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Na⁺/K⁺ATPase as a Signaling Molecule During Bovine Sperm Capacitation¹

Department of Animal and Poultry Science, University of Guelph, Ontario, Canada N1G 2W1

ABSTRACT

A heteromeric integral membrane protein, Na⁺/K⁺ATPase is composed of two polypeptides, alpha and beta, and is active in many cell types, including testis and spermatozoa. It is a well-known ion transporter, but binding of ouabain, a specific inhibitor of Na⁺/K⁺ATPase, to Na⁺/K⁺ATPase in somatic cells initiates responses that are similar to signaling events associated with bovine sperm capacitation. The objectives of the present study were to demonstrate the presence of Na⁺/K⁺ATPase in bovine sperm and to investigate its role in the regulation of bovine sperm capacitation. The presence of Na⁺/K⁺ATPase in sperm from mature Holstein bulls was demonstrated by immunoblotting and immunocytochemistry using a monoclonal antibody developed in mouse against the beta 1 polypeptide of Na⁺/K⁺ATPase. Binding of ouabain to Na⁺/K⁺ATPase inhibited motility (decreased progressive motility, average path velocity, and curvilinear velocity) and induced tyrosine phosphorylation and capacitation but did not increase intracellular calcium levels in spermatozoa. Furthermore, binding of ouabain to Na⁺/K⁺ATPase induced depolarization of sperm plasma membrane. Therefore, binding of ouabain to Na⁺/K⁺ATPase induced sperm capacitation through depolarization of sperm plasma membrane and signaling via the tyrosine phosphorylation pathway without an appreciable increase in intracellular calcium. To our knowledge, this is the first report concerning the signaling role of Na⁺/K⁺ATPase in mammalian sperm capacitation.

gamete biology, signal transduction, sperm, sperm capacitation, sperm maturation

INTRODUCTION

After ejaculation, mammalian spermatozoa reside in the female reproductive tract for a species-dependent period to attain fertilizing ability, during which time they undergo a series of structural and functional modifications collectively known as capacitation [1–4]. Although capacitation is a prerequisite for fertilization, the role of individual sperm proteins in the capacitation process is still under investigation. Understanding the specific role of individual sperm proteins in the capacitation and fertilization process is important for fertility investigations and for improving the success of assisted reproductive technologies.

A heteromeric integral membrane protein, Na⁺/K⁺ATPase belongs to the family of P-type ATPases; is composed of two polypeptides, alpha (110 kDa) and the glycosylated beta (55 kDa); and is active in many cell types, including testis and spermatozoa [5, 6]. The alpha polypeptide is the catalytic unit of the enzyme, containing the binding sites for cations, cardiac glycosides, and ATP. Four alpha polypeptide isoforms have been identified (alpha 1, alpha 2, alpha 3, and alpha 4), which are specific to different cell types and appear to regulate different functions [7]. The alpha 1 and testis-specific alpha 4 isoform of Na⁺/K⁺ATPase (ATP1AI and ATP1A4, respectively) have been isolated from the testis and are localized in spermatozoa [5, 8]. The inhibition of the activity of the ATP1A4 isoform has been shown to eliminate sperm motility [5]. The beta polypeptide is involved in the maturation of the enzyme, localization to the plasma membrane, and stabilization of the K⁺-occluded intermediate form of the protein [5]. Besides its role as an energy-transducing ion pump involved in the active extrusion of Na⁺ from the nerve cell [9], Na⁺/K⁺ATPase also acts as a sodium pump for the coupled active transports of Na⁺ and K⁺ across the plasma membrane of animal cells [10, 11]. This enzymatic activity results in the production of an electrochemical gradient that is required for many cellular processes, including maintenance of the resting membrane potential, regulation of the osmotic balance, and generation of the Na⁺ gradient that is necessary for the transport of many ions and other substrates across plasma membrane [12, 13]. The chemical gradient established by Na⁺/K⁺ATPase is important for the restoration of low intracellular Ca²⁺ concentrations after contraction of the cardiac muscle. Cardiac glycosides, such as ouabain and digoxin, can inhibit the activity of Na⁺/K⁺ATPase, which increases intracellular Ca²⁺ concentration and cardiac contractility [14]. Inhibition of Na⁺/K⁺ATPase disrupts cellular chemical gradients so that a drop in intracellular K⁺ levels occurs with a concomitant increase in Na⁺ levels [13]. Because the Na⁺/Ca²⁺-exchanger uses this gradient to pump Ca²⁺ from the cell, dampening this gradient acts as a rate-limiting step for the movement of Ca²⁺ out of the cell via the Na⁺/Ca²⁺-exchanger [7].

Recently, it was shown in cardiac myocytes and several other cell types that Na⁺/K⁺ATPase acts as a signal transducer in addition to its effect on ion transport [15]. Interaction of the endogenous cardiac glycoside ouabain [16, 17] with Na⁺/K⁺ATPase increases the production of reactive oxygen species and tyrosine phosphorylation in several proteins through a mechanism independent of changes in intracellular concentrations of Na⁺ and Ca²⁺ in cardiac myocytes [18] and epithelial cell lines [19]. The most proximal effects of ouabain binding of Na⁺/K⁺ATPase are Src activation, epidermal growth factor-receptor transactivation, and activation of protein kinase C and the extracellular signal-regulated kinase (ERK) family of the mitogen-activated protein kinase pathway (MAPK) [15]. Binding of ouabain to Na⁺/K⁺ATPase causes transcriptional regulation of several genes associated with cardiac hypertrophy through multiple signal transduction pathways, including MAPK [20].

In brief, studies of somatic cells suggest that interaction of ouabain with Na⁺/K⁺ATPase initiates a series of cellular responses, which are similar to the molecular events associated with mammalian sperm capacitation. For example, increase in intracellular Na⁺ and Ca²⁺ [3], tyrosine phosphorylation in a cohort of sperm proteins [21–27], generation of reactive oxygen species [4], and activation of the ERK family of the MAPK [28–30] are important events associated with sperm capacitation. Furthermore, Fraser et al. [31] observed that incubation of mouse sperm in the presence of ouabain increased the rate of capacitation in mouse spermatozoa, and we have demonstrated Na⁺/K⁺ATPase activity from the sperm head plasma membrane extracted from bovine spermatozoa [32]. Based on these observations, we hypothesized that Na⁺/K⁺ATPase signaling is involved in the regulation of bovine sperm capacitation.

The present study aimed to localize Na⁺/K⁺ATPase in bull spermatozoa and to investigate the role of this enzyme in the regulation of sperm motility, plasma membrane potential, tyrosine phosphorylation, and capacitation in bovine spermatozoa. To our knowledge, this is the first report concerning the role of Na⁺/K⁺ATPase in the regulation of bovine sperm capacitation.

MATERIALS AND METHODS

Materials

The following reagents were purchased from Sigma-Aldrich Canada Ltd.: Pisum sativum agglutinin conjugated to fluorescein isothiocyanate (FITC), 3-isobutyl-1-methylxanthine (IBMX), N⁶,2'-O-dibutyryl cAMP (dbcAMP), lysophosphatidylcholine (LPC), H89 (N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide), dithiothreitol, heparin sodium salt (from porcine intestinal mucosa, 180 USP U/mg), BSA (fraction V, catalog no. A-4503), paraformaldehyde (95%), luminol (97%, HPLC grade), p-coumaric acid, and 4-(6-carboxy-2-indolyl)-4'-methyl-2,2'-(ethylenedioxy) dianiline N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl) ester (Indo-1AM), dimethyl sulfoxide, pluronic acid, and ouabain. Propidium iodide (PI) and bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBac₄[3]) were purchased from Molecular Probes. Percoll was obtained from Amersham Pharmacia Biotech. The polyvinylidene fluoride membrane (pore size, 0.45 µm) was purchased from Millipore Corporation. Antiphosphotyrosine monoclonal antibody (clone 4G10) developed in mouse, goat anti-mouse IgG conjugated with horseradish peroxidase, rat kidney microsomal preparation containing Na⁺/K⁺ATPase beta 1 polypeptide (ATP1B1, previously known as Atpb or Atpb-1) in homogenized buffer (positive control), anti-ATP1B1 (mouse monoclonal IgG), goat anti-rabbit IgG-FITC conjugated (goat polyclonal IgG), goat anti-mouse IgG-FITC conjugated (goat polyclonal IgG), normal mouse IgG, and normal rabbit IgG were purchased from Upstate Biotechnology. Acrylamide/bis solution (30%, 29:1, 10x Tris/glycine/SDS buffer), Precision Plus Protein Kaleidoscope molecular mass standards, N,N,N',N'-tetramethyl ethylenediamine, and ammonium persulfate were purchased from Bio-Rad Laboratories. Ponceau red, glycine, and sodium vanadate were purchased from Fisher Scientific. Kodak Scientific Imaging X-OMAT LS film was obtained from Mandel Scientific. Kodak GBX developer and replenisher and Kodak GBX fixer and replenisher were purchased from Medtec.

Immunodetection and Immunolocalization of Na⁺/K⁺ATPase in Bovine Spermatozoa

A monoclonal antibody developed in mouse against the ATP1B1 polypeptide of Na⁺/K⁺ATPase was used to detect ATP1B1 in bovine sperm. Head plasma membrane of bovine sperm was prepared as described previously [32]. Protein extracts prepared from the head plasma membrane were used for SDS-PAGE and immunoblotting as described below. Rat brain extract provided by the manufacturer was used as the positive control.

For immunolocalization of ATP1B1, ejaculated bull spermatozoa (500 µl, 15 x 10⁶ sperm/ml washed in PBS [125 mM NaCl, 8 mM Na₂HPO₄, 2 mM NaH₂PO₄.H₂O, 5 mM KCl, and 5 mM dextrose; pH adjusted to 7.4]) were attached to poly-L-lysine-coated coverslips. Spermatozoa on coverslips were either fixed with 1% paraformaldehyde in PBS for 10 min at room temperature or permeabilized with cold (–20°C) ethanol for 30 sec. Spermatozoa were washed in PBS (twice for 5 min each wash). For immunostaining, spermatozoa were incubated in PBS containing 10% normal goat serum (blocking buffer) for 30 min at room temperature, followed by mouse anti-rabbit ATP1B1 clone (1:20 diluted in blocking buffer) for 1 h at 37°C. Following two 5-min washes in PBS, spermatozoa were incubated with FITC-conjugated goat anti-mouse IgG (1:50 in blocking buffer) for 1 h at 37°C. For control, spermatozoa were either incubated in blocking buffer or in mouse IgG instead of primary antibody.

Preparation of Incubation Media, Percoll Gradients, and Test Reagents

Modified Tyrode bicarbonate-buffered medium (Sp-TALP) and modified Tyrode Hepes-buffered medium (Sp-TALPH) were prepared as described previously [33, 34]. Percoll (90%, iso-osmotic, 280 mOsm) was prepared by mixing 100% Percoll with 10x Sp-TALPH at a ratio of 9:1 (v/v), and 45% Percoll was prepared by mixing isotonic Percoll as prepared above with an equal volume of Sp-TALPH (1x) as described previously [35]. Working solutions of ouabain and heparin were prepared in complete Sp-TALP medium [34] on the day of use. Stock solutions of IBMX (10 mM in dimethyl sulfoxide) and dbcAMP (100 mM in Milli-Q water) were prepared and stored at –20°C. On the day of use, the working solution of these reagents were prepared by mixing these stock solutions (1:1).

Preparation of Sperm for Motility Assessment, Tyrosine Phosphorylation, and Capacitation

Fresh semen ejaculates collected from mature Holstein-Friesian bulls were provided by Genetic Corporation (Gencor), Inc. Semen samples with at least 60%–65% motility were used. Semen samples were immediately diluted 1:5 with Sp-TALPH and transported to the laboratory within 30 min after collection in a thermos maintained at 35°C. Semen samples were subjected to Percoll wash on two-layer Percoll gradients (45%–90%) by centrifugation (700 x g, 30 min, 25°C; [35]). The resulting sperm pellet was resuspended in Sp-TALPH and washed to remove Percoll (380 x g, 10 min, 25°C). The sperm pellet was resuspended in Sp-TALP and washed again to remove Sp-TALPH (380 x g, 10 min, 25°C). The concentration of the resulting sperm pellet was determined using a hemocytometer and adjusted to 100 x 10⁶ sperm/ml with Sp-TALP.

Effect of Inhibition of Na⁺/K⁺ATPase on Sperm Motility, Tyrosine Phosphorylation, and Capacitation

Semen ejaculates obtained from different bulls (n = 3) were used for the evaluation of motility, capacitation, and tyrosine phosphorylation. The sperm pellet, prepared as described above, from each ejaculate was split into a series of 1.5-ml Eppendorf tubes and concurrently used for the evaluation of motility, tyrosine phosphorylation, and capacitation.

To study the effect of Na⁺/K⁺ATPase on motility, sperm samples (50 x 10⁶ sperm/ml) were incubated (39°C, 5% CO₂, high humidity) with either 0, 1, 10, 100, or 1000 µM ouabain for 5 h. At 0, 2, and 5 h of incubation, 10 µl of sperm preparation were drawn from each treatment group and adjusted to a final concentration of 5 x 10⁶ sperm/ml in egg yolk-Tris extender at 35°C [36], to avoid sperm sticking to the microscopic slides, for motility analysis. Eight microliters of semen were loaded onto a glass microscope slide, covered with a coverslip, and analyzed at 35°C with the Bovine Motility Program on a Hamilton-Thorne motility analyzer for percentage motility and percentage progressive motility with the following analytical setup: frame rate, 60 frames/sec; frames acquired, 50 frames/sec; minimum contrast, 55 pixels; minimum cell size, 7 pixels; threshold straightness, 80 microns/sec; medium average path velocity (VAP) cutoff, 100 microns/sec; low VAP cutoff, 20 microns/sec; static size limits, 0.60–2.99 pixels; static intensity limits, 0.59–1.41 pixels; static elongation limits, 0%–73%; nonmotile head size, 10 pixels; and nonmotile head intensity, 101. Slow cells (speed less than the above-stated VAP cutoff) were labeled as static or not moving.

For the evaluation of tyrosine phosphorylation and capacitation, sperm preparations were incubated (39°C, 5% CO₂, high humidity, 5 h) with or without heparin (10 µg/ml) in ouabain (1 µM, 10 µM, 100 µM, or 1 mM) or a combination of dbcAMP (1 mM) plus IBMX (0.1 mM). Each sample preparation for treatments contained 5 x 10⁶ sperm in a 100-µl volume. For capacitation experiments, two aliquots of sperm preparations were maintained per treatment group to evaluate capacitation status at 0 and 5 h of incubation. At each time point, sperm samples were incubated with either LPC (100 µg/ml) or Sp-TALPH for 30 min in an incubator at 39°C with 5% CO₂ under high humidity. The acrosomal status of spermatozoa (200 spermatozoa/slide) was determined using FITC-conjugated P. sativum agglutinin as described previously [24, 34]. The proportion of spermatozoa undergoing acrosome reaction was determined for each group treated with LPC and corrected for spontaneous acrosome reaction by subtracting the proportion of acrosome-reacted spermatozoa present in parallel samples incubated in Sp-TALPH alone.

For the evaluation of tyrosine phosphorylation, two aliquots of sperm preparations were maintained per treatment group, and sperm were processed at 0 and 5 h of incubation as described previously [34, 37] with the following modifications. Sperm were concentrated (10 000 x g, 3 min, room temperature) and the pellet washed (10 000 x g, 5 min, room temperature) in 1 ml of PBS containing 0.2 mM Na₂VO₃. The pellet was resuspended in 50 µl of PBS containing 0.2 mM Na₂VO₃ and 12.5 µl of 5x sample buffer (containing dithiothreitol and SDS). The preparation was mixed well and boiled for 5 min at 100°C, then centrifuged (10 000 x g, 5 min, room temperature), and the supernatants were used for SDS-PAGE and immunoblotting.

For SDS-PAGE and immunoblotting, sperm extracts prepared as described above were subjected to electrophoresis on 10% polyacrylamide gels for 15 min at 75 V for stacking, followed by electrophoresis at 100 V until the running front reached the bottom of the gel at room temperature. The gels were equilibrated in transfer buffer for 30 min at 4°C and electrotransferred to Immobilon P (G.E. Healthcare) using Tris-glycine buffer (pH 8.5) containing 20% methanol. The membranes were incubated with a solution of skim milk (5%, w/v) in Tris (20 mM, pH 7.8)-buffered saline containing Tween 20 (TTBS; 0.1% v/v, 1 h) and then treated with a monoclonal antibody developed in mouse against phosphotyrosine proteins (1:10 000 in TTBS supplemented with 0.1% w/v sodium azide) overnight at 4°C. After washing with TTBS (3 x for 5 min), membranes were incubated with goat anti-mouse IgG conjugated with horseradish peroxidase for 45 min at 20°C and then washed again with TTBS (3 x for 5 min). Positive immunoreactive bands were detected using the enhanced chemiluminiscence detection system. At the end of the experiments, blots were rinsed in distilled water and Ponceau stained [34] to ascertain that the amount of proteins loaded in each well was the same. The specificity of the antiphosphotyrosine antibody (clone 4G10) was determined as recommended by the manufacturer; the antibody recognized tyrosine phosphoproteins from the epidermal growth factor-stimulated cell lysate (positive control provided by the manufacturer). Parallel blots were incubated with secondary antibody alone to determine nonspecific bands.

Effects of Inhibition of Na⁺/K⁺ATPase on Intracellular Calcium Levels During Incubation under Capacitating Conditions

Percoll-washed sperm samples (n = 4) were loaded with a ratiometric calcium fluorescent probe, Indo-1AM, as described previously [38]. These sperm samples were incubated in capacitating conditions (39°C, 5% CO₂, high humidity) with Sp-TALP, heparin, or ouabain in the presence of 2 mM calcium and evaluated for intracellular calcium at 0 and after 4 h of incubation in capacitating conditions using a flow cytometer (Beckman-Coulter EPICS Elite). Immediately before flow-cytometric analysis, 500 µl of sperm samples (2 x 10⁶ sperm/ml) from each treatment were removed, and PI was added to a final concentration of 20 µM. The fluorescence intensities were recorded for a minimum of 10 000 spermatozoa at three wavelengths. For PI, conditions were excitation at 488 nm and emission at 610 nm. The fluorescent probe Indo-1AM was excited with ultraviolet light (325 nm), and fluorescence emissions were recorded for each sperm at two wavelengths using bandpass filters at 381 nm (Indo-1AM bound to calcium) and 525 nm (Indo-1AM unbound). Data were subsequently analyzed by FCS Express (De Novo Software) to calculate the relative intracellular calcium concentration of live cells. The PI-positive (dead) cells were first excluded, and the relative intracellular calcium of the remaining PI-negative cells were plotted based on the ratio of fluorescence at the two Indo-1AM wavelengths (381 nm and 525 nm for Indo-1AM bound and unbound, respectively, to calcium) for each individual sperm cell. This created two discrete populations. The number of sperm in each population was counted and expressed as a percentage of the total live cells analyzed, and mean percentages were calculated based on four replicates conducted with ejaculates from different bulls.

Effect of Inhibition of Na⁺/K⁺ATPase on Membrane Potential of Spermatozoa Incubated under Capacitating Conditions

Evaluations of the effect of inhibition of Na⁺/K⁺ATPase on intracellular calcium level as described above and on membrane potential were done using sperm preparations from the same ejaculate, and these experiments were replicated four times using ejaculates from different bulls. Percoll-washed spermatozoa were incubated with Sp-TALP alone, heparin (10 µg), or ouabain (100 µM) for 4 h under capacitating conditions, and the effect of inhibition of Na⁺/K⁺ATPase on sperm membrane was measured by flow cytometry (Beckman-Coulter EPICS Elite) using DiBac₄(3) as the fluorescent probe. The probe DiBac₄(3) is an anionic lipophilic potential-sensitive dye, with excitation at 488 nm and emission at 525 nm. With membrane depolarization, more dye enters the cytosol and the intensity of emitted fluorescence increases; with hyperpolarization, fluorescence decreases [39]. Stock solution of DiBac₄(3) was prepared in dimethyl sulfoxide according to the manufacturer's instructions. At 0 and 4 h of incubation, 25 µM DiBac₄(3) (based on the results of preliminary experiments) and 20 µM PI were added to sperm aliquots from each treatment group. The preparations were incubated at 37°C for 10 min, and a minimum of 10 000 events were evaluated by flow cytometry. The dead population of spermatozoa was excluded from analysis based on the uptake of PI. The DiBac₄(3) fluorescence of spermatozoa was plotted, and the mean intensity of fluorescence and mean proportion of live cells for each treatment group were derived using the FCS Express software.

Requirement of Extracellular Calcium for Tyrosine Phosphorylation and Capacitation Induced by Ouabain

To assess the need for exogenous calcium, sperm samples from the same ejaculate (n = 3) were evaluated for tyrosine phosphorylation and capacitation as described above, except that Percoll gradients and Sp-TALPH were prepared without adding calcium and incubation of spermatozoa was done in calcium-containing (2 mM CaCl₂) or calcium-deficient (no CaCl₂ added) Sp-TALP.

Statistical Analysis

Analysis of variance (two-tailed, unpaired values) was used to compare the effects of treatments on motility, capacitation, intracellular calcium levels, and membrane potential. Comparison of means between treatment groups was then done by the protected least-significant-difference test. A difference was considered to be statistically significant at P < 0.05.

RESULTS

Immunodetection and Immunolocalization of Na⁺/K⁺ATPase in Bovine Spermatozoa

A monoclonal antibody developed in mouse against the beta 1 polypeptide of Na⁺/K⁺ATPase recognized four bands (90, 60, 45, and 40 kDa) from an extract of proteins prepared from sperm head plasma membrane, and the antibody recognized two bands (45 and 40 kDa) from the control rat brain extract (Fig. 1). Incubation of parallel membranes with secondary antibody alone did not elicit any signal (data not shown). Immunostaining demonstrated a uniform distribution of Na⁺/K⁺ATPase on acrosomal, postacrosomal, and midpiece regions of fixed (nonpermeabilized) spermatozoa (Fig. 2A). However, the fluorescence pattern on the acrosomal and postacrosomal regions of permeabilized spermatozoa changed to irregular clusters (patchy) (Fig. 2B). Incubation of sperm preparations with FITC-conjugated secondary antibody or nonimmune mouse IgG alone did not elicit any fluorescent signal (data not shown).

View larger version (60K): [in this window] [in a new window]   FIG. 1. Immunodetection of Na+/K+ATPase in bovine spermatozoa. Immunoblotting using a mouse monoclonal antibody developed against the beta 1 polypeptide of Na+/K+ATPase detected four protein bands. Lanes 1–3: protein extract prepared from sperm head plasma membranes of three bulls; lane 4: positive control (rat brain extract) provided by the manufacturer. Immunoblotting of membrane using secondary antibody alone (control) did not demonstrate any specific signal (data not shown)

View larger version (7K): [in this window] [in a new window]   FIG. 2. Immunolocalization of Na+/K+ ATPase in bovine spermatozoa. The Na+/K+ ATPase beta 1 polypeptide was immunolocalized to sperm plasma membrane of acrosome (arrowhead) and the postacrosome (short arrow) and midpiece (long arrow) regions with even distribution in fixed (intact) spermatozoa (A). The distribution of Na+/K+ATPase was patchy in anterior acrosome (arrowhead) and postacrosomal (short arrow) regions of permeabilized spermatozoa (B). Incubation of spermatozoa with nonimmune mouse IgG or secondary antibody alone did not elicit a fluorescent signal (data not shown). Original magnification x600

Effect of Inhibition of Na⁺/K⁺ATPase on Sperm Motility

Inhibition of Na⁺/K⁺ATPase with ouabain did not affect the total percentage of motile sperm (Fig. 3A), which remained high throughout the incubation period. Ouabain reduced the percentage of progressively motile sperm, VAP, and curvilinear velocity of spermatozoa in a dose-dependent manner after incubation in capacitating conditions over a period of 5 h (Fig. 3, B, C, and D, respectively) but had no apparent effect on the amplitude of lateral head displacement (ALH) of spermatozoa (Fig. 3E). Head-to-head agglutination was noted in a proportion of spermatozoa incubated with ouabain, and these spermatozoa were excluded from evaluation based on size.

View larger version (23K): [in this window] [in a new window]   FIG. 3. Effect of ouabain on motility parameters of bovine sperm during incubation over a period of 5 h. Sperm preparations were incubated with ouabain at different concentrations (0–1000 µM), and its effects on total motility (A), progressive motility (B), VAP (C), curvilinear velocity (VCL; D), and ALH (E) of spermatozoa were evaluated using computer-assisted semen evaluation. Values are presented as the mean ± SEM of three independent experiments using semen samples from three bulls. *P < 0.05

Effect of Inhibition of Na⁺/K⁺ATPase on Tyrosine Phosphorylation and Capacitation

Incubation of spermatozoa in the presence of ouabain induced tyrosine phosphorylation in a cohort of sperm proteins (11, 50, 80, 100, 130, 200, and 250 kDa). The patterns of tyrosine phosphorylation for the samples that were incubated with ouabain were similar to that induced by a combination of dbcAMP plus IBMX (Fig. 4A), and these protein bands appeared to be more intense for sperm treated with ouabain compared to those treated with heparin (Fig. 4A). The intensity of the tyrosine phosphorylation in these proteins appeared to increase with increased amounts of ouabain. When parallel samples were assessed for capacitation (sensitivity to LPC-induced acrosome reaction), ouabain-induced capacitation occurred concurrently with tyrosine phosphorylation in spermatozoa. Incubation of sperm preparations with 10 µM, 100 µM, or 1 mM ouabain significantly increased the proportion of sperm undergoing acrosome reaction in response to LPC compared to sperm samples incubated in Sp-TALP alone (Fig. 4B). The proportion of sperm undergoing capacitation in response to 1 mM ouabain was similar to that in response to dbcAMP plus IBMX (27% ± 7% and 28.6% ± 1.3%, respectively), whereas the effects of lower concentrations of ouabain (10 or 100 µM) were similar to those of heparin (17% ± 2.5%, 17.6% ± 0.5%, and 14.3% ± 0.3% for sperm exposed to 10 or 100 µM ouabain or heparin, respectively) (Fig. 4B). No significant increase was found in non-LPC-induced acrosome reaction at 5 h compared to that at 0 h among different treatment groups (data not shown).

View larger version (34K): [in this window] [in a new window]   FIG. 4. Inhibition of Na+/K+ATPase induced tyrosine phosphorylation and capacitation in bovine spermatozoa. Percoll-washed sperm preparations (5 x 106 sperm in a 100-µl volume) were incubated in presence of ouabain at different concentrations, and the level of tyrosine phosphorylation (A) was evaluated at the beginning of incubation (0 h) and at the end of the incubation (5 h). Arrowheads at the right of the immunoblot indicate protein bands showing a change in the level of phosphorylation in different treatments. Parallel sperm preparations were evaluated for capacitation status (B) at 0 and 5 h of incubation. Percentage of sperm undergoing acrosome reaction in response to LPC was considered as the percentage capacitated spermatozoa. These values were corrected by subtracting the percentage of cells undergoing spontaneous acrosome reaction from the respective groups as described in Materials and Methods. The values in B are presented as the mean ± SEM of three independent experiments using semen samples from three bulls (abcP < 0.05)

Effects of Inhibition of Na⁺/K⁺ATPase on Intracellular Calcium Levels During Incubation under Capacitating Conditions

The proportion of dead sperm was identified based on PI uptake and excluded from analysis, as shown in Figure 5A. Incubation of spermatozoa in capacitating conditions resulted in two populations of live spermatozoa (Fig. 5B) with differing amounts of intracellular calcium (high and low) (Fig. 5, C and D, respectively). The actual mean amount of intracellular calcium within each population was not affected by treatments. More sperm were in the low-calcium population regardless of treatment, and only heparin affected the proportion of sperm in each population. After 4 h of incubation in the presence of heparin, fewer sperm were in the low-calcium population, and more were in the high-calcium population (Fig. 5, E and F).

View larger version (20K): [in this window] [in a new window]   FIG. 5. Effect of ouabain on intracellular calcium levels in spermatozoa. Sperm containing Indo-1AM, a ratiometric fluorescent calcium chelator, were incubated in Sp-TALP alone, 10 µg/ml of heparin, or 100 µM of ouabain . At 0 and 4 h of incubation, an aliquot of sperm from each treatment group (2 x 106 sperm/ml) were exposed to PI, and fluorescence intensity was assessed with bandpass filters at 610 nm (PI fluorescence), 381 nm (Indo-1AM bound to calcium), and 525 nm (Indo-1AM with no calcium bound). Sperm were separated on the basis of their PI fluorescence (A; R2: dead sperm), and those with low fluorescence (A; R1: live sperm) had the ratio of fluorescence intensity at 381/525 nm calculated (B). This resulted in two distinct populations, and the relative intracellular calcium content (as expressed by the ratio; C and D) and the numbers of sperm in each population (E and F) were quantified. The values in E and F are presented as the mean ± SEM of four independent experiments using semen samples from four bulls. FS, Forward scatter. *P < 0.05

Requirement of Extracellular Calcium for Tyrosine Phosphorylation and Capacitation Induced by Ouabain

Although ouabain did not significantly increase the proportion of spermatozoa with higher calcium levels, incubation of spermatozoa with ouabain did increase tyrosine phosphorylation levels in sperm proteins, with a concomitant increase in the proportion of spermatozoa undergoing capacitation as indicated by LPC-induced acrosome reaction (Fig. 4, A and B). Therefore, we investigated the requirement of extracellular calcium for tyrosine phosphorylation and capacitation in bovine sperm induced by ouabain. The intensity of tyrosine phosphorylation increased in a cohort of sperm proteins (50, 60, 80, 100, and 130 kDa) for the groups treated with heparin, dbcAMP plus IBMX, or ouabain, regardless of the level of extracellular calcium levels after 5 h of incubation in capacitating conditions, compared to spermatozoa incubated in Sp-TALP alone (Fig. 6A). In general, higher intensity of tyrosine phosphorylation was observed in sperm samples incubated with ouabain, including increased tyrosine phosphorylation in two additional protein bands at 200 and 250 kDa (Fig. 6A). Interestingly, sperm samples incubated with heparin, dbcAMP plus IBMX, or ouabain demonstrated that an 11-kDa protein underwent tyrosine phosphorylation only in the presence of extracellular calcium, and the intensity of this protein band was higher for the sperm samples incubated in presence of ouabain or dbcAMP plus IBMX. Capacitation assay demonstrated that extracellular calcium was required for heparin- or for dbcAMP plus IBMX-induced capacitation based on acrosome reaction data (Fig. 6B), but not for the ouabain-induced increase in the proportion of sperm undergoing acrosome reaction, suggesting that extracellular calcium is not mandatory for the ouabain-induced acrosome reaction.

View larger version (39K): [in this window] [in a new window]   FIG. 6. The requirement of extracellular calcium for tyrosine phosphorylation and capacitation in bovine spermatozoa. Sperm preparations (5 x 106 sperm in a 100-µl volume) were incubated in Sp-TALP deficient in calcium and Sp-TALP with 2 mM calcium. Tyrosine phosphorylation (A) and capacitation (B) were evaluated as described for Figure 4B. The values in B are presented as the mean ± SEM of three independent experiments using semen samples from three bulls (abP < 0.05)

Inhibition of Na⁺/K⁺ATPase Depolarizes Sperm Plasma Membrane

Incubation of spermatozoa in the presence of ouabain increased DiBac₄(3) fluorescence, indicating that inhibition of Na⁺/K⁺ATPase depolarized the plasma membrane of living spermatozoa (Fig. 7, A–C). Heparin did not alter DiBac₄(3) fluorescence. The proportion of live spermatozoa did not differ among treatment groups during the period of incubation (Fig. 7D).

View larger version (25K): [in this window] [in a new window]   FIG. 7. Effect of inhibition of Na+/K+ATPase on sperm membrane potential. Sperm preparations were incubated in Sp-TALP alone, 100 µM ouabain, or 10 µg/ml of heparin. At 0 and 4 h of incubation, sperm samples were loaded with 25 µM DiBac4(3) and PI. Sperm that were PI-positive (A; R2) were not analyzed for DiBac4(3) fluorescence. The DiBac4(3) fluorescence of live spermatozoa (A; R1) was evaluated (B). The ouabain-treated sperm at 4 h of incubation had significantly greater fluorescence than all others (*P < 0.05), indicating that this population of spermatozoa had undergone membrane depolarization (C). The proportion of live spermatozoa did not differ among treatment groups (D). The values in C and D are presented as the mean ± SEM of four independent experiments using semen samples from four bulls

DISCUSSION

In addition to the well-known activity of Na⁺/K⁺ATPase as an ion transporter involved in capacitation-related depolarization of the sperm membrane, we demonstrate here, to our knowledge for the first time, that this enzyme has characteristics of a signaling molecule responsible for tyrosine phosphorylation in the earliest stages of capacitation. In addition, we clarify the location of the enzyme on the sperm head and its mechanism of action. The enzyme must be inactivated to induce membrane depolarization and the signaling cascade that are hallmarks of capacitation induction. The signaling cascade appears to be independent of calcium, although we have identified at least one low-molecular-mass protein for which tyrosine phosphorylation requires incubation in capacitating conditions in the presence of ouabain or dbcAMP plus IBMX and is greatly enhanced by calcium.

Immunodetection and Immunolocalization of Na⁺/K⁺ATPase in Bovine Sperm

A monoclonal antibody identified ATP1B1 in four protein bands (90, 60, 45, and 40 kDa) in the proteins of head plasma membrane from bull sperm. The molecular masses of two protein bands (45 and 40 kDa) were similar to those in the positive control (rat brain extract), indicating the presence of ATP1B1 in bovine sperm. Lack of specific signal from control membrane (i.e., incubated with secondary antibody alone) confirmed the specificity of monoclonal antibody to recognize ATP1B1 in bull spermatozoa and positive control. However, the antibody also recognized two additional protein bands (90 and 60 kDa) in the head plasma membrane of bull sperm, which may represent total or partial dimerization of ATP1B1 or ATP1B1 with different levels of glycosylation. The beta polypeptides are heavily glycosylated, and glycosylation patterns of different isoforms of beta polypeptides may differ between species [12]. Moreover, organ-specific glycoforms of Na⁺/K⁺ATPase have been reported [40, 41]. Our immunolocalization study suggests that Na⁺/K⁺ATPase is uniformly distributed in the plasma membrane of sperm head (acrosomal and postacrosomal regions) and the midpiece of fixed, nonpermeabilized spermatozoa (Fig. 2A). The change in fluorescence pattern in permeabilized spermatozoa (Fig. 2B) suggests that Na⁺/K⁺ATPase also is present intracellularly or on the outer acrosomal membrane. Rat epididymal sperm possess both ATP1A1 and ATP1A4 localized to the midpiece of the flagellum [5]. In human sperm, ATP1A4 is localized to the principal piece of flagellum. In the present study, the localization of Na⁺/K⁺ATPase over the midpiece region indicates its possible role in the regulation of bull sperm motility [5]. These differences among species in the location of Na⁺/K⁺ATPase suggest different roles for this protein in sperm physiology [42].

Na⁺/K⁺ATPase Affects Sperm Motility

Ouabain, a specific inhibitor of Na⁺/K⁺ATPase [16, 17, 20], reduced the percentage of progressively motile sperm and the velocity at which ejaculated bull spermatozoa moved in a dose- and time-dependent manner. When 10 µM ouabain inhibited Atp1A4 alone in freshly isolated epididymal rat sperm, it reduced the total percentage of motile sperm to the same level as when 10 mM ouabain inhibited all the Na⁺/K⁺ATPase [5]. Here, with ejaculated bovine sperm, 100 µM or 1 mM ouabain reduced the percentage of progressively motile, but not the percentage of total motile, spermatozoa. These differences in results between bull and rat spermatozoa [5] may be caused by the differences in species (rat vs. bull) and sources of spermatozoa (epididymal vs. ejaculated). It has been reported that 1 mM ouabain inhibited progressive motility of bovine sperm within 30 min [43]. In our study, a Hamilton-Thorn motility analyzer was used for motility evaluation, which detected a statistically significant reduction in progressive motility after 5 h of incubation under capacitating conditions, thus confirming the previously reported [43] impact of Na⁺/K⁺ATPase impact on sperm motility parameters.

No evident change was observed in ALH, which is contrary to the expectation that ALH increases with capacitation. However, many sperm underwent head-to-head agglutination during incubation in the presence of ouabain, preventing the measurement of their motility characteristics. Agglutination of spermatozoa during incubation in the presence of known capacitating agents has been reported [33]. Therefore, agglutination of spermatozoa during incubation in the presence of ouabain suggests that ouabain binding to the Na⁺/K⁺ATPase induces head plasma membrane modifications associated with capacitation.

Na⁺/K⁺ATPase Affects Tyrosine Phosphorylation and Capacitation

A role of cAMP/protein kinase A-dependent protein tyrosine phosphorylation during sperm capacitation has been reported in several species [21–27]. However, the specific plasma membrane proteins involved in the regulation of protein tyrosine phosphorylation remain to be identified. Recent studies with somatic cells demonstrated that inhibition of Na⁺/K⁺ATPase induces tyrosine phosphorylation in proteins [19]. Furthermore, Fraser et al. [31] observed that incubation of mouse epididymal sperm with ouabain increased the rate of capacitation, and we previously demonstrated Na⁺/K⁺ATPase activity in the head plasma membrane of bovine spermatozoa [32]. Now, we show that ouabain induces tyrosine phosphorylation in ejaculated spermatozoa in a dose-dependent manner. In general, the pattern of ouabain-induced tyrosine phosphorylation was similar, albeit more intense, than that of dbcAMP plus IBMX (a phosphodiesterase inhibitor) in bovine sperm, as has been shown previously [27, 34]. We also show a cohort of sperm proteins with higher molecular masses (130, 200, and 250 kDa) and a low-molecular-mass protein (11 kDa) that appeared to be undergoing tyrosine phosphorylation.

Ouabain also induced capacitation (measured as the ability to undergo LPC-induced acrosomal exocytosis) in a dose-dependent manner, suggesting that Na⁺/K⁺ATPase inhibition-associated increase in tyrosine phosphorylation occurs in synchrony with capacitation. Although the ability to fertilize an oocyte remains the best indicator of capacitation status [3], the association between tyrosine phosphorylation and LPC-induced acrosome reaction links the fusion ability of sperm membranes with the molecular events associated with capacitation in bovine [24, 27, 34] and human [26, 44] spermatozoa.

Inhibition of Na⁺/K⁺ATPase Did Not influence Intracellular Calcium Levels in Spermatozoa

Increase in intracellular calcium levels is one of the major events occurring during sperm capacitation in several species [4, 26]. In most mammalian cells, Na⁺/K⁺ATPase is an energy-transducing ion pump that actively transports Na⁺ and K⁺ across the plasma membrane. Inhibition of Na⁺/K⁺ATPase can lead into a small increase in intracellular Na⁺ concentration, which in turn can increase intracellular Ca²⁺ concentration through the Na⁺/Ca²⁺ exchanger [45, 46]. The proportion of live spermatozoa did not differ among treatment groups, but incubation of sperm with heparin increased the proportion of spermatozoa with higher intracellular calcium levels. However, incubation with ouabain for 4 h did not significantly increase the proportion of spermatozoa with higher calcium levels. Therefore, Na⁺/K⁺ATPase inhibition leads to tyrosine phosphorylation and sperm capacitation without increasing intracellular calcium levels, similar to the calcium-independent signaling mechanisms leading to the activation of MAPK, tyrosine phosphorylation, and generation of reactive oxygen species following ouabain inhibition of Na⁺/K⁺ATPase in somatic cells [47].

Requirement of Extracellular Calcium for Tyrosine Phosphorylation and Capacitation Induced by Heparin and Ouabain

Because ouabain did not increase intracellular calcium concentration, we tested the calcium-dependence of the ouabain-induced tyrosine phosphorylation and capacitation. All the proteins but one were tyrosine phosphorylated equally with or without 2 mM calcium when sperm were incubated with heparin, dbcAMP plus IBMX, or ouabain, suggesting that extracellular calcium levels did not influence the level of tyrosine phosphorylation in spermatozoa. The requirement of extracellular calcium for tyrosine phosphorylation is controversial and still under investigation [20]. Previous studies have demonstrated that an increase in tyrosine phosphorylation could be achieved in mouse [21] and human [48] sperm by increasing the extracellular concentration of Ca²⁺. Contrary to these observations, a recent study [49] demonstrated that the presence of calcium in the external medium decreases tyrosine phosphorylation in both human and mouse spermatozoa by decreasing the availability of intracellular ATP. Similarly, an inhibitory effect of extracellular calcium on tyrosine phosphorylation in a 55-kDa protein was reported in epididymal bovine sperm [50]. Therefore, the predominant calcium-independence of tyrosine phosphorylation in the present study is not inconsistent with other published results. The significance of calcium-dependent tyrosine phosphorylation of the 11-kDa protein observed in the present study remains to be elucidated.

Calcium was required for heparin- and for dbcAMP plus IBMX-induced acrosomal exocytosis, as expected given the well-established requirement of extracellular Ca²⁺ for mammalian sperm capacitation and acrosome reaction [51–54]. However, ouabain-induced capacitation occurred in calcium-deficient medium, suggesting that plasma membrane modifications in spermatozoa induced by Na⁺/K⁺ATPase inhibition were sufficient to support LPC-stimulated acrosomal exocytosis in bovine sperm. Also, the incubation medium was calcium-deficient but not Ca²⁺-free; therefore, extracellular Ca²⁺ could still be available for membrane fusion. In addition, internal stores of Ca²⁺ localized in the acrosomal vesicle [54] may be a possible source of calcium for the membrane fusion. Conversely, extracellular calcium is not an absolute requirement for acrosome reaction in response to certain agonists [55]. The difference in the calcium dependence of the heparin- and ouabain-induced capacitation suggests that ouabain and heparin induces acrosomal exocytosis through different mechanisms.

Inhibition of Na⁺/K⁺ATPase Depolarizes Sperm Plasma Membrane

The enzymatic activity of Na⁺/K⁺ATPase results in the production of an electrochemical gradient that is required for many cellular processes, including maintenance of the resting membrane potential [12]. Inhibition of the Na⁺/K⁺ATPase disrupts cellular chemical gradients so that a drop in intracellular K⁺ levels occurs with a concomitant increase in Na⁺ levels, which leads to the depolarization of plasma membrane [56]. Membrane potential can couple external signals to cellular responses, and this process is particularly relevant in transcriptionally inactive spermatozoa, in which many physiological processes are controlled by ion fluxes [57]. Incubation of spermatozoa in the presence of ouabain for a period of 4 h increased the uptake of DiBac₄(3), an anionic fluorescent probe, indicating that ouabain depolarizes the plasma membrane of spermatozoa; depolarization was not observed in heparin-treated sperm. This difference between heparin- and ouabain-induced capacitation further supports the suggestion that these two agents induce acrosomal exocytosis through different mechanisms. Interestingly, Zeng et al. [58] observed that epididymal mouse sperm and ejaculated bovine spermatozoa undergo hyperpolarization during incubation under capacitating conditions. These differences in results may be caused by the fact that ouabain incubation would inhibit Na⁺/K⁺ATPase, leading to persistent depolarization of sperm plasma membrane, resulting in tyrosine phosphorylation and capacitation in spermatozoa as reported based on studies in somatic cells [19].

Although tyrosine phosphorylation was induced in spermatozoa incubated with ouabain, it is not clear whether spermatozoa complete the capacitation process before initiating biochemical changes associated with acrosome reaction. This is particularly relevant considering that inhibition of Na⁺/K⁺ATPase induces depolarization of sperm membrane, an event associated with acrosome reaction [43], and that acrosomal exocytosis of spermatozoa following incubation in the presence of ouabain is independent of extracellular calcium levels. Therefore, inhibition of Na⁺/K⁺ATPase and associated tyrosine phosphorylation may "prematurely" induce acrosome reaction before sperm complete the capacitation process.

The physiological relevance of ouabain-induced functional modification of spermatozoa remains to be elucidated. Although ouabain is not identified from reproductive tissues, its synthesis from the adrenal gland [17, 59] and its presence in blood plasma [60] suggests that spermatozoa may be exposed to this compound during its passage through the female reproductive tract, with a possible physiological role in the fertilization process by inducing capacitation and/or acrosome reaction.

In summary, the present study demonstrated that Na⁺/K⁺ATPase is involved in the regulation of signaling mechanisms leading to tyrosine phosphorylation and capacitation in bovine spermatozoa, which appeared to be occurring without an appreciable increase in intracellular calcium. The level of tyrosine phosphorylation and membrane depolarization following Na⁺/K⁺ATPase inhibition appeared to be sufficient to support membrane fusion and acrosomal exocytosis in spermatozoa. The localization of Na⁺/K⁺ATPase in spermatozoa and its role in the regulation of sperm capacitation demand further studies to investigate its role in the fertilization process per se.

FOOTNOTES

¹ Supported by the Natural Sciences and Engineering Research Council of Canada and the L'Alliance Boviteq, Saint-Hyacinthe, Quebec.

² Correspondence. FAX: 519 824 0870; mbuhr{at}uoguelph.ca

Received: 26 September 2005.

First decision: 31 October 2005.

Accepted: 8 May 2006.

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