Sodium and Epithelial Sodium Channels Participate in the Regulation of the Capacitation-associated Hyperpolarization in Mouse Sperm
2005; Elsevier BV; Volume: 281; Issue: 9 Linguagem: Inglês
10.1074/jbc.m508172200
ISSN1083-351X
AutoresEnrique O. Hernández‐González, Julian Sosnik, Jennifer L. Edwards, Juan José Acevedo‐Fernández, Irene Mendoza-Lujambio, Ignacio López‐González, Ignacio A. Demarco, Eva Wertheimer, Alberto Darszon, Pablo E. Visconti,
Tópico(s)Ovarian function and disorders
ResumoIn a process called capacitation, mammalian sperm gain the ability to fertilize after residing in the female tract. During capacitation the mouse sperm plasma membrane potential (Em) hyperpolarizes. However, the mechanisms that regulate sperm Em are not well understood. Here we show that sperm hyperpolarize when external Na+ is replaced by N-methyl-glucamine. Readdition of external Na+ restores a more depolarized Em by a process that is inhibited by amiloride or by its more potent derivative 5-(N-ethyl-N-isopropyl)-amiloride hydrochloride. These findings indicate that under resting conditions an electrogenic Na+ transporter, possibly involving an amiloride sensitive Na+ channel, may contribute to the sperm resting Em. Consistent with this proposal, patch clamp recordings from spermatogenic cells reveal an amiloride-sensitive inward Na+ current whose characteristics match those of the epithelial Na+ channel (ENaC) family of epithelial Na+ channels. Indeed, ENaC-α and -δ mRNAs were detected by reverse transcription-PCR in extracts of isolated elongated spermatids, and ENaC-α and -δ proteins were found on immunoblots of sperm membrane preparations. Immunostaining indicated localization of ENaC-α to the flagellar midpiece and of ENaC-δ to the acrosome. Incubations known to produce capacitation in vitro or induction of capacitation by cell-permeant cAMP analogs decreased the depolarizing response to the addition of external Na+. These results suggest that increases in cAMP content occurring during capacitation may inhibit ENaCs to produce a required hyperpolarization of the sperm membrane. In a process called capacitation, mammalian sperm gain the ability to fertilize after residing in the female tract. During capacitation the mouse sperm plasma membrane potential (Em) hyperpolarizes. However, the mechanisms that regulate sperm Em are not well understood. Here we show that sperm hyperpolarize when external Na+ is replaced by N-methyl-glucamine. Readdition of external Na+ restores a more depolarized Em by a process that is inhibited by amiloride or by its more potent derivative 5-(N-ethyl-N-isopropyl)-amiloride hydrochloride. These findings indicate that under resting conditions an electrogenic Na+ transporter, possibly involving an amiloride sensitive Na+ channel, may contribute to the sperm resting Em. Consistent with this proposal, patch clamp recordings from spermatogenic cells reveal an amiloride-sensitive inward Na+ current whose characteristics match those of the epithelial Na+ channel (ENaC) family of epithelial Na+ channels. Indeed, ENaC-α and -δ mRNAs were detected by reverse transcription-PCR in extracts of isolated elongated spermatids, and ENaC-α and -δ proteins were found on immunoblots of sperm membrane preparations. Immunostaining indicated localization of ENaC-α to the flagellar midpiece and of ENaC-δ to the acrosome. Incubations known to produce capacitation in vitro or induction of capacitation by cell-permeant cAMP analogs decreased the depolarizing response to the addition of external Na+. These results suggest that increases in cAMP content occurring during capacitation may inhibit ENaCs to produce a required hyperpolarization of the sperm membrane. Mammalian sperm are not able to fertilize after ejaculation. They acquire this ability only after residing in the female uterine tract for a finite period of time that varies depending on the species. The molecular, biochemical, and physiological changes that occur in sperm while in the female tract are collectively referred to as capacitation (1Yanagimachi R. The Physiology of Reproduction.in: Knobil E. Neill J.D. Vol. 1. Raven Press, Ltd., New York1994: 189-317Google Scholar). Capacitation is associated with changes in membrane properties, enzyme activities, and motility that prepare the sperm for the acrosome reaction and for penetration of the egg vestments prior to fertilization. The molecular basis of capacitation has been partially defined and includes: the removal of cholesterol from the sperm plasma membrane by cholesterol acceptors such as bovine serum albumin (2Visconti 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, 3Visconti P.E. Ning X. Fornes M.W. Alvarez J.G. Stein P. Connors S.A. Kopf G.S. Dev. Biol. 1999; 214: 429-443Crossref PubMed Scopus (237) Google Scholar), modifications in plasma membrane phospholipids, fluxes of HCO–3 (4Demarco 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 (174) Google Scholar) and other intracellular ions, and increased tyrosine phosphorylation of proteins (5Visconti P.E. Bailey J.L. Moore G.D. Pan D. Olds-Clarke P. Kopf G.S. Development. 1995; 121: 1129-1137Crossref PubMed Google Scholar, 6Visconti P.E. Moore G.D. Bailey J.L. Leclerc P. Connors S.A. Pan D. Olds-Clarke P. Kopf G.S. Development. 1995; 121: 1139-1150Crossref PubMed Google Scholar, 7Ficarro S. Chertihin O. Westbrook V.A. White F. Jayes F. Kalab P. Marto J.A. Shabanowitz J. Herr J.C. Hunt D. Visconti P.E. J. Biol. Chem. 2003; 278: 11579-11589Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar). These events are likely to play a role in the induction of hyperactivated motility and the ability of the sperm to undergo a regulated acrosome reaction (for review see Ref. 8Visconti 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). Bovine and mouse sperm capacitation is also accompanied by a plasma membrane hyperpolarization. Em decreases in mouse sperm from –38 to –55 mV (4Demarco 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 (174) Google Scholar, 9Zeng Y. Clark E.N. Florman H.M. Dev. Biol. 1995; 171: 554-563Crossref PubMed Scopus (176) Google Scholar, 10Muñoz-Garay C. De la Vega-Beltran J.L. Delgado R. Labarca P. Felix R. Darszon A. Dev. Biol. 2001; 234: 261-274Crossref PubMed Scopus (82) Google Scholar) and in bovine sperm from –33 to –66 mV (9Zeng Y. Clark E.N. Florman H.M. Dev. Biol. 1995; 171: 554-563Crossref PubMed Scopus (176) Google Scholar). Because capacitation prepares sperm for the acrosome reaction, the capacitation-associated hyperpolarization may regulate the ability of sperm to generate transient Ca2+ elevations during the acrosome reaction induced by physiological agonists (e.g. zona pellucida) (11Florman H.M. Arnoult C. Kazam I.G. Li C. O'Toole C.M. Biol. Reprod. 1998; 59: 12-16Crossref PubMed Scopus (166) Google Scholar). In this respect, low voltage-activated T-type Ca2+ channels have been detected in mouse spermatogenic cells (12Lievano A. Santi C.M. Serrano C.J. Trevino C.L. Bellve A.R. Hernandez-Cruz A. Darszon A. FEBS Lett. 1996; 388: 150-154Crossref PubMed Scopus (151) Google Scholar, 13Arnoult C. Villaz M. Florman H.M. Mol. Pharmacol. 1998; 53: 1104-1111PubMed Google Scholar), and these channels are also present in mature mouse sperm (14Arnoult C. Cardullo R.A. Lemos J.R. Florman H.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13004-13009Crossref PubMed Scopus (206) Google Scholar, 15Treviño 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). One unique property of low voltage-activated Ca2+ channels is that they inactivate at the resting Em of sperm prior to capacitation (around –35 mV) (12Lievano A. Santi C.M. Serrano C.J. Trevino C.L. Bellve A.R. Hernandez-Cruz A. Darszon A. FEBS Lett. 1996; 388: 150-154Crossref PubMed Scopus (151) Google Scholar, 14Arnoult C. Cardullo R.A. Lemos J.R. Florman H.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13004-13009Crossref PubMed Scopus (206) Google Scholar). Thus, if low voltage-activated Ca2+ channels are involved in the regulation of the acrosome reaction, the capacitation-associated sperm hyperpolarization may be required to remove this inactivation (11Florman H.M. Arnoult C. Kazam I.G. Li C. O'Toole C.M. Biol. Reprod. 1998; 59: 12-16Crossref PubMed Scopus (166) Google Scholar, 16Arnoult C. Kazam I.G. Visconti P.E. Kopf G.S. Villaz M. Florman H.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6757-6762Crossref PubMed Scopus (189) 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). Although the molecular mechanisms by which the sperm Em hyperpolarizes during capacitation are not clear, there exist several potential candidates. Muñoz-Garay et al. (10Muñoz-Garay C. De la Vega-Beltran J.L. Delgado R. Labarca P. Felix R. Darszon A. Dev. Biol. 2001; 234: 261-274Crossref PubMed Scopus (82) Google Scholar) demonstrated with patch clamp techniques that inward rectifying K+ channels are expressed in mouse spermatogenic cells and proposed that these channels may contribute to the capacitation-associated sperm membrane hyperpolarization. An increase in sperm K+ permeability should lead to an Em hyperpolarization, according to the K+ equilibrium potential (18Wright S.H. Adv. Physiol. Educ. 2004; 28: 139-142Crossref PubMed Scopus (111) Google Scholar). Alternatively, the sperm plasma membrane may become less permeable to Na+. The relatively depolarized mammalian sperm resting Em before capacitation could be explained, at least in part, by a relatively high Na+ permeability. A capacitation-dependent decrease of this permeability would result in a sperm hyperpolarization. It has been reported that human sperm suspended in 0 Ca2+ medium undergo a Na+-dependent depolarization. Li+ can replace Na+; thus, these findings suggest that a Na+- and Li+-permeable electrogenic pathway may be present in mammalian sperm (19Gonzalez-Martinez M.T. J. Biol. Chem. 2003; 278: 36304-36310Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The present work explores this later possibility. We observed that replacement of external Na+ by nonpermeable cations resulted in sperm Em hyperpolarization. The addition of external Na+ to these sperm produced a depolarization that was potently inhibited by amiloride and its analog EIPA, 2The abbreviations used are: EIPA, 5-(N-ethyl-N-isopropyl)-amiloride hydrochloride; ENaC, epithelial Na+ channel(s); BCECF, 2′,7′-bis-(2-carboxyethyl)-5-carboxyfluorescein; AM, acetoxymethyl ester; WH, Whitten's HEPES-buffered; PBS, phosphate-buffered saline; PC, polycystin; m-chlorophenylhydrazone, carbonyl cyanide 3-chlorophenylhydrazone; Sp-cAMPS, Sp-adenosine 3′,5′-cyclic monophosphorothioate. 2The abbreviations used are: EIPA, 5-(N-ethyl-N-isopropyl)-amiloride hydrochloride; ENaC, epithelial Na+ channel(s); BCECF, 2′,7′-bis-(2-carboxyethyl)-5-carboxyfluorescein; AM, acetoxymethyl ester; WH, Whitten's HEPES-buffered; PBS, phosphate-buffered saline; PC, polycystin; m-chlorophenylhydrazone, carbonyl cyanide 3-chlorophenylhydrazone; Sp-cAMPS, Sp-adenosine 3′,5′-cyclic monophosphorothioate. high pH, and the incubation of mouse sperm under capacitating conditions. Moreover, high pH and amiloride were also capable of hyperpolarizing sperm in the presence of Na+. Altogether, these results suggest that epithelial Na+ channels (ENaCs) are present in mouse sperm and that they may contribute to the capacitation-associated hyperpolarization. Consistent with this hypothesis, we detected the transcripts for both ENaC-α and ENaC-δ subunits in mouse spermatogenic cells and the respective proteins in mature sperm. This is the first time ENaC-δ is reported in mouse. Furthermore, we used electrophysiological techniques to demonstrate the presence of ENaC type currents in spermatogenic cells, the precursors of sperm. Finally, we present evidence that reduction of the Na+ sperm permeability does contribute to the capacitation-associated Em hyperpolarization. Materials—Amiloride, dibutyryl cAMP, m-chlorophenylhydrazone (carbonyl cyanide 3-chlorophenylhydrazone), valinomycin, choline chloride (choline+Cl–), choline bicarbonate (choline+HCO–3), N-methyl-d-glucamine, and water for embryo transfer (used to make Whitten's medium) were purchased from Sigma. EIPA, 3,3′-dipropylthiadicarbocyanine iodide (DiSC3) (5Visconti P.E. Bailey J.L. Moore G.D. Pan D. Olds-Clarke P. Kopf G.S. Development. 1995; 121: 1129-1137Crossref PubMed Google Scholar), 2′,7′-bis-(2-carboxyethyl)-5-carboxyfluorescein-acetoxymethyl ester (BCECF-AM), and Sodium Green tetraacetate were obtained from Molecular Probes (Eugene, OR). Sp-cAMPS, and 3-isobutyl-1-methylxanthine were purchased from Biomol (Butler Pike, PA). Polyclonal antibodies against ENaC-α and ENaC-δ were purchased from Chemicon International (Temecula, CA). Donkey anti-rabbit IgG biotin-conjugated and Avidin fluorescein isothiocyanate-conjugated antibodies were from Pierce. The following compounds were prepared in Me2SO at the stock concentrations noted between parentheses and stored at –20 °C except when otherwise stated: DiSC3 (5Visconti P.E. Bailey J.L. Moore G.D. Pan D. Olds-Clarke P. Kopf G.S. Development. 1995; 121: 1129-1137Crossref PubMed Google Scholar), BCECF-AM, and m-chlorophenylhydrazone and valinomycin (1 mm stocks). Other compounds were prepared on the day of the experiment and dissolved in Whitten's medium and added at the final concentration indicated between parentheses: Sp-cAMPS (100 μm), Rp-cAMPS (100 μm), dibutyryl cAMP (1 mm), and 3-isobutyl-1-methylxanthine (100 μm). Sperm Preparation—Experimental protocols were approved by the University of Massachusetts Animal Care Committee. In most experiments, cauda epididymal mouse sperm were collected from CD1 retired male breeders by placing minced cauda epididymis in 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). This medium, which does not support capacitation, was first prepared in the absence of bovine serum albumin and NaHCO3 and contains 1 mm polyvinyl pyrrolidone (average molecular weight, 40,000). After 5 min, sperm in suspension were washed in 10 ml of the same medium by centrifugation at 800 × g for 10 min at room temperature. The 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 of bovine serum albumin and 24 mm of NaHCO3 were added. The pH was maintained at 7.2 except when the role of extracellular pH (pHe) was evaluated. To study the role of Na+ in capacitation and in the regulation of Em, NaCl was replaced by either choline+Cl– or N-methyl-d-glucamine+Cl– up to the concentration indicated in the respective experiment. Membrane Potential Assay in Sperm Populations—Em was measured as previously described (4Demarco 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 (174) Google Scholar). Briefly, sperm were collected as indicated above and, after dilution in the appropriate medium, capacitated for different time periods depending on the experiment. Eight min before the measurement, 1 μm DiSC3 (5Visconti P.E. Bailey J.L. Moore G.D. Pan D. Olds-Clarke P. Kopf G.S. Development. 1995; 121: 1129-1137Crossref PubMed Google Scholar) (final concentration) was added to the sperm suspension and further incubated for 5 min at 37 °C. One μm m-chlorophenylhydrazone (final concentration) was then added to collapse mitochondrial potential, and the sperm was incubated for 2 additional 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 (4Demarco 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 (174) Google Scholar) by adding 1 μm valinomycin and sequential additions of KCl (21Espinosa F. Darszon A. FEBS Lett. 1995; 372: 119-125Crossref PubMed Scopus (62) Google Scholar). To analyze changes provoked by the addition of Na+, sperm were recovered as described above and incubated for different time periods depending on the experiment. The cells were then transferred to a gently stirred cuvette at 37 °C, and the fluorescence (620/670 nm excitation/emission) was recorded continuously. After reaching steady state fluorescence, different Na+ concentrations were added while the fluorescence was recorded. After a new fluorescent steady state was reached, calibration was performed as indicated above (4Demarco 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 (174) Google Scholar). The changes in Em elicited by NaCl were quantified taking into consideration the calibration curve and the initial steady state fluorescent before NaCl addition. Intracellular pH and Na+ Measurements in Sperm Populations— [Na+]i and pHi measurements were conducted as described before (4Demarco 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 (174) Google Scholar). Briefly, sperm (1 × 106 cell/ml) in WH medium were incubated at 37 °C for 30 min in 10 μm Sodium Green tetraacetate or 4 μm BCECF-AM, the cell-permeant nonfluorescent precursor of Sodium Green and BCECF, respectively. After incubation, the cells were washed in fresh medium once (400 × g for 5 min) and resuspended, and 1.5 ml of this suspension was placed in a gentle stirring cuvette for fluorescence measurements. Changes in fluorescent were expressed in arbitrary units of fluorescence. When the effects of [Na+]e on pHi or [Na+]i were assayed, sperm were collected, loaded, and washed in Na+-free WH medium. RNA Isolation and Reverse Transcription-PCR Experiments—Total RNA was prepared from isolated mouse elongated spermatids (22Bellve A.R. Methods Enzymol. 1993; 225: 84-113Crossref PubMed Scopus (296) Google Scholar) using TRIzol reagent (Sigma) according to the manufacturer's instructions. cDNA was synthesized from total RNA samples by 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 ENaC-α subunit primers were designed using the mouse reported nucleotide sequence for this gene (Scnn1a, NM_011324). Primers for the ENaC-δ subunit were designed using the mouse genomic clone sequence AL670236. Primer sequences are ENaC-α, forward, 5′-AAG CCC AAG GGT GTA GAG T-3′, and reverse, 5′-GAT GAG CCG AAC CAC AGG-3′; ENaC-δ, forward, 5′-CCC AGC CAT AAA CTC-3′, and reverse, 5′-ATC TCC ACC ATC AGC-3′. The absence of genomic contamination in the RNA samples was confirmed with reverse transcription-negative controls (no cDNA) for each experiment (not shown). Amplified products were analyzed by DNA sequencing to confirm their identity. SDS-PAGE and Immunoblotting—Mouse sperm membranes were obtained by the method described by Hernández-González et al. (23Hernández-González E.O. Lecona-Valera A.N. Escobar-Herrera J. Mujica A. Cell. Motil. Cytoskeleton. 2000; 46: 43-58Crossref PubMed Scopus (46) Google Scholar). The sperm membranes were concentrated by centrifugation (100,000 × g) and resuspended in sample buffer (23Hernández-González E.O. Lecona-Valera A.N. Escobar-Herrera J. Mujica A. Cell. Motil. Cytoskeleton. 2000; 46: 43-58Crossref PubMed Scopus (46) Google Scholar) without 2-mercaptoethanol and boiled for 5 min. After centrifuging, the supernatants were saved, and 2-mercaptoethanol was added to a final concentration of 5% (v/v); the sample was boiled for 5 min, and then subjected to 10% SDS-PAGE (24Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207018) Google Scholar). Electrophoretic transfer of proteins to Immobilon P (Bio-Rad) and immunodetection of ENaC subunits were carried out as previously described (25Kalab P. Visconti P. Leclerc P. Kopf G.S. J. Biol. Chem. 1994; 269: 3810-3817Abstract Full Text PDF PubMed Google Scholar). Immunoblots were developed with the appropriate secondary antibody conjugated to horseradish peroxidase (Sigma) and an ECL kit (Amersham Biosciences) according to the manufacturer's instructions. Indirect Immunofluorescence—Sperm suspensions were fixed in formaldehyde (1.5% 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 1 h 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 1 h at 37°C. Finally, the samples were washed and mounted in PBS-glycerol (SlowFade, Molecular Probes) and examined using an epifluorescence microscope. Single Cell Fluorescence Analysis of Changes in [Na+]i—Cauda epididymal mouse sperm incubated in WH medium or Na+-free WH medium were loaded with 10 μm Sodium Green tetraacetate for 30 min at 37 °C and immobilized on a poly-l-lysine-coated glass. To withdraw the dye excess, sperm were washed three times with fresh medium. Fluorescence images were collected for 1 s every 10 s using the excitation/emission pair 470/490 nm on an inverted microscope (IX-70 Olympus) through a 40× objective with a digital CCD camera (Hamamatsu C4742–95, MA). The experiments were performed at 37 °C employing a heating chamber regulated on-line with the system acquisition control. Off-line analysis of the collected data were performed using Open Lab (Improvision). At least 30 cells were analyzed in each experiment. Electrophysiology—Spermatogenic cells were obtained following the procedure described by Santi et al. (26Santi C.M. Darszon A. Hernandez-Cruz A. Am. J. Physiol. 1996; 271: C1583-C1593Crossref PubMed Google Scholar). In ∼3-month-old mice individual meiotic pachytene spermatocytes and spermatids or their symplasts mainly constitute the spermatogenic cell suspension. Recordings were performed only on these symplasts. Na+ currents were recorded according to the whole cell patch clamp technique (27Hamill O.P. Marty A. Neher E. Sakmann B. Sigworth F.J. Pfluegers Arch. Eur. J. Physiol. 1981; 391: 85-100Crossref PubMed Scopus (15138) Google Scholar). In brief, all of the recordings were done at room temperature using an Axopatch 200A amplifier (Axon Instruments) and 2–4 MOhm micropipettes. The cells were clamped at a holding potential of –50 mV. Currents were evoked by 200-ms voltage steps to test potentials ranging from –100 to +40 mV. The currents were captured on-line and digitized at a sampling rate of 10 kHz following filtering of the current record (5 kHz) using a computer attached to a Digidata 1200 interface (Axon). Pulse protocols, data capture, and analysis of recordings were performed using pCLAMP software (Axon). To isolate Na+ currents, the cells were bathed in a solution containing 130 mm NaCl, 3 mm KCl, 10 mm CaCl2, 2 mm MgCl2, 1 mm NaHCO3, 0.5 mm NaH2PO4, 5 mm HEPES, 10 mm glucose (pH 7.4). The internal solution consisted of 110 mm CsMeSO3, 10 mm CsF, 15 mm CsCl, 4.6 mm CaCl2, 10 mm EGTA, HEPES buffer 5 (pH 7.3/CsOH). ENaC channel blockers amiloride and EIPA were prepared as 100 mm stock solutions in Me2SO and diluted in the bath solution for each experiment (Me2SO final concentration < 0.1%). Statistical Analysis—The data are expressed as the means ± S.E. The means were compared using paired Student's t test, and p < 0.05 was considered to be statistically significant. Mouse Sperm Resting Membrane Potential Is Na+-dependent—To investigate whether the Na+ permeability contributes to the resting Em, Na+ in the incubation medium was replaced by nonpermeant cations such as choline+ or N-methyl-d-glucamine+. Sperm were then diluted in WH medium with differing final Na+ concentrations; the sum of the concentrations of either Na+ and choline+ or Na+ and N-methyl-d-glucamine+ were maintained constant in all cases. Reduction of the extracellular Na+ concentration ([Na+]e) in the incubation medium leads to an Em hyperpolarization in a concentration-dependent manner (Fig. 1, A and B). These results indicate that Na+ participates in the regulation of the resting Em in mouse sperm. To directly assay how [Na+]e influences sperm Em, these cells were recovered in Na+-free WH medium, and increasing concentrations of Na+ were added while the Em was recorded continuously as described under "Experimental Procedures" (Fig. 1, C and D). Under these conditions a Na+ concentration-dependent depolarization was observed, consistent with the hypothesis that electrogenic Na+ uptake occurs in mouse sperm. The Na+-induced Depolarization Is Inhibited by Amiloride and Regulated by pHe—The electrogenic Na+ uptake that seems to occur in noncapacitated mouse sperm could be due to Na+-permeable channels or to the Na+/Ca2+ antiporter. Tetrodotoxin and pyrethroid, which both affect voltage-dependent Na+ channels (28Narahashi T. Mini. Rev. Med. Chem. 2002; 2: 419-432Crossref PubMed Scopus (81) Google Scholar, 29Yu F.H. Catterall W.A. Genome Biol. 2003; 4: 207.1-7Crossref Scopus (514) Google Scholar), did not alter the Na+-induced depolarizing current when used at 1 and 50 μm, respectively (data not shown). Similarly, a well known inhibitor of Na+/Ca2+ antiporters, KB-R7943 (30Linck B. Qiu Z. He Z. Tong Q. Hilgemann D.W. Philipson K.D. Am. J. Physiol. 1998; 274: C415-C423Crossref PubMed Google Scholar), at concentrations up to 10 μm, did not inhibit the Na+ permeability (data not shown). Therefore, it is unlikely that voltage-dependent Na+ channels or the Na+/Ca2+ antiporter are responsible for the Na+-induced depolarization in noncapacitated sperm. On the other hand, amiloride and the amiloride analog EIPA inhibited the Na+ depolarizing current in a concentration-dependent manner (Fig. 2, A and B). Amiloride and EIPA are known to potently inhibit the ENaC family of Na+ channels with IC50 values similar to those obtained for the inhibition of the Na+-induced depolarization (31Inagaki A. Yamaguchi S. Ishikawa T. Am. J. Physiol. 2004; 286: C380-C390Crossref Scopus (21) Google Scholar, 32Schreiber R. Konig J. Sun J. Markovich D. Kunzelmann K. J. Membr. Biol. 2003; 192: 101-110Crossref PubMed Scopus (18) Google Scholar). Although known Na+/H+ antiporters are not electrogenic (33Aronson P.S. Annu. Rev. Physiol. 1985; 47: 545-560Crossref PubMed Google Scholar, 34Grinstein S. Rothstein A. J. Membr. Biol. 1986; 90: 1-12Crossref PubMed Scopus (535) Google Scholar) and cannot be directly responsible for the Na+-induced depolarization, an intracellular pH (pHi) change could modulate other channels. This possibility was discarded by showing that addition of 20 mm [Na+]e to sperm loaded with a pH-sensitive dye (BCECF) and incubated in Na+-free WH medium did not alter pHi (Fig. 2C, left panel). As expected, the addition of NH4Cl increased pHi (Fig. 2C, right panel). Altogether these findings are consistent with the hypothesis that a member of the ENaC family is present in mature mouse sperm. The activity of ENaC family members is dependent on pHe (35Awayda M.S. Boudreaux M.J. Reger R.L. Hamm L.L. Am. J. Physiol. 2000; 279: C1896-C1905Crossref PubMed Google Scholar, 36Ji H.L. Benos D.J. J. Biol. Chem. 2004; 279: 26939-26947Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 37Yamamura H. Ugawa S. Ueda T. Nagao M. Shimada S. J. Biol. Chem. 2004; 279: 12529-12534Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), and thus we investigated the effect of pHe on the Na+-induced depolarizing current. Mouse sperm were incubated for 10 min in Na+-free WH medium buffered at different pH (6.8–7.6), and the Na+-induced depolarization was recorded (Fig. 2, D and E). The observation that the Na+ depolarizing current is significantly activated at low pHe (6.8) is consistent with ENaC being present in sperm. To confirm that the depolarization induced by Na+ is due to Na+ influx, sperm were loaded with Sodium Green, a Na+-specific fluorescent dye. The addition of Na+ resulted in an increase in the concentration of intracellular Na+ ([Na+]i) in the sperm population (Fig. 3, A and B) that could be inhibited by 1 μm EIPA (Fig. 3C). More so, the addition of Li+ did not increase cell fluorescence (Fig. 3D), even though, as shown below, it causes a larger Em depolarization than Na+ (Fig. 4). Furthermore, [Na+]i was examined in individual Sodium Green-loaded sperm suspended in Na+-free WH medium before (Fig. 3E) and after (Fig. 3E′) adding 50 mm NaCl. The relative fluorescence (Rf) was quantified independently in the heads and flagella and expressed as F/F0 (F = fluorescence intensity after Na+ addition, F0 = basal fluorescence intensity). The addition of NaCl increased th
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