816-9821Crossref PubMed Scopus (169) Google Scholar); this group hypothesized that in addition to its role as a Cl– transporter, CFTR also transports HCO3-. As CFTR is primarily a Cl– channel and other anion transporters are likely to be involved in capacitation (4Hernandez-Gonzalez E.O. Trevino C.L. Castellano L.E. de la Vega-Beltran J.L. Ocampo A.Y. Wertheimer E. Visconti P.E. Darszon A. J. Biol. Chem. 2007; 282: 24397-24406Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), the present work focuses on the role of Cl– on sperm function. epithelial Na+ channel Na+/K+/Cl– cotransporter cystic fibrosis transmembrane regulator zona pellucida Sp-diastereomer of adenosine 3′,5′-cyclic monophosphothiorate 3-isobutyl-1-methylxanthine diphenylamine-2-carboxylate 5-[(4-carboxyphenyl)methylene]-2-thioxo-3-[(3-trifluoromethyl)phenyl-4-thiazolidinone 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid disodium 4-acetamido-4′-isothiocyanato-stilben-2,2′-disulfonate dimethyl sulfoxide bovine serum albumin phosphate-buffered saline in vitro fertilization phosphotyrosine computer-assisted semen analysis Cl–-selective ion channel K+/Cl– cotransporter soluble adenylyl cyclase protein kinase A Cl– can be transported through various systems across the plasma membrane including different types of Cl– channels and a series of specialized carriers. Intracellular Cl– levels are determined by the relative contributions of all Cl– transporters present in the plasma membrane of a given cell type. Cl– channels are a subset of ion channels selective to Cl– and often permeable to other small monovalent anions. Four structural Cl– channel families have been identified to date: 1) Cl–-selective ion channels (ClCs); 2) CFTR channels; 3) the γ-aminobutyric acid- and glycine-gated neurotransmitter receptors; and 4) Ca2+-activated Cl– channels. In addition, functional data suggest that other not yet identified Cl– channels may exist. The Cl– carrier proteins include: 1) the electroneutral and electrogenic Cl-/HCO3- exchanger families (6Steward M.C. Ishiguro H. Case R.M. Annu Rev. Physiol. 2005; 67: 377-409Crossref PubMed Scopus (198) Google Scholar) and 2) the electroneutral cation-Cl– cotransporter family. All of these are secondary active transporters, which means that the translocation of one ion is coupled to the translocation of another ion in either the opposite direction (antiporter) or the same direction (cotransporter or symporter). The flow of the ion translocated up the electrochemical gradient is coupled to the flow of a second ion down its electrochemical gradient, and thus the energy required does not come directly from ATP (7Akabas M.H. Encyclopedia of Life Sciences. ed. Macmillan Reference Ltd., London2001: 1-7Google Scholar). In the present work we have analyzed whether Cl– ions regulate different events in the capacitation process. The central observations of this work are that: 1) Cl– is necessary for the capacitation-associated increase in cAMP and tyrosine phosphorylation; 2) sperm incubated in a medium without Cl– do not acquire either responsiveness to zona pellucidae (ZP) for the induction of acrosome reaction nor hyperactivated motility; 3) fertilization is inhibited if sperm are incubated under capacitating medium without Cl–; 4) in the absence of Cl–, cAMP-permeable analogs can induce both the increase in tyrosine phosphorylation as well as hyperactivated motility; 5) bumetanide and furosemide, two Na+/K+/Cl– cotransporter (NKCC) inhibitors, block capacitation and the capacitation-associated processes; and 6) at least one member of the NKCC family is present in mature sperm. Materials—Chemicals were obtained from the following sources. Bovine serum albumin (BSA, fatty acid-free), sodium methanesulfonate, sodium gluconate, potassium gluconate, dibutyryl cyclic AMP, Sp-cAMPS, 8-bromo-cAMP, 3-isobutyl-1-methylxanthine (IBMX), carbonyl cyanide m-chlorophenylhydrazone, valinomycin, chlorotoxin, diphenylamine-2-carboxylate (DPC), 5-[(4-carboxyphenyl)methylene]-2-thioxo-3-[(3-trifluoromethyl)phenyl-4-thiazolidinone (inh-172), bumetanide, furosemide, 9-(hydroxymethyl)anthracene, R(+)-butylindazone, (+)-bicuculline, 5-nitro-2-(3-phenylpropylamino)benzoic acid, indanyloxyacetic acid 94, hydrochlorothiazide, picrotoxin, DIDS, and SITS were purchased from Sigma. The fluorescent dyes 3,3-dipropylthiadicarbocyanine iodide (DiSC3-(5)) and Hoestch 33342 were obtained from Invitrogen. Niflumic acid was from Calbiochem. Cyclic AMP analogs and inhibitors were prepared fresh the day of the experiment in either Milli-Q water or DMSO depending on solubility. Anti-phosphotyrosine (PY) monoclonal antibody (clone 4G10) was purchased from Upstate Biotechnology (Lake Placid, NY). In addition, for immunodetection of NKCC and β-tubulin, an anti-NKCC1 monoclonal antibody (clone T4) developed by Lytle et al. (8Lytle C. Xu J.C. Biemesderfer D. Forbush III, B. Am. J. Physiol. 1995; 269: C1496-C1505Crossref PubMed Google Scholar) and an anti-β-tubulin monoclonal antibody (Clone E7) developed by Chu and Klymkowsky (9Chu D.T. Klymkowsky M.W. Dev. Biol. 1989; 136: 104-117Crossref PubMed Scopus (128) Google Scholar) were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institutes of Health, NICHD, and maintained by The University of Iowa Department of Biological Sciences, Iowa City, IA. For negative control donkey anti-mouse affinity-purified IgG was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Mouse Sperm Preparation—Cauda epididymal mouse sperm were collected from CD1 retired male breeders (Charles River Laboratories, Wilmington, MA) and sacrificed in accordance with the Institutional Animal Care and Use Committee guidelines. Minced cauda epididymis from each animal was placed in 500 μl of a modified Krebs-Ringer medium (Whitten's HEPES-buffered medium) (10Moore G.D. Ayabe T. Visconti P.E. Schultz R.M. Kopf G.S. Development (Camb.). 1994; 120: 3313-3323Crossref PubMed Google Scholar). This medium does not support capacitation unless supplemented with 5 mg/ml BSA (fatty acid-free) and 15 mm NaHCO3. After 10 min, the sperm suspension was washed by adding 1 ml of noncapacitating medium and posterior 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 medium depending on the experiment performed. In experiments where capacitation was investigated, 5 mg/ml BSA and 15 mm NaHCO3 were added, and sperm were incubated at 37 °C for at least 1 h. To study the role of Cl– in capacitation, NaCl and KCl in the media were replaced either by sodium gluconate and potassium gluconate or by sodium methanesulfonate and KOH. In all cases pH was maintained at 7.2. When different Cl– concentrations were assessed, the total NaCl plus sodium gluconate (or sodium methanesulfonate) was maintained at 100 mm. For the experiments in K+-free media, KCl and KH2PO4 were replaced by NaCl and NaH2PO4, respectively. To test the effect of the different inhibitors in capacitation, they were pre-incubated with sperm for 15 min preceding the beginning of the capacitating incubation. For the in vitro fertilization (IVF) assays, sperm were obtained and incubated for capacitation in Whitten's medium without HEPES containing 22 mm NaHCO3 and 15 mg/ml BSA and equilibrated in a humidified atmosphere of 5! CO2 in air (11Tutuncu L. Stein P. Ord T.S. Jorgez C.J. Williams C.J. Dev. Biol. 2004; 270: 246-260Crossref PubMed Scopus (42) Google Scholar). Sperm Membrane Purification—The preparation of sperm fractions was carried out as described previously (12Visconti P.E. Olds-Clarke P. Moss S.B. Kalab P. Travis A.J. de las Heras M. Kopf G.S. Mol. Reprod. Dev. 1996; 43: 82-93Crossref PubMed Scopus (43) Google Scholar). Briefly, sperm (20 × 107 cells) were homogenized using 10 strokes with a 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 indicated by the manufacturer plus 0.4 mm leupeptin, 0.4 mm aprotinin, 0.1 mm pepstatin, 0.3 m benzamidine, and 0.32 mg/ml calpain I and II inhibitor). After homogenization, the sample was 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 the supernatant was centrifuged at 10,000 × g for 10 min at 4 °C. Again, the resultant pellet was saved, and the supernatant then was centrifuged at 100,000 × g for 1 h at 4 °C. The final pellet, which contained the membrane fraction, was resuspended in sample 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), resuspended in Laemmli sample buffer (13Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207208) Google Scholar) without β-mercaptoethanol, and boiled for 5 min. After centrifugation, the supernatants were saved, and β-mercaptoethanol was added to a final concentration of 5!. The samples were boiled for 5 min and subjected to SDS-PAGE using 8–10! mini-gels; protein extracts 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-phosphorylated proteins were carried out using PY monoclonal antibodies as described previously (14Kalab P. Visconti P. Leclerc P. Kopf G.S. J. Biol. Chem. 1994; 269: 3810-3817Abstract Full Text PDF PubMed Google Scholar). For loading controls, membranes were stripped, and an anti-β-tubulin monoclonal antibody was used at a 5.2 ng/ml. For immunodetection of the NKCC, an anti-NKCC monoclonal antibody was used at a concentration of 2.3 μg/ml. Immunoblots were developed with the appropriate secondary antibody conjugated to horseradish peroxidase (Jackson ImmunoResearch Laboratories) and ECL chemiluminescence reagents. Kidneys from CD1 male retired breeders were collected, and proteins were extracted using radioimmune precipitation assay buffer (10 mm Tris, pH 7.2, 150 mm NaCl, 0.1! SDS, 1! Triton X-100, 1! deoxycholate, 5 mm EDTA, and protease and phosphatase inhibitors). Protein concentration was assayed using the BCA kit from Pierce. In each lane 50 μg of total protein was loaded in an SDS-8! polyacrylamide gel. Proteins were transferred onto polyvinylidene difluoride membranes, and Western blotting 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 for 1 h. After incubation, the sperm suspension was loaded on a 20-μm chamber slide (Leja slide, Spectrum Technologies) and placed on a microscope stage heated to 37 °C. Sperm movement was examined using the CEROS computer-assisted semen analysis (CASA) system (Hamilton Throne Research, Beverly, MA). The parameters used were: frames acquired = 30, frame rate = 60 Hz, minimum cell size = 4 pixels, low average path velocity cutoff = 5 mm/s, static head size = 0.2–2.99, static head intensity = 0.26–1.31, and static head elongation = 0–100. Sperm with hyperactivated motility, defined as motility with high amplitude thrashing patterns and short distance of travel, were sorted using the criteria established by Bray et al. (15Bray C. Son J.H. Kumar P. Meizel S. Biol. Reprod. 2005; 73: 807-814Crossref PubMed Scopus (59) Google Scholar): 1) curvilinear velocity (VCL, velocity calculated from the sum of track-point to track-point velocity) > 180 μm/s; 2) amplitude of lateral head displacement (ALH, mean width of the sperm head oscillation as the cell swims), > 9.5 μm; 3) linearity (LIN, departure of the cell from a straight line) < 38!. Acrosome Reaction Assay—Capacitation was measured indirectly by determining the zona pellucidae-induced acrosome reaction based on the premise that only capacitated sperm will undergo exocytosis. Zona pellucidae were prepared from homogenized ovaries of virgin female 22-day-old outbred CD1 mice (Charles River Laboratories) as described (16Ward C.R. Storey B.T. Kopf G.S. J. Biol. Chem. 1992; 267: 14061-14067Abstract Full Text PDF PubMed Google Scholar, 17López-González I. De La Vega-Beltran J.L. Santi C.M. Florman H.M. Felix R. Darszon A. Dev. Biol. 2001; 236: 210-219Crossref PubMed Scopus (36) Google Scholar) and solubilized by the procedures outlined previously (18Visconti P.E. Bailey J.L. Moore G.D. Pan D. Olds-Clarke P. Kopf G.S. Development (Camb.). 1995; 121: 1129-1137Crossref PubMed Google Scholar). The percentage of acrosome reaction was measured using Coomassie Blue G-250 staining as described by Visconti et al. (19Visconti P.E. Galantino-Homer H. Ning X. Moore G.D. Valenzuela J.P. Jorgez C.J. Alvarez J.G. Kopf G.S. J. Biol. Chem. 1999; 274: 3235-3242Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Briefly, following a 45-min incubation at 37 °C under the conditions mentioned for each experiment, 5 zona pellucida equivalents/μl were added. After an additional 30 min of incubation at 37 °C, a fixative solution consisting of 5! final concentration of formaldehyde in PBS was added to each tube. Following fixation, 10-μl aliquots of sperm suspension were spread onto glass slides and air-dried. The slides were then stained with 0.22! Coomassie Blue G-250 in 50! methanol and 10! glacial acetic acid for 3–5 min, gently rinsed with deionized H2O, air-dried, and mounted with 50! (v/v) glycerol in phosphate-buffered saline. To calculate the percentage of acrosome reaction, at least 100 sperm were assayed per experimental condition for the presence or absence of the characteristic dark blue acrosomal crescent. The percentage of acrosome-reacted spermatozoa was calculated for each experimental condition dividing the number of acrosome-reacted spermatozoa by the total number of spermatozoa scored (sum of acrosome-reacted and non-acrosome-reacted) and multiplying this ratio by 100. Mouse Eggs Collection and IVF Assays—Egg collection was performed as described previously (20McAvey B.A. Wortzman G.B. Williams C.J. Evans J.P. Biol. Reprod. 2002; 67: 1342-1352Crossref PubMed Scopus (65) Google Scholar). Briefly, metaphase II-arrested eggs were collected from 6–8-week-old superovulated CD1 female mice (Charles River Laboratories) at 13 h after human chorionic gonadotropin (Sigma) injection. Cumulus cells were removed by brief incubation (<5 min) in Whitten's HEPES-buffered medium with 7 mm NaHCO3, 5 mg/ml BSA, and 0.02! type IV-S hyaluronidase (Sigma). After cumulus cell removal, eggs were placed in a drop of Whitten's medium containing 22 mm NaHCO3 and 15 mg/ml BSA and then allowed to recover for 30 min in an incubator with 5! CO2 at 37 °C (11Tutuncu L. Stein P. Ord T.S. Jorgez C.J. Williams C.J. Dev. Biol. 2004; 270: 246-260Crossref PubMed Scopus (42) Google Scholar). Fertilization drops (200 μl 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 conditions. After 4 h of insemination, eggs were washed through three drops of Whitten's medium containing 22 mm NaHCO3 and 15 mg/ml BSA using a thin bore pipette to detach any loosely attached sperm. After another 3 h of incubation, eggs were fixed with 3.7! paraformaldehyde/PBS for 15 min, washed, and stained with Hoestch 33342 (Sigma; 10 μg/ml final concentration) in PBS for 10 min at room temperature. To assess fertilization the three following criteria were considered: 1) the formation of the male and female pronuclei, 2) the emission of the second polar body, and 3) the presence of the sperm tail. The percentage of fertilization was calculated for each fertilization drop by dividing the number of fertilized eggs by the total number of eggs in that drop (sum of fertilized and nonfertilized eggs) and multiplying this ratio by 100. RNA Isolation and Reverse Transcription-PCR—Total RNA was prepared from isolated mouse pachytene, round and elongated spermatids (60Bellve A.R. Methods Enzymol. 1993; 225: 84-113Crossref PubMed Scopus (299) Google Scholar) using TRIzol reagent (Sigma) according to the manufacturer's instructions. cDNA was synthesized from total RNA samples with random hexamer-primed reverse transcription (Superscript II RNase H-reverse transcriptase; Invitrogen). cDNA was then subjected to PCR amplification using Taq DNA polymerase (Invitrogen). The NKCC1 primers were designed using the mouse-reported nucleotide sequence for these genes (mouse NKCC GenBank™ accession number NM_009194.2). Primer sequences for mouse NKCC1 were as follows: forward, 5′-CCT GCT TTA CTT CAT C-3′; reverse, 5′-GTC AAA CCT CCA TCA-3′. The absence of genomic contamination in the RNA samples was confirmed with reverse transcription negative controls (no reverse transcriptase) for each experiment. Amplified products were analyzed by DNA sequencing in order to confirm their identity. Indirect Immunofluorescence—Sperm obtained by the swim-up method in Whitten's HEPES-buffered medium were washed once, resuspended in PBS at a concentration of 1–2 × 105 sperm/ml, and seeded on 8-well glass slides. After air-drying, sperm were fixed with 3.7! paraformaldehyde in PBS for 15 min at room temperature, washed with PBS (four washes each for 5 min), and permeabilized with 0.5! Triton X-100 for 5 min. Following permeabilization, sperm were treated with 10! BSA in PBS for 1 h at room temperature and then incubated either with the respective primary antibody (1:50–1:250) diluted in PBS containing 1! BSA or with the same concentration of the corresponding affinity-purified IgG. Incubations were then carried out at 4 °C overnight. After incubation, sperm were washed thoroughly with PBS and incubated with the corresponding 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 peanut agglutinin (1:100) for staining acrosomes. Incubation with the secondary antibody was followed by four washes in PBS, mounted using SlowFade Light reagents (Molecular Probes, Eugene, OR), and observation by epifluorescence microscopy using a TE300 Eclipse microscope (magnification ×60) (Nikon). Differential interference contrast images were taken in parallel and served as control for sperm morphology. Negative controls using secondary antibody alone were also used to check for antibody specificity (not shown). cAMP Measurements—Sperm (5 × 106 sperm/ml) were incubated for 1 h in noncapacitating (without BSA and NaHCO3) or capacitating medium (with BSA and NaHCO3) in the presence or absence of Cl– or bumetanide (1 mm). All treatments were supplemented with IBMX (0.1 mm). Afterward, the sperm were centrifuged for 2 min at 800 × g, the pellet was resuspended in 200 μl of HCl (0.1 mm), vortexed twice for 2 s, and incubated for 20 min at room temperature. Next, sperm were centrifuged at 5000 × g for 5 min, and the supernatant was used to measure cAMP levels by using the BIOMOL format A cyclic AMP "PLUS" EIA kit (BIOMOL International, Plymouth Meeting, PA). A standard curve was run for each assay, and the unknown cAMP concentrations were obtained utilizing a weighted four-parameter logistic curve fitting (as recommended 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 control values, which were considered as 100!. In order to assume normal distribution, percentages were converted to ratios and all data subjected to the arcsine square root transformation (21Sokal R.R. Rolf J.F. Biometry: The Principles and Practice of Statistics in Biological Research. 3rd Ed. W. H. Freeman, New York1995Google Scholar). Statistical analysis was performed with the aid of GraphPad software using the parametric t test for simple comparisons and either the Tukey test following one-way analysis of variance or the Bonferroni post-tests after two-way analysis of variance for multiple comparisons. Cl– Is Necessary for Capacitation—Cl– is involved in the regulation of the capacitation-associated hyperpolarization of the sperm Em (4Hernandez-Gonzalez E.O. Trevino C.L. Castellano L.E. de la Vega-Beltran J.L. Ocampo A.Y. Wertheimer E. Visconti P.E. Darszon A. J. Biol. Chem. 2007; 282: 24397-24406Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). However, it has not been established whether Cl– acts upstream, downstream, or independently of other signaling pathways involved in capacitation. To analyze the role of Cl– in capacitation, media with different Cl– concentrations were prepared by replacing this anion with gluconate as described under "Experimental Procedures." Initially, the capacitation-associated increase in tyrosine phosphorylation was analyzed by Western blot using anti-PY antibodies. As shown previously (18Visconti P.E. Bailey J.L. Moore G.D. Pan D. Olds-Clarke P. Kopf G.S. Development (Camb.). 1995; 121: 1129-1137Crossref PubMed Google Scholar), in the absence of BSA and HCO3-, either with or without Cl–, there is no increase in tyrosine phosphorylation (Fig. 1A). When BSA and HCO3- were present, the increase in tyrosine phosphorylation was observed only in the presence of Cl– (Fig. 1A). Moreover, the increase in tyrosine phosphorylation was dependent on the Cl– concentration (Fig. 1B). Because hexokinase (116 kDa) is a protein that is constitutively phosphorylated in tyrosine residues (18Visconti P.E. Bailey J.L. Moore G.D. Pan D. Olds-Clarke P. Kopf G.S. Development (Camb.). 1995; 121: 1129-1137Crossref PubMed Google Scholar), it is commonly used as a loading control for Western blots with anti-PY antibodies. To confirm this understanding, as an additional loading control each membrane was stripped and reproved with an anti-tubulin antibody (described under "Experimental Procedures"). Capacitation is linked to sperm flagellum hyperactivation and to sperm preparation for agonist-induced acrosome reaction. To investigate the effect of Cl– on hyperactivated motility, CASA of sperm incubated in capacitating media with or without Cl– was carried out. Although the percentage of motile sperm did not differ in either medium (with or without Cl–), the percentage of hyperactivated cells was significantly reduced in the absence of Cl– (Fig. 1C). To assure that the absence of Cl– did not affect sperm viability, sperm membrane integrity was tested using propidium iodide, a nonpermeable dye commonly used to assay this parameter. No difference in the percentage of viable sperm was observed between sperm incubated in media with and without Cl– (data not shown). Second, to test whether the prese
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