Involvement of Cystic Fibrosis Transmembrane Conductance Regulator in Mouse Sperm Capacitation
2007; Elsevier BV; Volume: 282; Issue: 33 Linguagem: Inglês
10.1074/jbc.m701603200
ISSN1083-351X
AutoresEnrique O. Hernández‐González, Claudia L. Treviño, Laura E. Castellano, José Luis de la Vega‐Beltrán, Ana Y. Ocampo, Eva Wertheimer, Pablo E. Visconti, Alberto Darszon,
Tópico(s)Pluripotent Stem Cells Research
ResumoMammalian sperm acquire fertilizing ability in the female tract during a process known as capacitation. In mouse sperm, this process is associated with increases in protein tyrosine phosphorylation, membrane potential hyperpolarization, increase in intracellular pH and Ca2+, and hyperactivated motility. The molecular mechanisms involved in these changes are not fully known. Present evidence suggests that in mouse sperm the capacitation-associated membrane hyperpolarization is regulated by a cAMP/protein kinase A-dependent pathway involving activation of inwardly rectifying K+ channels and inhibition of epithelial sodium channels (ENaCs). The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel that controls the activity of several transport proteins, including ENaCs. Here we explored whether CFTR is involved in the regulation of ENaC inhibition in sperm and therefore is essential for the capacitation-associated hyperpolarization. Using reverse transcription-PCR, Western blot, and immunocytochemistry, we document the presence of CFTR in mouse and human sperm. Interestingly, the addition of a CFTR inhibitor (diphenylamine-2-carboxylic acid; 250 μm) inhibited the capacitation-associated hyperpolarization, prevented ENaC closure, and decreased the zona pellucida-induced acrosome reaction without affecting the increase in tyrosine phosphorylation. Incubation of sperm in Cl--free medium also eliminated the capacitation-associated hyperpolarization. On the other hand, a CFTR activator (genistein; 5-10 μm) promoted hyperpolarization in mouse sperm incubated under conditions that do not support capacitation. The addition of dibutyryl cyclic AMP to noncapacitated mouse sperm elevated intracellular Cl-. These results suggest that cAMP-dependent Cl- fluxes through CFTR are involved in the regulation of ENaC during capacitation and thus contribute to the observed hyperpolarization associated with this process. Mammalian sperm acquire fertilizing ability in the female tract during a process known as capacitation. In mouse sperm, this process is associated with increases in protein tyrosine phosphorylation, membrane potential hyperpolarization, increase in intracellular pH and Ca2+, and hyperactivated motility. The molecular mechanisms involved in these changes are not fully known. Present evidence suggests that in mouse sperm the capacitation-associated membrane hyperpolarization is regulated by a cAMP/protein kinase A-dependent pathway involving activation of inwardly rectifying K+ channels and inhibition of epithelial sodium channels (ENaCs). The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel that controls the activity of several transport proteins, including ENaCs. Here we explored whether CFTR is involved in the regulation of ENaC inhibition in sperm and therefore is essential for the capacitation-associated hyperpolarization. Using reverse transcription-PCR, Western blot, and immunocytochemistry, we document the presence of CFTR in mouse and human sperm. Interestingly, the addition of a CFTR inhibitor (diphenylamine-2-carboxylic acid; 250 μm) inhibited the capacitation-associated hyperpolarization, prevented ENaC closure, and decreased the zona pellucida-induced acrosome reaction without affecting the increase in tyrosine phosphorylation. Incubation of sperm in Cl--free medium also eliminated the capacitation-associated hyperpolarization. On the other hand, a CFTR activator (genistein; 5-10 μm) promoted hyperpolarization in mouse sperm incubated under conditions that do not support capacitation. The addition of dibutyryl cyclic AMP to noncapacitated mouse sperm elevated intracellular Cl-. These results suggest that cAMP-dependent Cl- fluxes through CFTR are involved in the regulation of ENaC during capacitation and thus contribute to the observed hyperpolarization associated with this process. Sperm capacitation is a complex phenomenon required for fertilization. In mouse sperm, capacitation includes reorganization of the plasma membrane, increase in protein tyrosine phosphorylation, hyperpolarization of the plasma membrane potential (Em), 2The abbreviations used are: Em, plasma membrane potential; AR, acrosome reaction; ZP, zona pellucida; ENaC, epithelial sodium channel; PKA, protein kinase A; CFTR, cystic fibrosis transmembrane conductance regulator; Bt2cAMP, dibutyryl cyclic AMP; IBMX, 3-isobutyl-1-methylxantine; MQAE, N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide; DiSC3(5), 3,3′-dipropylthiadicarbocyanine iodide; SITS, 4-acetomido-4′-isothiocyanatostilbene-2,2′-disulfonic acid; TRITC, tetramethylrhodamine isothiocyanate; DPC, diphenylamine-2-carboxylic acid; WH, Whitten's HEPES-buffered. and increases in intracellular pH (pHi) and Ca2+ ([Ca2+]i) (reviewed in Refs. 1Darszon A. Nishigaki T. Wood C. Trevino C.L. Felix R. Beltran C. Int. Rev. Cytol. 2005; 243: 79-172Crossref PubMed Scopus (139) Google Scholar and 2Visconti P.E. Westbrook V.A. Chertihin O. Demarco I. Sleight S. Diekman A.B. J. Reprod. Immunol. 2002; 53: 133-150Crossref PubMed Scopus (290) Google Scholar). Capacitation is also associated with the appearance of hyperactivated motility (3Yanagimachi R. Knobile E. Neill J.D. The Physiology of Reproduction. 1. 1994: 189-317Google Scholar). All of these changes prime sperm to effectively reach and penetrate the outer layers of the egg and to undergo the acrosome reaction (AR) (4Florman H.M. Arnoult C. Kazam I.G. Li C. O'Toole C.M. Biol. Reprod. 1998; 59: 12-16Crossref PubMed Scopus (166) Google Scholar). Among the changes observed during mouse sperm capacitation, the hyperpolarization that takes place during this process has been proposed to play the important role of removing inactivation from voltage-dependent Ca2+ channels such that they open upon a zona pellucida (ZP)-induced depolarization and trigger the AR. Little is known about the molecular entities that participate in this hyperpolarization and how all of the capacitation-associated changes are combined to promote capacitation. Several candidates have been proposed to participate in the capacitation-induced hyperpolarization. Studies from our group have demonstrated that inwardly rectifying K+ channels contribute to this hyperpolarization (5Munoz-Garay C. De la Vega-Beltran J.L. Delgado R. Labarca P. Felix R. Darszon A. Dev. Biol. 2001; 234: 261-274Crossref PubMed Scopus (83) Google Scholar, 6Acevedo J.J. Mendoza-Lujambio I. de la Vega-Beltran J.L. Trevino C.L. Felix R. Darszon A. Dev. Biol. 2006; 289: 395-405Crossref PubMed Scopus (45) Google Scholar). In addition, we documented that an electrogenic Na+/HCO3− cotransporter hyperpolarizes mouse sperm when external HCO3− is elevated (7Demarco I.A. Espinosa F. Edwards J. Sosnik J. De La Vega-Beltran J.L. Hockensmith J.W. Kopf G.S. Darszon A. Visconti P.E. J. Biol. Chem. 2003; 278: 7001-7009Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Furthermore, we recently reported the presence of epithelial Na+ channels (ENaCs) in mouse sperm and showed that a reduction in the sperm Na+ permeability is essential for the capacitation-associated hyperpolarization (8Hernandez-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 (102) Google Scholar). This later work strongly suggests that mouse sperm (ENaCs) are constitutively active in noncapacitated sperm and close during capacitation, resulting in Em hyperpolarization. Interestingly, experiments in this work suggest that closing of ENaCs is regulated directly or indirectly by a cAMP/protein kinase A (PKA)-dependent pathway (8Hernandez-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 (102) Google Scholar). The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-modulated Cl- channel and a regulator of several transporters and proteins, including K+ channels, such as ROMK1 and ROMK2 (9Kunzelmann K. Schreiber R. J. Membr. Biol. 1999; 168: 1-8Crossref PubMed Scopus (95) Google Scholar, 10Liu X. Singh B.B. Ambudkar I.S. J. Biol. Chem. 1999; 274: 25121-25129Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), anion exchangers, aquaporins, and ENaCs (11Briel M. Greger R. Kunzelmann K. J. Physiol. (Lond.). 1998; 508: 825-836Crossref Scopus (115) Google Scholar). In salt-absorbing sweat ducts, activation of CFTR causes ENaC stimulation (12Nagel G. Szellas T. Riordan J.R. Friedrich T. Hartung K. EMBO Rep. 2001; 2: 249-254Crossref PubMed Scopus (51) Google Scholar). In other tissues that are absorptive or secretory, depending on the physiological need, activation of CFTR reciprocally inhibits ENaCs (13Schwiebert E.M. Benos D.J. Egan M.E. Stutts M.J. Guggino W.B. Physiol. Rev. 1999; 79: 145-166Crossref PubMed Scopus (379) Google Scholar). Cystic fibrosis, the most prevalent human genetic disease, is caused by CFTR mutations (14Guggino W.B. Stanton B.A. Nat. Rev. Mol. Cell. Biol. 2006; 7: 426-436Crossref PubMed Scopus (350) Google Scholar). Additionally, the involvement of CFTR in male and female infertility has long been recognized (15Jarzabek K. Zbucka M. Pepinski W. Szamatowicz J. Domitrz J. Janica J. Wolczynski S. Szamatowicz M. Reprod. Biol. 2004; 4: 119-129PubMed Google Scholar). Nearly all men with cystic fibrosis are infertile due to a congenital bilateral absence of the vas deference. Recently, Chan et al. (16Chan H.C. Shi Q.X. Zhou C.X. Wang X.F. Xu W.M. Chen W.Y. Chen A.J. Ni Y. Yuan Y.Y. Mol. Cell. Endocrinol. 2006; 250: 106-113Crossref PubMed Scopus (62) Google Scholar) proposed that HCO3− permeation through CFTR (17Poulsen J.H. Fischer H. Illek B. Machen T.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5340-5344Crossref PubMed Scopus (365) Google Scholar) present in endometrial cells plays a role during in vivo sperm capacitation and may account for some cases of female cystic fibrosis infertility. This finding is consistent with the requirement of HCO3− for capacitation. Several modes of regulation of CFTR have been explored, including phosphorylation, Cl- and ATP levels, Em, and direct protein-protein interaction (12Nagel G. Szellas T. Riordan J.R. Friedrich T. Hartung K. EMBO Rep. 2001; 2: 249-254Crossref PubMed Scopus (51) Google Scholar, 18Kunzelmann K. Kiser G.L. Schreiber R. Riordan J.R. FEBS Lett. 1997; 400: 341-344Crossref PubMed Scopus (130) Google Scholar, 19Reddy M.M. Quinton P.M. Am. J. Physiol. 2006; 291 (-C129): C122Crossref Scopus (15) Google Scholar). Interestingly, CFTR and ENaC colocalize and may interact in several tissues (14Guggino W.B. Stanton B.A. Nat. Rev. Mol. Cell. Biol. 2006; 7: 426-436Crossref PubMed Scopus (350) Google Scholar). In this work, we document the presence of CFTR in both mouse and human sperm and demonstrate that diphenylamine-2-carboxylic acid (DPC), an inhibitor of CFTR, blocks the capacitation-associated hyperpolarization. Moreover, genistein, an activator of CFTR channels, hyperpolarized sperm under conditions that do not support capacitation. These genistein-induced changes in Em were inhibited by DPC. Since CFTR is a Cl- channel, we used the Cl- fluorescent probe N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) to determine the intracellular Cl- concentration ([Cl-]i) of noncapacitated mouse sperm and explored the influence of CFTR regulators on this parameter. The addition of genistein, as well as cAMP analogues, promoted Cl- influx in noncapacitated sperm that was blocked by DPC. Considering that closing of ENaCs in sperm is regulated by a cAMP pathway, we hypothesized that CFTR regulation of the sperm Em might be mediated by ENaCs. Consistent with this hypothesis, genistein diminished the amiloride-induced hyperpolarization and the amiloride-sensitive Na+ permeability in noncapacitated sperm that we had reported earlier (8Hernandez-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 (102) Google Scholar), suggesting that CFTR regulates the capacitation-associated hyperpolarization in mouse sperm through the inhibition of ENaCs. Materials—Amiloride, dibutyryl cyclic AMP (Bt2cAMP), 3-isobutyl-1-methylxantine (IBMX), carbonyl cyanide m-chlorophenylhydrazone, valinomycin, nigericin, sodium gluconate, and potassium gluconate were purchased from Sigma. MQAE, 3,3′-dipropylthiadicarbocyanine iodide (DiSC3(5)), 4-acetomido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS) were obtained from Invitrogen. Polyclonal antibody against CFTR was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mouse anti-goat IgG biotin-conjugated and Avidin TRITC-conjugated were from Pierce. DPC was from Sigma. DPC (10 mm stock), genistein, DiSC3(5), carbonyl cyanide m-chlorophenylhydrazone, and valinomycin (1 mm stocks) were prepared in Me2SO and stored at -20 °C until use. Bt2cAMP (1 mm), H-89, and IBMX (100 μm) were prepared on the day of the experiment using a modified Krebs-Ringer medium (Whitten's HEPES-buffered (WH) medium) (20Moore G.D. Ayabe T. Visconti P.E. Schultz R.M. Kopf G.S. Development. 1994; 120: 3313-3323Crossref PubMed Google Scholar) and used at the indicated concentration. Sperm Preparation—Experimental protocols were approved by the local Animal Care and Bioethics Committee of the Instituto de Biotecnologiía-Universidad Nacional Autónoma de México. In most experiments, cauda epididymal mouse sperm were collected from CD1 retired male breeders by placing minced cauda epididymis in WH medium. This medium does not support capacitation unless supplemented with 5 mg/ml bovine serum albumin and 24 mm NaHCO3. After 10 min, the sperm suspension was washed in 10 ml of the same medium by centrifugation at 800 × g for 10 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. To study the role of Cl- in capacitation and in the regulation of Em, NaCl and KCl were replaced by sodium gluconate and potassium gluconate, respectively. Human Sperm Preparation—Semen was obtained from normal, fertile volunteers by masturbation after at least 2 days of abstinence. After liquefaction, 1 ml of Ham's F-10 was applied to 1 ml of semen to allow the motile sperm to swim up into the upper layer of the suspension (1 h at 37 °C). Swim-up sperm were collected and adjusted to 1 × 106 cells/ml. Samples were used for Western blot analysis and indirect immunofluorescence analysis as described for mouse sperm. RNA Isolation and Reverse Transcription-PCR Experiments—Total RNA was prepared from isolated mouse elongated spermatids (21Bellve A.R. Methods Enzymol. 1993; 225: 84-113Crossref PubMed Scopus (300) Google Scholar) or ejaculated human sperm, 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 TaqDNA polymerase (Invitrogen). The CFTR subunit primers were designed using the human and mouse reported nucleotide sequence for these genes (human CFTR NM_020038 and mouse CFTR NM_021050, respectively). Primer sequences for human CFTR are as follows: forward, 5′-ATG ATT ATG GGA GAA CTG G-3; reverse, 5′-ATG AGA AAC GGT GTA AGG T-3′. Primer sequences for mouse CFTR are as follows: forward, 5′-GGA GCA AAC CCA AAC A-3′; reverse, 5′-AGC AGC CAC CTC AAC C-3′. The absence of genomic contamination in the RNA samples was confirmed with reverse transcription-negative controls (no reverse transcription) for each experiment. Amplified products were analyzed by DNA sequencing in order to confirm their identity. SDS-PAGE and Immunoblotting—Human and mouse sperm extracts were obtained by the method described by Ref. 22Hernandez-Gonzalez E.O. Lecona-Valera A.N. Escobar-Herrera J. Mujica A. Cell Motil. Cytoskeleton. 2000; 46: 43-58Crossref PubMed Scopus (46) Google Scholar. Sperm were resuspended in sample buffer containing protease inhibitors without 2-mercaptoethanol, and boiled for 5 min. Sperm samples were centrifuged at 10,000 × g for 15 min. After centrifugation, the supernatants were collected, and 2-mercaptoethanol was added to a final concentration of 5% (v/v). The samples were boiled for an additional 5 min and then subjected to 10% SDS-PAGE (23Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). Electrotransfer of proteins to Immobilon P (Bio-Rad) and immunodetection of CFTR was carried out as previously described (24Kalab P. Visconti P. Leclerc P. Kopf G.S. J. Biol. Chem. 1994; 269: 3810-3817Abstract Full Text PDF PubMed Google Scholar). Immunoblots were incubated with anti-CFTR and developed with the appropriate secondary antibody conjugated to horseradish peroxidase (Sigma) and the chemoluminescent ECL kit (Amersham Biosciences) according to the manufacturer's instructions. Indirect Immunofluorescence—Sperm suspensions were fixed in paraformaldehyde (4% final concentration) for 30 min at room temperature, washed by centrifugation at 800 × g for 5 min, permeabilized in PBS-Triton X-100 (0.05% final concentration) for 15 min at room temperature, and washed three times with PBS. Specific primary antibodies were added to sperm samples and incubated overnight at 4 °C, washed three times with PBS, and then incubated with the appropriate secondary antibody (Biotin-conjugated anti-rabbit IgG) for 1h at 37 °C. The secondary antibody was then subjected to three consecutive washes with PBS and developed by incubation with avidin-fluorescein isothiocyanate diluted in HEPES-saline buffer (20 mm HEPES and 100 mm NaCl, pH 8.2) for 1h at 37 °C. Finally, the samples were washed and mounted in PBS-glycerol (SlowFade, Molecular Probes) and examined using a confocal microscope. Membrane Potential Assay in Sperm Populations—Em was measured as previously described (7Demarco I.A. Espinosa F. Edwards J. Sosnik J. De La Vega-Beltran J.L. Hockensmith J.W. Kopf G.S. Darszon A. Visconti P.E. J. Biol. Chem. 2003; 278: 7001-7009Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Briefly, sperm were collected as indicated above, diluted in the appropriate medium and capacitated for different time periods depending on the experiment. Eight min before the measurement, 1 μm DiSC3(5) (final concentration) was added to the sperm suspension and further incubated for 5 min at 37 °C. One μm carbonyl cyanide m-chlorophenylhydrazone (final concentration) was then added to collapse mitochondrial potential, and sperm were incubated for an additional 2 min. After this period, 1.5 ml of the suspension was transferred to a gently stirred cuvette at 37 °C, and the fluorescence (620/670 nm excitation/emission) was recorded continuously. Calibration was performed as described before by adding 1 μm valinomycin and sequential additions of KCl. Na+-induced Depolarization and Amiloride-induced Hyperpolarization—After reaching steady state fluorescence, 50 mm NaCl was added while fluorescence was recorded in the presence or absence of different compounds as indicated in each figure. After a new fluorescent steady state was reached, calibration was performed as indicated above. Em changes elicited by NaCl were quantified, taking into consideration the calibration curve and the initial steady state fluorescence before NaCl addition. Controls to determine if amiloride or its solvent (Me2SO) altered the fluorescence of DiSC3(5) were performed with dye only and did not show significant changes in fluorescence (data not shown). Intracellular Cl- Measurements in Sperm Populations—[Cl-]i was measured in sperm populations using a Cl--sensitive fluorescent dye (MQAE) (25Verkman A.S. Sellers M.C. Chao A.C. Leung T. Ketcham R. Anal. Biochem. 1989; 178: 355-361Crossref PubMed Scopus (179) Google Scholar). Sperm were incubated with 10 mm MQAE for 30 min at 37 °C. Excess MQAE was removed by diluting sperm 10-fold with WH-Cl--free medium (100 mm sodium gluconate, 4.4 mm potassium gluconate, 1.2 mm KH2PO4, 1.2 mm MgSO4, 5.4 mm glucose, 0.8 mm pyruvic acid, 4.8 mm lactic acid, 20 mm HEPES, pH 7.2) and centrifuging for 3 min at 300 × g. The sperm pellet was resuspended in aliquots of 2 × 106 sperm/ml with medium under the following conditions. 1) To obtain the maximum fluorescence (Fmax), WH-Cl--free medium with nigericin (10 μm) and tributyltin (10 μm) was used. 2) To determine the fluorescence at defined Cl- concentrations (FCl-), two media were mixed (WH containing different NaCl concentrations (10-100 mm) and WH-Cl--free media keeping Cl- + gluconate- = 100 mm); nigericin (10 μm) and tributyltin (10 μm) were also added. 3) For minimum fluorescence (Fmin), 150 mm KSCN buffered with 10 mm HEPES (pH 7.2) and 5 μm valinomycin was used. Samples were illuminated at 350 nm, and emission was collected at 450 nm. The relationship between Cl- concentration and MQAE fluorescence intensity expressed as the ratio F0/FCl- provides a straight line with a slope equal to Kq, the Stern-Volmer constant, where the total quenchable signal F0 is defined by Fmax - Fmin. [Cl-]i was estimated using the constructed plot (25Verkman A.S. Sellers M.C. Chao A.C. Leung T. Ketcham R. Anal. Biochem. 1989; 178: 355-361Crossref PubMed Scopus (179) Google Scholar, 26Inglefield J.R. Schwartz-Bloom R.D. Methods. 1999; 18: 197-203Crossref PubMed Scopus (28) Google Scholar, 27Jayaraman S. Biwersi J. Verkman A.S. Am. J. Physiol. 1999; 276 (-C757): C747Crossref PubMed Google Scholar). The sperm [Cl-]i was also evaluated according to Garcia and Meizel (28Garcia M.A. Meizel S. J. Androl. 1999; 20: 88-93PubMed Google Scholar). Mouse sperm were incubated for 30 min in WH medium containing different Cl- concentrations (0-100 mm) and 10 mm MQAE. Excess MQAE was removed as mentioned above, and emission fluorescence intensity data from sperm suspensions (2 × 106 sperm/ml) were recorded for 120 s. The intracellular and extracellular [Cl-] were equilibrated by permeabilizing the sperm membrane with digitonin (10 μm), and the fluorescence was recorded for another 120 s. The [Cl-]i was calculated as described by Garcia and Meizel (28Garcia M.A. Meizel S. J. Androl. 1999; 20: 88-93PubMed Google Scholar). It is important to note that the values of the [Cl-]i are to be taken cautiously, since they are obtained from a heterogeneous sperm population. Cl- determinations require a calibration, which assumes that the dye behaves in sperm as in other cells (24Kalab P. Visconti P. Leclerc P. Kopf G.S. J. Biol. Chem. 1994; 269: 3810-3817Abstract Full Text PDF PubMed Google Scholar, 25Verkman A.S. Sellers M.C. Chao A.C. Leung T. Ketcham R. Anal. Biochem. 1989; 178: 355-361Crossref PubMed Scopus (179) Google Scholar, 26Inglefield J.R. Schwartz-Bloom R.D. Methods. 1999; 18: 197-203Crossref PubMed Scopus (28) Google Scholar) and does not correct for residual external dye, and some differences in experimental conditions. It is only possible to wash mouse sperm once by centrifugation, since they loose viability if centrifuged further. The influence of different drugs (i.e. genistein, cAMP/IBMX, DPC, H-89, and SITS) on [Cl-]i was determined using sperm suspensions loaded with MQAE as described above, after recording the basal fluorescence for 1-3 min, and measuring for a further 5-10 min. Two controls were performed: 1) drug solvents (Me2SO or water) were added while the fluorescence was recorded, and 2) MQAE fluorescence without cells was recorded, and the drugs were added. No significant fluorescence changes were observed after performing both controls (data not shown). [Cl-]i changes were estimated as described above. Acrosome Reaction Assay—Caudal epididymal mouse sperm were collected from CD1 mice and placed in capped 1.5-ml microcentrifuge tubes containing medium 199 (Sigma) supplemented with bovine serum albumin (0.4%, w/v), Na+ pyruvate (30 mg/liter), and NaHCO3 (2.2 g/liter) at 37 °C (4-5 × 106 cells/ml). The swim-up method (29Henkel R.R. Schill W.B. Reprod. Biol. Endocrinol. 2003; 1: 108Crossref PubMed Scopus (380) Google Scholar) was used to separate sperm with >90% motility. The sperm suspension was incubated 10 min, and the top ∼1 ml was separated and capacitated for 30 min at 37 °C (30Visconti 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 (271) Google Scholar). AR was induced after capacitation (in the presence or absence of DPC) in a 30-μl aliquot by adding 5 ZP eq/μl or 15 μm A23187. ZP was obtained from mouse oocytes (31Cross N.L. Meizel S. Biol. Reprod. 1989; 41: 635-641Crossref PubMed Scopus (173) Google Scholar). The percentage of AR was determined in a 30-μl aliquot by adding an equal volume of 10% formaldehyde in phosphate-buffered saline. Following fixation, 10-μl aliquots of the sperm suspension were spread onto glass slides and air-dried. The slides were stained with 0.22% Coomassie Blue G-250 in 50% methanol and 10% glacial acetic acid for ∼5 min, rinsed, and mounted with 50% (v/v) glycerol in phosphate-buffered saline (5Munoz-Garay C. De la Vega-Beltran J.L. Delgado R. Labarca P. Felix R. Darszon A. Dev. Biol. 2001; 234: 261-274Crossref PubMed Scopus (83) Google Scholar). At least 100 sperm were assayed per experimental condition to calculate the percentage of AR. Statistical Analysis—The data are expressed as mean ± S.E. The means were compared using an unpaired Student's t test, and p < 0.05 was considered to be statistically significant. CFTR Is Present in Mammalian Sperm—CFTR modulates several cellular processes, interacting with various channels and ionic transporters, particularly with ENaCs (14Guggino W.B. Stanton B.A. Nat. Rev. Mol. Cell. Biol. 2006; 7: 426-436Crossref PubMed Scopus (350) Google Scholar, 32Sheppard D.N. Welsh M.J. Physiol. Rev. 1999; 79 (-S45): S23Crossref PubMed Scopus (813) Google Scholar). We reported the presence of αENaC and δENaC subunits in mouse sperm (8Hernandez-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 (102) Google Scholar) and have now found the αENaC subunit in human sperm (data not shown). We examined if CFTR is present in mouse and human sperm. First, the presence of CFTR transcripts was analyzed using reverse transcription-PCR; cDNA was synthesized from total RNA extracted from human sperm ejaculates and purified mouse spermatogenic cells (33Trevino C.L. Felix R. Castellano L.E. Gutierrez C. Rodriguez D. Pacheco J. Lopez-Gonzalez I. Gomora J.C. Tsutsumi V. Hernandez-Cruz A. Fiordelisio T. Scaling A.L. Darszon A. FEBS Lett. 2004; 563: 87-92Crossref PubMed Scopus (66) Google Scholar). Specific primers for CFTR were designed using the reported human and mouse nucleotide sequences for these genes. CFTR fragments of the expected length were detected from human sperm (476 bp) and mouse spermatogenic cells (581 bp) cDNA (Fig. 1A), and their identities were confirmed by DNA sequencing. Because expression of CFTR transcripts in mouse spermatids and in human sperm does not imply the presence of the CFTR protein in mature sperm, Western blot and immunofluorescence experiments were conducted using an anti-CFTR polyclonal antibody. This antibody detected a protein band at the expected molecular weight in both human and mouse sperm, as well as in whole extracts from mouse lung that were included as a positive control (Fig. 1B). In all of these cases, preincubation with the CFTR antigenic peptide eliminated the Western blot signal (Fig. 1B). Using the same antibody, CFTR was immunolocalized to the midpiece of human (Fig. 1C) and mouse sperm (Fig. 1E). It is worth noting that αENaC localized to the same sperm region (8Hernandez-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 (102) Google Scholar). Previous incubation of the antibody with the respective antigenic peptide blocked the immunofluorescence signal (Fig. 1, D and F). CFTR Is Involved in the Capacitation-associated Hyperpolarization—Since CFTR is a Cl- channel, it may contribute to the mouse sperm resting Em and to the changes it undergoes during capacitation. Interestingly, 250 μm DPC, which inhibits CFTR, was able to block the capacitation-associated hyperpolarization without affecting the Em of sperm incubated under conditions that do not support capacitation (Fig. 2). On the other hand, noncapacitated sperm incubated 10 min with 10 μm genistein, a compound that activates CFTR, undergo a hyperpolarization that was greater than that which accompanies capacitation. Notably, the genistein-induced hyperpolarization was blocked by DPC. These results indicate that CFTR activation influences the sperm Em (Fig. 2, A and B). CFTR Participates in Capacitation—Taking into consideration that DPC blocked the capacitation-associated hyperpolarization, we explored whether CFTR inhibition was also able to block the capacitation process. Two experimental protocols were used. 1) Mouse sperm were capacitated in the presence or absence of 250 μm DPC, and then the AR was induced adding ZP. 2) Sperm were capacitated in the absence of DPC and then incubated for 5 min with DPC, just prior to the induction o
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