Mol. Hum. Reprod. Advance Access originally published online on July 22, 2006
Molecular Human Reproduction 2006 12(9):565-576; doi:10.1093/molehr/gal062
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The Na,K-ATPase 
4 isoform from humans has distinct enzymatic properties and is important for sperm motility
Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS, USA
1 To whom correspondence should be addressed at: Department of Molecular and Integrative Physiology, 3901 Rainbow Boulevard, Kansas City, KS 66160, USA. E-mail: gblanco{at}kumc.edu
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Abstract |
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In the rat, the Na,K-ATPase
4 isoform exhibits unique enzymatic characteristics and is important for sperm motility. In this work, we studied expression, localization and function of 4 in human spermatozoa. We show two catalytically active Na,K-ATPase polypeptides with different ouabain affinity and identified expression of 1, 4, ß1 and ß3 isoforms in the gametes. In addition, human sperm presented two Na,K-ATPases composed of 4, 4ß1 and 4ß3. Kinetic analysis of these isozymes produced in insect cells showed that, compared with human 1ß1, 4ß1 and 4ß3 exhibit higher Na+ and lower K+ affinity and higher sensitivity to ouabain. These particular enzymatic properties suggested a role for 4 in sperm function. Using computer-assisted sperm analysis (CASA), we found that ouabain inhibition of 4 significantly decreased percentage sperm motility. In contrast, ouabain did not affect linearity of forward progression, amplitude of lateral head displacement, beat cross frequency and sperm straight-line, curvilinear or average path velocities. This suggests a primary role of 4 in flagellar motility. Accordingly, we found 4 in the sperm tail, predominating in the mid-piece of the flagellum. Therefore, similar to the rat ortholog, human Na,K-ATPase 4 isoform has a distinct activity that is essential for sperm function.
Key words:
4 isoform/Na,K-ATPase/ouabain/sperm motility
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Introduction |
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The Na,K-ATPase is an enzyme of the plasma membrane of most animal cells that uses the free energy from the hydrolysis of ATP to mediate the exchange of cytoplasmic Na+ for extracellular K+ in a 3:2 ratio (Kaplan, 2002
). The Na,K-ATPase plays a key role in numerous cell processes that depend directly or indirectly on the transmembrane gradients of Na+ and K+. In this manner, the enzyme is essential in maintaining cell osmotic balance, volume and pH; in maintaining the cell resting membrane potential; and in providing the chemical energy for the secondary Na+-coupled transport of other ions, solutes and water across the cell membrane (Skou and Esmann, 1992).
The Na,K-ATPase consists of multiple isozymes, each composed of the association of distinct molecular forms of two main polypeptides, the and ß subunits (Kaplan, 2002). Four structural variants of the (1, 2, 3 and 4) and three different ß (ß1, ß2 and ß3) isoforms have been discovered in mammalian tissues (Mobasheri et al., 2000; Blanco, 2005). The polypeptides constitute the catalytic subunit of the Na,K-ATPase, directly participating in the ion translocation and hydrolytic activity of the enzyme. All isoforms are 10-membrane-spanning proteins that contain the binding sites for Na+, K+ and ATP (Jorgensen et al., 2003). In addition, the isoforms bind cardiotonic steroids, such as ouabain, which inhibits the catalytic and transport activity of the enzyme (Blaustein et al., 1998). The ß subunits are single-membrane-spanning glycoproteins that are required for the proper folding and trafficking of the Na,K-ATPase from intracellular stores to the plasma membrane (Geering, 2001).
Each Na,K-ATPase isozyme is characterized by a particular pattern of expression that is regulated in a cell-type-specific, developmentally controlled manner (Orlowski and Lingrel, 1988). While 1 in association with ß1 is found in most cells, the other and ß polypeptides are more limited in their expression (Blanco, 2005). In addition, different Na,K-ATPase isozymes exhibit unique kinetic properties that primarily depend on the subunit composition of the enzyme (Mobasheri et al., 2000; Blanco, 2005). The characteristic expression and activity of the Na,K-ATPase isoforms suggest that the molecular heterogeneity of the enzyme is of physiological relevance. Information on the biological role of the Na,K-ATPase isoforms is now coming to light through studies in transgenic animals (Dostanic et al., 2005; Looney et al., 2005; Moseley et al., 2005; Zhang et al., 2005), and through the identification of mutations of the transporter in humans (Vanmolkot et al., 2003; Wessman et al., 2004).
A distinctive Na,K-ATPase isoform expression profile has been found in the mammalian testis. The male gonad shows the selective expression of the 4 polypeptide, which is abundant in the male germ cells (Shamraj and Lingrel, 1994; Underhill et al., 1999; Blanco et al., 2000). Besides 4, the 1 isoform, and two ß subunits, ß1 and ß3, are also present in testis (Shamraj and Lingrel, 1994; Arystarkhova and Sweadner, 1997; Blanco et al., 2000). We have shown that in the rat, 4 is able to associate with the ß1 and ß3 subunits to produce two catalytically competent Na,K-ATPases, 4ß1 and 4ß3 (Blanco et al., 1999). These Na,K-ATPase isozymes are functionally different from the other Na,K-ATPases. They have a high affinity for Na+, a low affinity for K+, an intermediate affinity for ATP, and a high sensitivity to ouabain (Blanco et al., 1999; Woo et al., 1999). The unique enzymatic properties of 4 suggest that the isoform is not redundant, but rather plays a specific role in sustaining the ion gradients, membrane potential and excitability of male germ cells. In support of this, ouabain inhibition of 4 has been shown to impair rat sperm motility (Woo et al., 2000).
Most of the information regarding 4 derives from studies in the rat, and at present, little is known about the isoform from other species. Recently, the nucleotide sequence of the Na,K-ATPase 4 from humans has been determined (Keryanov and Gardner, 2002) and the encoded polypeptide has been shown to be expressed in human testis (Hlivko et al., 2006). Interestingly, 4 is absent from sections of immature human testes and its expression is coincident with the appearance of spermatozoa in the gonad (Hlivko et al., 2006). This suggests that 4 is playing a role in the physiology of human sperm.
To better understand the function of the Na,K-ATPase 4 isoform from humans, we have investigated the enzymatic properties, ß subunit association, cell distribution and the role of the human 4 isoform in sperm motility. Our results show that, similar to the rat isoform, the human 4 polypeptide has properties different from those of 1, and that its activity is essential for sperm function. These results support the physiological relevance of 4 for human male fertility. A preliminary report of some of these findings has been previously presented in abstract form (Sanchez et al., 2005).
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Human sperm samples
Semen samples were collected from healthy adult donors showing a normal spermiogram. Samples were collected by masturbation according to the protocols approved by the Institutional Review Board at University of Kansas Medical Center. Samples were allowed to liquefy for 30 min at 37°C and were diluted 1:4 with Ham’s F10 medium, pH 7.4. Cells were separated by centrifugation for 7 min at 330 x g. The pellet was resuspended in 3 ml of Ham’s F10 plus 3 mg/ml bovine serum albumin (F10-BSA) and recentrifuged for 3 min at 330 x g. After discarding the supernatant and adding 1.5 ml of F10-BSA, samples were incubated at 37°C in 5% CO2 in air in a 45° rack to allow the spermatozoa to swim up. After 45 min of incubation, 1 ml aliquots were removed from the supernatant to obtain the highly motile swim-up population of cells. For Na,K-ATPase activity assays, the spermatozoa were resuspended in 0.32 M sucrose, 30 mM Tris–HCl (pH 7.4), 1 mM EGTA and homogenized using a glass–glass homogenizer. For measurement of sperm motility, the cells were used after treating them in the absence and presence of different ouabain concentrations.
Human 4, ß1 and ß3 cDNAs
The cDNA corresponding to the human Na,K-ATPase 4 isoform was obtained from a human testes cDNA library purchased from Clontech (Palo Alto, CA, USA) using polymerase chain reaction (PCR). PCR was performed using high fidelity polymerase, Klentag LA (Sigma Chemical, St. Louis, MO, USA), and oligonucleotides that were designed based on the 4 isoform sequence published by Keryanov and Gardner (2002). This allowed us to obtain two overlapping fragments that were combined by overlap extension PCR to obtain the full-length isoform. Integrity of the clone was confirmed by oligonucleotide sequencing. The obtained sequence was in agreement with that reported previously (Keryanov and Gardner, 2002; Hlivko et al., 2006). The cDNAs for the human Na,K-ATPase 1 and 3 isoforms (I.M.A.G.E. ID number 3506311 and 6574355 respectively) were obtained from the Mammalian Genome Collection of ATCC (Manassas, VA, USA). The 4, ß1 and ß3 isoforms were subcloned into the pBluebac TOPO expression vector (Invitrogen, Carlsbad, CA, USA). Baculovirus preparation and selection was performed according to the procedures recommended by the supplier (Invitrogen).
Insect cells and viral infections
Sf-9 cells were grown in Grace’s medium (JRH Biosciences, Lenexa, KS, USA) with 3.3 g/l lactalbumin hydrolysate, 3.3 g/l yeastolate, and supplemented with 10% (v/v) fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin and 0.25 µg/ml Fungizone. Infections were performed in 150 mm petri dishes as previously described (Blanco et al., 1999). After 72 h at 27°C, cells were scraped from the culture plates, centrifuged at 1500 x g for 10 min and washed twice in 10 mM imidazole hydrochloride (pH 7.5) and 1 mM EGTA. Cells were then suspended in the same solution and used for assays. Before determination of Na,K-ATPase activity, the cells were permeabilized with the ionophore alamethicin as described (Blanco et al., 1999).
Reverse transcriptase–polymerase chain reaction analysis
Total RNA from each cell type was isolated using TRIzol reagent (Invitrogen). Complementary DNA was generated by reverse transcription using the SuperScriptTM First-Strand Synthesis System (Invitrogen) and oligo (dT) primers as described (Wagoner et al., 2005). The resulting first-strand cDNA was amplified using Na,K-ATPase isoform-specific primers, and under PCR conditions that assured no cross-reactivity among the Na,K-ATPase isoforms. The sequences of the primers used, their annealing properties and the size of the amplified cDNAs are described in Table I. One microlitre of DNA was added to 50 µl of a PCR mixture containing 100 mM Tris–HCl (pH 8.3), 500 mM KCl, 15 mM MgCl2, 200 µM dNTPs, 500 nmoles of each primer, and 2.5 units of Taq DNA polymerase. The conditions for PCR included a first cycle of 30 s at 94°C, followed by 30 cycles of: (i) denaturation for 30 s at 94°C, (ii) an annealing step that varied depending on the primers used (Table I) and (iii) an elongation step for 50 s at 72°C. Finally, an additional elongation step of 5 min was performed at 72°C. The amplified DNA fragments were identified by electrophoresis in a 1% agarose gel stained with ethidium bromide.
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Antibodies
For the human Na,K-ATPase 4 isoform, two different antibodies were used. One was raised in rabbits at Covance Immunology Services, Denver, CO, USA. The other was raised in chickens at Aves Labs, Tigard, OR, USA. The rabbit antiserum was directed against a 24-amino-acid synthetic peptide (KLTLEELSTKYSVDLTKGHSHQRA), and the chicken antibody against an 18-amino-acid synthetic peptide (KMVKREKQKRNMEELKKE), corresponding to amino acids 53–76 and 29–46, respectively, of the specific N-terminal portion of the human 4 polypeptide. After confirming the purity of the peptides by standard reverse-phase high-performance liquid chromatography (HPLC), they were conjugated to keyhole limpet haemocyanin and used to immunize the rabbits and hens respectively. The effectiveness of immunization was tested with enzyme-linked immunoabsorbent assay (ELISA) with the peptide absorbed on the solid phase. The IgY from the immune eggs was purified by passage over a peptide affinity column. The obtained antibodies specifically recognized the human 4 polypeptide, showing no cross reactivity to the 1, 2 and 3 isoforms (see Results).
For the human 1 isoform, the monoclonal 6F antibody (Mobasheri et al., 2001), obtained from The Developmental Studies Hybridoma Bank, University of Iowa, was used. For 2, the monoclonal MCB2 (Arystarkhova and Sweadner, 1996), kindly provided by Dr Kathleen Sweadner (Massachusetts General Hospital), was used. The 3 isoform was detected with monoclonal antibodies MA3–915 (Arystarkhova and Sweadner, 1996) and the ß1 subunit with M17–P5–F11 (Sun and Ball, 1992), both purchased from Affinity Bioreagents, Golden, CO, USA. For ß2, an anti–ß2 antiserum (Mobasheri et al., 2001), generously provided by Dr P. Martin–Vasallo, was used (Universidad de La Laguna, Tenerife, Spain). Finally, for ß3, a monoclonal antibody (Malik et al., 1996), from BD Biosciences (San Jose, CA, USA), was used.
Polyacrylamide gel electrophoresis and immunoblot analysis
Protein expression was analysed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE, 7.5% gel) and immunoblotting. After separation by SDS–PAGE, proteins were transferred onto nitrocellulose membranes (Nitrobind, Osmonics, Minnetonka, MN, USA) and immunobloted as described previously (Blanco et al., 1999). Primary antibodies specific for each of the human Na,K–ATPase isoforms were used to identify the corresponding polypeptides. The dilution used for each antibody was the following: anti–1 6F, 1:100; anti–2 MCB2, 1:250; anti–3 MA3–915, 1:500; rabbit anti–4 antiserum, 1:100; anti–ß1 M17–P5–F11, 1:250; anti–ß2, 1:200; and anti–ß3, 1:200. Horse radish peroxidase anti–mouse and anti–rabbit conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) added in a 1:5000 dilution and chemiluminescence were used for detection.
Immunoprecipitations
Human spermatozoa (
3 x 106 cells) were lysed with 1% 3–[(3–cholamidopropyl)dimethyl ammonio]–1–propanesulfonate (CHAPS) in 150 mM NaCl, 25 mM HEPES (pH 7.4). After incubation on ice for 30 min, the insoluble material was removed by centrifugation at 15 000 x g for 10 min, and samples were subjected to immunoprecipitation as previously described (Blanco et al., 1994). For this, either 30 µl (0.7 mg/ml) of the anti–human 4 antiserum generated in rabbit or 50 µl (1 mg/ml) of anti–ß1 or anti–ß3 antibodies were used. To pull down the Na,K–ATPase subunit–antibody complexes, 70 µl (1 mg/ml) of goat anti–mouse or goat anti–rabbit coated magnetic beads were used (BioMag; Qiagen, Stanford, CA, USA). After overnight incubation on a rocking table at 4°C, beads were isolated by holding the microcentrifuge tube to a magnet and aspirating the supernatant. The beads were washed three times in the lysis buffer. The precipitated protein was eluted by resuspending the beads in sample buffer (100 mM Tris–HCl, pH 6.8, 2% SDS, 33% glycerol, 100 mM DTT) and incubating for 5 min at 95°C. Eluted proteins were separated by SDS–PAGE (9% gel), transferred to nitrocellulose and immunobloted with the anti–ß1 and anti–ß3 antibodies when the anti–4 antiserum was used for the immunoprecipitation step—or with anti–4, when the anti–ß antibodies were used in the pull–down assays.
Immunocytochemistry and confocal microscopy
Immunocytochemistry was performed on Sf–9 cells expressing the Na,K-ATPase 4ß1 and 4ß3 isozymes, and on human spermatozoa. Sf–9 cells were plated in 24–well culture plates on 11 mm glass coverslips and infected with the corresponding baculoviruses. Forty–eight hours after infection, cells were treated with 100 µg/ml of cycloheximide, an inhibitor of protein synthesis, to allow detection of the expressed polypeptides at their final destination. For human spermatozoa, cells were plated on 11 mm glass coverslips in 24-well tissue culture plates and centrifuged (3000 x g for 3 min) to help the cells attach. Sf-9 cells and spermatozoa were fixed in 4% paraformaldehyde (buffered formalin phosphate, Fisher Scientific, Pittsburgh, PA, USA). Samples were then processed for immunocytochemistry as described (Sanchez and Blanco, 2004). Briefly, cells were permeabilized with 0.3% Triton X100 in 25 mM HEPES, pH 7.4, 150 mM NaCl and 1 mM EGTA (HBS). After blocking for 2 h at room temperature with 0.2% BSA and 2% normal goat serum in HBS, the primary antibodies against specific Na,K-ATPase isoforms were applied. The dilutions used were anti-1 6F, 1:5; chicken anti-4, 1:100; anti-ß1 M17-P5-F11, 1:30; and anti-ß3, 1:30. Following overnight incubation at 4°C, samples were washed 3 x 15 min each, and treated with the corresponding secondary antisera, conjugated to Alexa fluor 488 or Alexa fluor 594 (Molecular Probes, Eugene, OR, USA) in a 1:1000 dilution. After washing as mentioned before, samples were mounted on slides using SlowFade mounting solution (Molecular Probes), which contains 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI), to stain the cells nuclei. To stain the mitochondria, MitoFluor Red 589, a marker specific for mitochondria (Molecular Probes), was used. This was applied after the primary antibody in a 1:100 dilution. Fluorescent digital images were obtained using a Zeiss LSM510 confocal microscope. Images were acquired in Multitrack channel mode (sequential excitation/emission) with LSM510 (v 3.0) software and a Plan-Apochromat 63X/1.4 Oil DIC objective with a frame size of 1024 x 1024 pixels and a zoom factor of 3 (field size of 0.048 mm x 0.048 mm). Detector gain was set initially to cover the full range of all the samples and background was corrected by setting the amplifier gain in comparison to the relevant control slides, and all images were then collected under the same photomultiplier detector conditions and pinhole diameter. Control slides consisted of (i) mounted cells only, without the antibodies, to check for auto-fluorescence, (ii) single colour stained samples to check for bleed-through into all the other channels and (iii) secondary antibodies only to check for non-specific binding.
Biochemical assays
Protein assays were performed using the dye-binding assay based on the method of Bradford, from Bio-Rad (Hercules, CA, USA). Na,K-ATPase activity was assayed on cell homogenates through determination of the initial rate of release of 32Pi from 
[32P]-ATP as described (Blanco et al., 1995). The ATPase activity of 10 µg total protein samples for sperm cells or 30 µg for Sf-9 cells was measured in a final volume of 0.25 ml in medium containing 120 mM NaCl, 30 mM KCl, 3 mM MgCl2, 0.2 mM EGTA, 30 mM Tris–HCl (pH 7.4), 3 mM ATP with 0.2 µCi [32P]-ATP in the presence and absence of the indicated ouabain concentrations. For the cation activation curves, Na+ was varied between 0 and 120 mM, and K+ from 0 to 30 mM. Incubation was performed at 37°C for 30 min. Released 32Pi-Pi was converted to phosphomolybdate, extracted with isobutanol, and radioactivity of 170 µl of the organic phase was measured by liquid scintillation counting. The ATP hydrolysed never exceeded 15% of the total ATP present in the sample and hydrolysis was linear over the incubation time. Specific activity was determined as the difference in ATP hydrolysis in the absence and presence of 1 mM ouabain.
Data analysis
Curve fitting of the experimental data was performed using a Marquardt least-squares non-linear regression computing program (Sigma Plot, Jandel Scientific, San Rafael, CA, USA). Na+ and K+ activation curves were best fitted according to a cooperative model for ligand binding as previously described (Blanco et al., 1995). Dose–response relations for the inhibition of Na,K-ATPase by ouabain were best fitted assuming the presence of one (Sf-9 cells) or two (human spermatozoa) enzyme populations with different affinities for ouabain, applying the equations described previously (Blanco et al., 2000). The validity of using a two-component versus a single-component model for ouabain binding was statistically supported by applying the Snedecor’s F test (Blanco et al., 1995). Statistical significance of the differences between ouabain treated and untreated groups was analysed by Student’s t-test. Statistical significance was defined as P < 0.05.
Sperm motility assays
Approximately 1 x 106 cells were incubated in 100 µl of F10-BSA medium without and with 1 x 10–8 M or 1 x 10–3 M ouabain for 5, 10, 20, 30, 40, 50, 60, 90 and 120 min. Aliquots of 10 µl were placed into a cell chamber 32 µm in depth prepared as described previously (Bracho et al., 1997). The chamber was sealed with a plastic coverslip to ensure uniform well depth and distribution of sperm. Experiments were performed at room temperature as previously described (Robertson et al., 1988; Bracho et al., 1997). Samples were viewed through a Nikon Optiphot microscope with a 20x phase objective. Viewing areas on each slide were videotaped using a VK-M24 Hitachi solid-state video camera and a video recorder. Each sample was recorded for a total of 240 s and the field of view on the motility chamber was changed every 15 s. Different sperm motility parameters were analysed including percentage motility, straight line, curvilinear, average path velocity, linearity of forward progression, amplitude of lateral head displacement and beat cross frequency. This was performed using computer-assisted sperm analysis (CASA) and the CellTrack/S system (version 5.00, Motion Analysis Corporation, Santa Rosa, CA, USA) with the following set-up parameters: frame rate, 30 frames/s; duration of data capture, 23 frames; minimum motile speed, 8 microns/s; maximum burst speed, 200 microns/s; distance scale factor, 2.16 microns/pixel; minimum cell size, 4 pixels; maximum cell size, 40 pixels; and number of cells/well, 200. Beat cross frequency was obtained using the CellSoft system and the same setup parameters described above, as previously described (Robertson et al., 1988). Results were expressed as percentage of control without ouabain, and represented the mean ± SE of determinations performed on 16 fields per experiment.
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Na,K-ATPase ouabain sensitivity of human spermatozoa
In rodents, one of the most obvious functional differences among the Na,K-ATPase isozymes is their reactivity to cardiotonic steroids. This primarily depends on the
isoform composition of the enzyme, and while the 1 isoform is highly resistant to ouabain, 2, 3 and 4 are progressively more sensitive (Blanco, 2005). Differences in ouabain affinity have been used as a tool to distinguish the activity of Na,K-ATPase isoforms and to estimate their relative contribution in a sample. In humans, however, differences in the ouabain response among the 1, 2 and 3 isoforms are much more modest (Crambert et al., 2000; Wang et al., 2001), and, at present, the ouabain kinetics of the human 4 polypeptide have not been determined. To explore this, we performed dose–response analysis for the inhibition of Na,K-ATPase activity by ouabain in human spermatozoa. As shown in Figure 1, human sperm homogenates exhibited a heterogeneous inhibition profile to ouabain. The curve was best fit by using a two-component model that assumed the presence of two Na,K-ATPase populations with different affinities for ouabain. Approximately 56% of the total Na,K-ATPase exhibited a higher resistance to ouabain, with an inhibition constant (Ki) for ouabain coinciding with that reported for the 1 isoform (Ki = 4.6 ± 1.5 x 10–7). Interestingly, the remaining 44% of the enzyme showed an ouabain sensitivity that was 100-fold higher, with a Ki of 0.5 ± 0.3 x 10–9 M. This result suggests that two isoforms of the Na,K-ATPase with different affinities for ouabain are expressed in human spermatozoa.
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Na,K-ATPase isoform expression in human spermatozoa
To ascertain the isoform composition underlying the functional heterogeneity of the Na,K-ATPase of spermatozoa, we determined the expression profile of the catalytic and ß subunits of the enzyme. As a first approach, we investigated this at the RNA level by RT-PCR. As spermatozoa are terminally differentiated cells that are transcriptionally inactive, we used total RNA from human testes (Ambion, Austin, TX, USA). Figure 2A and B shows the RT-PCR results for the and ß isoforms, respectively. As shown, 1, 4, ß1 and ß3 were found to be present in the gonad. The specificity of the Na,K-ATPase primers used is demonstrated by the lack of cross reactivity with cDNAs of isoforms other than that containing the corresponding complementary sequence. In addition, PCR reactions performed on the samples in the absence of reverse transcription yielded no Na,K-ATPase isoform products, indicating the lack of genomic DNA contamination in the RNA isolation step (data not shown). Besides 1, 4, ß1 and ß3, very low levels of the 3 isoform could also be detected in testis. The Na,K-ATPase 3 isoform has been shown to be expressed in neuronal cells (Levenson, 1994). Autonomic innervation is present in the testis (Prince, 1996); therefore, the appearance of some 3 in our RT-PCR analysis is likely to be related to the existence of nerve fibres and terminals in the gonad. Although these results do not indicate the and ß subunit composition present in spermatozoa, it provided some preliminary information for the pattern of Na,K-ATPase isoform expression in the human male gametes, and suggested the absence of 2 and ß2 in the cells.
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To directly determine the expression of Na,K-ATPase isoforms in human spermatozoa, we explored for the presence of the different and ß polypeptides in the cells. For this, total proteins from cell homogenates were separated by SDS-PAGE, transferred to nitrocellulose and immunoblotted using Na,K-ATPase isoform-specific antibodies. Figure 2C shows that only 1 and 4 were present in the gametes, with 2 and 3 being absent. For the ß subunits, ß1 and ß3, but not ß2, were found. As expected, the isoforms migrated as single bands of approximately 112 KD, while the ß1 and ß3 subunits were resolved in various bands ranging from 30 to 55 KD. This complex electrophoretic pattern, typical of the ß subunits, corresponds to different degrees of glycosylation of the polypeptides in the cells (Geering, 2001). As a control, the various isoforms and the ß1 and ß3 subunits produced in Sf-9 insect cells were used. For ß2, human brain protein, purchased from BD Biosciences, was used (control lanes in Figure 2C). While the baculovirus produced Na,K-ATPase isoforms co-migrated with the corresponding isoforms from spermatozoa, the ß subunits synthesized in insect cells showed a higher electrophoretic mobility than their native counterparts. This is due to the limited protein glycosylation characteristic of the invertebrate cells (Blanco et al., 1995). The specificity of the primary antibodies employed, with the exception of the anti-4 antibody, has been shown previously (Sun and Ball, 1992; Arystarkhova and Sweadner, 1996; Mobasheri et al., 2001). The antibodies against the human 4 isoform we generated only recognized the expected polypeptide and did not crossreact with any of the other Na,K-ATPase isoforms. The results for the 4 antibody raised in rabbits are shown in Figure 2D. Similar results were obtained with the anti-4 antibody generated in chicken (data not shown).
Our results indicate that human spermatozoa exhibit a Na,K-ATPase isoform profile consisting of the 1, 4, ß1 and ß3 isoforms.
ß -subunit association of 4 in human spermatozoa
The presence of the ß1 and ß3 subunits of the Na,K-ATPase in human spermatozoa raised the possibility for the existence of two different isozymes of the 4 isoform in the cells. To explore the ability of the 4 subunit to associate with different ß polypeptides, immunoprecipitation assays were performed. As shown in Figure 3A, when anti-ß1 and anti-ß3 were used as the immunoprecipitating antibodies, 4 was identified in the immunoprecipitates indicating that the isoform is able to assemble with both the ß1 and ß3 subunits. In contrast, 4 was not found in samples in which the anti-ß1 or anti-ß3 antibodies were omitted. As a control, the 4 isoform from human sperm not subjected to immunoprecipitation is shown (control lane in Figure 3A). Association between 4 and the ß1 and ß3 subunits was also demonstrated by using as immunoprecipitating antibody the anti-4 antiserum (Figure 3B and C). As shown, ß1 and ß3 were detected in the immunoprecipitates only in the samples in which the anti-4 antiserum was present. Interestingly, the precipitated ß polypeptides were primarily detected as single bands that comigrated with the upper bands of ß1 and ß3 from human sperm samples not subjected to immunoprecipitation. This suggests that the main species of the ß subunits that associate with 4 are those corresponding to the fully glycosylated forms of the polypeptides, and agrees with results showing that the ß subunit forming part of the Na,K-ATPase at the plasma membrane is completely glycosylated (Geering, 2001). Altogether, these experiments suggest that human spermatozoa express two different Na,K-ATPase isozymes composed of the 4 polypeptide, 4ß1 and 4ß3.
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Immunolocalization of Na,K-ATPase isoforms in human spermatozoa
Another important goal in the analysis of Na,K-ATPase of human male gametes is the localization of the isoforms in the cells. We determined this by immunofluorescence using the 1, 4, ß1 and ß3 antibodies and Alexa fluor 488-conjugated secondary antibodies. The confocal microscopy images obtained for the and ß isoforms are shown in Figure 4A and B, respectively. Samples treated with the corresponding carrier media, instead of the primary antibodies, were used as a control. In all cases, DAPI was included to stain the cells nuclei. As shown, the anti-1, anti-ß1 and anti-ß3 antibodies primarily labelled the sperm flagellum, exhibiting little reactivity in the sperm head. Label for the 1, ß1 and ß3 isoforms showed an even distribution along the sperm tail, covering most of the sperm flagellum. The 4 isoform was also localized at the sperm flagellum and was virtually undetectable in the sperm head. Interestingly, signal for 4 was not evenly distributed, but was stronger in the portion of the flagellum closest to the sperm head, suggesting higher levels of 4 in that area (Figure 4A). Co-staining of the spermatozoa with MitoFluor Red 589, a marker specific for mitochondria, showed a spatial correspondence of the segment of the flagellum where the mitochondria are present and the region where 4 expression is the highest. The individual labels for 4, the mitochondria and the merge of both images are shown in Figure 5. These results suggest that although 4 expression is not limited to the mid-piece of the flagellum, it predominates in that region. The differences in localization of the 1 and 4 catalytic subunits of the Na,K-ATPase in human spermatozoa are reminiscent of those found for the corresponding isoforms of the rat gametes, and suggests that 1 and 4 may play different roles in the cells.
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Expression of the human Na,K-ATPase 4ß1 and 4ß3 isozymes in insect cells
After establishing the expression, cell localization, and ß subunit pairing of the human 4 isoform, our next goal was to study the functional properties of the 4ß1 and 4ß3 isozymes. However, the presence of more than one isoform of the Na,K-ATPase in human spermatozoa makes it difficult to individually analyse their enzymatic properties. Therefore, to determine the kinetic characteristics of the Na,K-ATPase isozymes composed of 4, we used the baculovirus expression system. We have successfully used this system in the past for the study of Na,K-ATPase isozymes, because it provides catalytically competent molecules of the enzyme in an environment almost free of endogenous Na,K-ATPase activity (Blanco, 2005). Insect cells were coinfected with viruses containing the cDNA coding for the 4 isoform and either the ß1 or the ß3 subunits. In addition, the other isoform present in human sperm, 1 was coexpressed in the cells with ß1. To determine expression of the corresponding virally induced polypeptides, 72 h after infection, cells were harvested and proteins were subjected to SDS–PAGE and immunoblotting. As shown in Figure 6A, antibodies specific to the Na,K-ATPase and ß polypeptides detected high amounts of the corresponding proteins in the infected cells.
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To determine the cellular distribution of the human 4ß1 and 4ß3 isozymes, infected cells were analysed by immunocytochemistry and confocal microscopy. For this, cells coinfected with the 4 and the ß subunits were grown for 48 h, treated with cycloheximide and fixed. Cells were probed with the anti- and ß antisera. The antibody was identified using Alexa fluor 488-conjugated goat anti-rabbit, while the ß antibodies were detected with Alexa fluor 594-conjugated goat anti-mouse secondary. As shown in Figure 6B, the antibodies only recognized the Na,K-ATPase polypeptides in the baculovirus-infected cells. The left panels show the expression of the 4 polypeptide, while the right panels show expression of the ß1 and ß3 subunits. As has been previously described for other Na,K-ATPases, the human isozymes containing 4 are localized to the plasma membrane of the cells. Therefore, the insect cells are able to synthesize and deliver the human 4ß1 and 4ß3 Na,K-ATPases to the surface of the cells.
Enzymatic properties of the human 4ß1 and 4ß3 isozymes expressed in insect cells
Expression of the human Na,K-ATPase 1ß1, 4ß1 and 4ß3 isozymes in insect cells also resulted in catalytically competent molecules. This allowed us to study the enzymatic properties of the Na,K-ATPases. To determine the affinity of the 4 containing Na,K-ATPases to Na+, K+ and ouabain, dose–response curves to each ligand were performed. These were compared with the corresponding kinetic behaviour of the human 1ß1 isozyme. The Na+ dependency of Na,K-ATPase activity was determined at varying concentrations of Na+ and constant saturating K+ (30 mM). The obtained activation curves are shown in Figure 7A. The values for the apparent affinities for Na+ (K0.5 values) are presented in Figure 7D. As shown, the 4ß1 and 4ß3 isozymes exhibit half activation constants for Na+ that are significantly lower than that of the 1ß1 enzyme.
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To determine the requirement for K+, Na,K-ATPase activity was measured at varying concentrations of K+, with Na+ fixed at 120 mM. The obtained dose–response curves and the calculated K0.5 values for K+ are presented in Figure 7B and E, respectively. As shown, both 4ß1 and 4ß3 isozymes have similar reactivity to K+, but have significantly less affinity to the cation than 1ß1.
We also explored the kinetics of the 4-containing isozymes to ouabain, determining the inhibition profile of Na,K-ATPase activity to different concentrations of the cardiotonic steroid under saturating concentrations of Na+, K+ and ATP. As shown in Figure 7C, both preparations were best fitted, as expected, by an equation that considered one population of Na,K-ATPase, indicating the presence of a single ouabain-interacting enzyme species. Both 4ß1 and 4ß3 isozymes exhibited a high affinity for ouabain, with Kis of 1.0 ± 0.3 x 10–8 M and 4.9 ± 1.7 x 10–9 M, respectively. These values were significantly lower than that of the 1ß1 enzyme, which showed a Ki for ouabain of 2.0 ± 0.6 x 10–7 M (Figure 7F). Altogether these results demonstrate that human Na,K-ATPases containing the 4 catalytic subunit respond to ligands with kinetics that are different from those of the 1ß1 isozyme. In addition, the differences in ouabain affinity of the baculovirus-directed Na,K-ATPases strongly support that the bimodal response to the steroid observed in human spermatozoa results form the activity of 1 and 4.
Role of the Na,K-ATPase 4 isoform in human sperm motility
The higher affinity to ouabain we encounter for Na,K-ATPases containing 4 polypeptides provides the opportunity to preferentially inhibit this isoform and determine whether 4 plays a role in the function of human spermatozoa. Based on the experiments in the native cells (Figure 1), it is apparent that an ouabain concentration of 1 x 10–8 M should be sufficient to completely inhibit the activity of 4, without having significant effect on the 1 polypeptide. An ouabain concentration of 1 x 10–3 M, however, will cause inactivation of both the 1 and 4 isoforms. We used these ouabain concentrations to explore if the function of 4 in particular or also that of 1 is important for the motility of human spermatozoa. As shown in Figure 8 in the control medium without ouabain, sperm motility parameters remained constant during the 120-min incubation period. In contrast, ouabain inhibition of the 4 isoform alone was sufficient to impair the percent of motile spermatozoa in a time-dependent manner (Figure 8A). Additional ouabain inhibition of the 1 isoform with 1 x 10–3 M ouabain did not result in further changes of sperm movement. This suggests that the 4 isoform is important for sperm movement, and that it is this isoform, and not 1, that is primarily involved in motility of the gametes. Interestingly, ouabain did not have a significant effect on the straight line, curvilinear, average path velocity, linearity, amplitude of lateral head displacement or beat cross frequency of sperm (Figure 8B–G). These results suggest that the Na,K-ATPase, and in particular the 4 isoform, plays an important role in the motility of human sperm, but is not involved in regulating other mobility parameters in the cells.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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To understand the catalytic and functional properties of the Na,K-ATPase
4 isoform from humans, we studied the expression, cell localization and activity of the 4 isoform from humans in spermatozoa and after expression in insect cells using the baculovirus expression system. Using antibodies specific for the human 4, we identified the polypeptide in human spermatozoa, confirming previous observations (Hlivko et al., 2006). Expression of 4 is not exclusive to the gametes, as the cells also express the 1, ß1 and ß3 subunits. This shows that the expression pattern of Na,K-ATPase isoforms in human spermatozoa is similar to what we have previously reported for the rat (Wagoner et al., 2005).
Immunoprecipitation analysis shows that 4 assembles with both ß polypeptides of spermatozoa. Expression of the and ß isoforms of human sperm is mainly localized to the cell flagellum. The co-expression of the ß subunits and 4 in the sperm flagellum supports the conclusion that 4 associates with ß1 and ß3. The ability of 4 to associate with different ß subunits is not surprising, because pairing of ß isoforms in different combinations has been shown to be a promiscuous event (Lemas et al., 1994; Blanco, 2005). In addition, the presence of 1 throughout the sperm tail raises the possibility that this isoform also constitutes two other Na,K-ATPase isozymes (1ß1 and 1ß3). At present, the physiological relevance of the expression of isozymes composed of different ß subunits in spermatozoa is unknown.
Importantly, we found that the 4 isoform is catalytically active in human spermatozoa, as suggested by the heterogeneous dose–response of Na,K-ATPase activity to ouabain inhibition. Expression in insect cells confirmed functional competency of the human 4 isoform, and showed that the polypeptide is active in the presence of both ß1 and ß3 subunits. To our knowledge, this is the first demonstration for an Na+- and K+-dependent hydrolysis of ATP by the human 4 isoform. Moreover, the baculovirus-directed expression of human 4 isoform resulted in a catalytically functional enzyme with unique kinetic properties. Thus, human 4 has a high affinity for ouabain, and showed a Ki for the steroid that was lower than that of the 1 isoform. The different ouabain affinities of the human 1 and 4 isoforms agree with the bimodal profile of Na,K-ATPase inhibition we observed in spermatozoa. Calculation of the Kis (inhibition constants) for ouabain for each isoform showed slight differences between the insect and the native cells. This may reflect dissimilarities in the lipid environment of the plasma membrane of each cell type, as has been suggested previously (Crambert et al., 2000). In both the spermatozoa and the Sf-9 cells, however, a higher sensitivity of 4 to ouabain compared with 1 was apparent. The elevated reactivity of human 4 to ouabain agrees with previous results (Hlivko et al., 2006). However, in that study, 4 was reported to have a lower sensitivity to the steroid (Ki of 1 x 10–7 M) than the one we found (Ki of 1 x 10–9 M), and was indistinguishable from the 1 isoform. These discrepancies are likely due to differences in the type of cells used for in vitro expression of Na,K-ATPase isoforms, or in the methods used to determine ouabain-Na,K–ATPase interaction. In the work by Hlivko et al. (2006), ouabain affinity of human 4 was assessed indirectly, through the ability of the steroid to cause death of HeLa cells expressing the isoform. On the other hand, our determination was based on direct kinetic determinations of inhibition of Na,K-ATPase activity by ouabain.
Previous work has shown slight differences among the ouabain inhibition constants of the Na,K-ATPase 1, 2 and 3 isoforms from humans (Crambert et al., 2000; Muller-Ehmsen et al., 2001; Wang et al., 2001). The ouabain kinetics we found for 4 suggest that this isoform is the catalytic subunit of the human Na,K-ATPase with the highest sensitivity to the steroid. The differences in affinity of 1 and 4 in sperm cells raise the question regarding the functional relevance of this specific property. Differences in reactivity to cardiotonic steroids have been shown to be physiologically important (Blaustein et al., 1998). Ouabain exists as an endogenous hormone that is released by the adrenal glands of several mammals, including man (Blaustein, 1996; Schoner and Scheiner-Bobis, 2005). Endogenous ouabain-like compounds have also been detected in seminal fluid of humans (Vadazs et al., 1983). Therefore, ouabain could act as a regulator of the ion homeostasis of spermatozoa by selectively binding to the highly ouabain sensitive 4 isoform.
Ouabain has been shown to affect motility of human spermatozoa (Kocak-Toker et al., 2002). This suggested an important role for the Na,K-ATPase in maintaining the electrochemical ion gradient and membrane potential of the male gametes. Nevertheless, because information concerning Na,K-ATPase isoform expression and sensitivity to ouabain was not available for human spermatozoa, these authors analysed the effect of ouabain on human sperm motility using concentrations of ouabain that were relatively high. In this manner, those experiments were not able to distinguish the role of different isoforms of the enzyme in sperm motility. We show that selective inactivation of 4 with ouabain was sufficient to decrease the motility of human spermatozoa. Moreover, the use of ouabain at concentrations that maximally inhibited the 1 and 4 isoforms did not further affect sperm movement. This shows that the ion gradients generated by the 4 isoform are critical for the function of the gametes. Although ouabain inhibition of 4 impaired sperm motility, it did not affect other motility parameters in the motile cells. This suggests that 4 activity is important for triggering flagellar movement and general motility of the cells, but it does not participate in modulating linearity, amplitude of lateral head displacement, beat cross frequency and speed of sperm. The role of 4 in flagellar motility is consistent with the localization of the isoform in the proximal region of the flagellum. The importance of the 4 isoform in sperm function is evidenced by the fact that almost half of the hydrolysis of ATP catalysed by the Na,K-ATPase of the human male gamete is dependent on the activity of 4.
The human 4 isoform also exhibited unique kinetics to the transported ions. Thus, 4 has a higher apparent affinity for Na+ and a lower affinity for K+ than 1. These functional properties are shared with those of the Na,K-ATPase 4 isoform from rats and their conservation between species suggest their importance. The low affinity of 4 for extracellular K+ (4 < 1) may correlate with the high K+ environment the male germ cells face before being released. Thus, the high K+ concentration of the testis tubules and epididymus (Muffly et al., 1985) will not affect the activity of the 1 isoform, but will be able to regulate the function of 4, influencing the electrochemical balance of the cells. Once the spermatozoa are released into the female tract, they face drastic changes in the ion concentrations. Importantly, extracellular Na+ becomes, high showing concentrations ranging between 114 and 156 mM depending on the specific region of the female tract considered. (Borland et al., 1977). The influx of Na+ into the cells will then preferentially stimulate 4, which has a higher affinity for the cation (4 > 1). The resulting increase in 4 activity will then be essential to provide the ion gradients necessary to sustain membrane excitability according to the demands of sperm motility. In this manner, the 4 isoform may represent an important modulator of the basal ion homeostasis maintained by 1, thus providing the male gametes with the Na+ and K+ electrochemical gradients necessary for the specific requirements of the cells.
Consistent with the role of human 4 in sperm motility, we found the isoform primarily localized to the sperm flagellum. Distribution of 4 slightly differs from that of 1. While the 1 isoform is evenly present along the plasma membrane of the sperm flagellum, 4 is more abundant in the proximal portion of the sperm flagellum, corresponding to the mid-piece of the sperm tail. In the rat, 4 has been found to specifically localize to the middle piece of the sperm flagellum (Woo et al., 2000; Wagoner et al., 2005). In this portion of the flagellum, the function of 4 appears to be linked to that of the Na+/H+ exchanger (NHE), with the Na+ gradient generated by the Na,K-ATPase providing the energy that drives the secondary movement of protons, released from the mitochondria, out of the cell (Woo et al., 2002). This mechanism is supported by the finding of a co-localization of the Na,K-ATPase and NHE transporters in the mid-piece of the flagellum, and by the ability of the ionophores nigericin and monensin that allow leakage of H+ out of the cells, to reinitiate the motility in spermatozoa that were treated with ouabain (Woo et al., 2002). Although not limited to the mid-piece, our observation indicating a high expression of 4 in that area of the flagellum is in agreement with the notion that activation of the isoform may be important to prevent the rise of H+ in the cytoplasm of the actively moving spermatozoa (Woo et al., 2002).
In conclusion, in this report we have characterized the Na,K-ATPase isoform expression in human spermatozoa, and show for the first time that the Na,K-ATPase human 4 isoform has distinct functional properties and plays a primary role in sperm flagellar motility. Future studies on the mechanisms of action of 4 will help elucidate the role of the isoform in male gamete fertility. In addition, the relevance of 4 in sperm motility provides the impetus for the search of specific 4 inhibitors that could be used as male contraceptives.
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Acknowledgements |
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This work was supported by National Institutes of Health grant HD043044 and KUMC Center of Excellence grant. We thank Kathleen Sweadner and Pablo Martin-Vasallo for providing the anti-
2 and anti-ß2 Na,K-ATPase antibodies. We are grateful for the help of Robert W. Mercer for his pertinent comments on the manuscript, and to Elizabeth Petrosky for the analysis of the immunocytochemical images. Confocal images were aquired at KUMC core facility (http://www.kumc.edu/cic), supported by NIH Shared Resource Grant (NCRR RR14637-01) and the Kansas Biomedical Research Infrastructure network. |
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Submitted on May 4, 2006; resubmitted on May 30, 2006; accepted on June 8, 2006.
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