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Abstract

In addition to the three isoforms of the catalytic subunit of the Na,K-ATPase originally identified (α1, α2, and α3), a fourth α polypeptide (α4) has recently been found in mammalian cells. This novel α-subunit of the Na,K-ATPase is selectively expressed in male gonadal tissues. In the testes, α4 is functionally active and comprises approximately half of the Na,K-ATPase activity of the organ. At present, the pattern of expression of the α4 polypeptide within the cells of the male gonad is unknown. By in situ hybridization, immunocytochemistry, and the ouabain inhibition profile of Na,K-ATPase activity, we show that the α4-subunit is expressed in the germ cells of rat testes. The highest amounts of the isoform are found in spermatozoa, where it constitutes two thirds of the Na,K-ATPase activity of the gametes. The other Na pump present in the cells is the ubiquitously expressed α1 polypeptide. The characteristic localization of α4 in the gonad is further supported by the drastic reduction of the polypeptide in mice that are infertile as a consequence of arrest in maturation of the germ cells. In addition, GC-1spg cells, a murine cell line derived from testis spermatogonia, also contain the Na,K-ATPase α4 polypeptide. However, the level of expression of the isoform in these cells is much lower than in the spermatozoa, a fact that may depend on the limited ability of the GC-1spg cells to differentiate in vitro. The particular expression of the Na,K-ATPase α4 isoform we encounter and the specific enzymatic properties of the polypeptide suggests its importance for ionic homeostasis of the germ cells of the testes. (J Histochem Cytochem 48:1023–1032, 2000)

Keywords

Na,K-ATPase isozymes α4 isoform testes spermatozoa

The Na,K-ATPase or Na pump exists as multiple isozymes consisting of different α- and β-subunits (reviewed in Sweadner 1989; Lingrel and Kuntzweiler 1994; Levenson 1994; Blanco and Mercer 1998). At present, four distinct α-subunits (α1, α2, α3, and α4) and three different β-subunits (β1, β2, and β3) have been identified in vertebrates (Schneider et al. 1985; Shull et al. 1986; Sverdlov et al. 1987; Martin-Vasallo et al. 1989; Gloor et al. 1990; Malik et al. 1996; Besirli et al. 1997). The highly regulated pattern of expression of the α and β polypeptides and their ability to associate in different pairs provides each cell with a particular array of Na,K-ATPase isozymes (reviewed in Lingrel et al. 1990; Lingrel and Kuntzweiler 1994; Ewart and Klip 1995; Blanco and Mercer 1998). The structural heterogeneity and functional diversity of the various αβ complexes (Jewell and Lingrel 1991; Munzer et al. 1994; O'Brien et al. 1994; Blanco et al. 1995a,b; Therien et al. 1996; Crambert et al. 2000) suggest that, rather than being redundant, the Na pump isozymes perform specific physiological roles (reviewed in Blanco and Mercer 1998). The least-characterized Na,K-ATPase isozymes are those composed of the α4-subunit. Recently, studies of the function of this polypeptide have confirmed that it can function as a catalytic subunit of the Na,K-ATPase. Thus, the α4 isoform is able to bind [³H]-ouabain, become phosphorylated from ATP, hydrolyze ATP, and transport ⁸⁶Rb, in a manner typical for a Na,K-ATPase (Blanco et al. 1999; Woo et al. 1999), although clearly an Na pump α isoform, α4, exhibits enzymatic characteristics that distinguish it from the other α isoforms. For example, α4 is very sensitive to ouabain, has a high apparent affinity for Na⁺, a low affinity for K⁺, and an intermediate affinity for ATP (Blanco et al. 1999). Another property unique to the α4 polypeptide is its limited tissue localization. The isoform appears to be selectively expressed in the testes and epididymis (Shamraj and Lingrel 1994; Underhill et al. 1999). The Na,K-ATPase from the testes consists exclusively of the α1 and α4 isoforms (Shamraj and Lingrel 1994; Blanco et al. 1999). In the gonadal tissues, the Na,K-ATPase has been identified in the epithelial cells of the seminiferous and epididymal tubules and in the germ cells (Uesugi and Yamazoe 1966; Quinn and White 1967; McGrady 1979; Ashraf et al. 1982; Byers and Graham 1990; Kenneth and Hess 1992). In the epithelial cells, the enzyme helps create the appropriate medium inside the tubule lumen for normal development of the spermatozoa (Byers and Graham 1990). In the germ cells, Na,K-ATPase activity maintains the cell membrane potential and motility of the spermatozoa (Nelson and McGrady 1981). At present, the functional relevance of the α4 isoform in the physiology of the testes is unknown. An important goal in understanding the role of this isoform is to determine the distribution of the polypeptide within the cells of the gonad. On the basis of in situ hybridization analysis, immunocytochemistry, and the ouabain inhibition profiles of the Na,K-ATPase activity, we present evidence that, in the testes, the α4 polypeptide is expressed in the germ cells and predominantly in spermatozoa.

Materials and Methods

Tissue and Cells

Testes and sperm cells or the germ cell line GC-1spg were used in this study. Testes were obtained from adult Sprague-Dawley rats weighing 200–240 g or from adult mice. In the case of mice, normal and transgenic mice deficient in the translation factor Egr4 (Tourtellotte et al. 1999) were used. Spermatozoa were collected from rat epididymis by standard techniques (Vermouth et al. 1986). Briefly, whole epididymis was dissected, minced, and suspended in 100 mM KCl, 12 mM KH₂PO₄, 3 mM MgCl₂, 0.4 mM EDTA, and 20 mM Tris, pH 7.4. After stirring for 20 min, the suspension was filtered through a fine mesh cloth and centrifuged at 800 × g for 10 min. The sperm pellet was resuspended and recentrifuged as before. Finally, the sample was resuspended in 320 mM sucrose, 25 mM imidazole (pH 7.4), and 0.1 mM EGTA. GC-1spg cells, derived from germ cells of mouse testis, were purchased from the American Type Tissue Culture Collection (ATCC; Rockville, MD). These cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 1 mM sodium pyruvate, 10% fetal bovine serum, penicillin (100 U/ml), streptomycin (100 mg/ml), and Fungizone (0.25 mg/ml).

Tissue and Cell Preparations

Membrane fractions were prepared from adult rat and mouse testes according to the protocol described previously (Blanco et al. 1999). Briefly, samples were homogenized on ice in 320 mM sucrose, 25 mM imidazole (pH 7.4), and 0.1 mM EGTA, using a glass-glass homogenizer. The lysate was then centrifuged at 1000 × g for 10 min. The pellet was re- suspended, homogenized again, and recentrifuged at 1000 × g for 10 min. The supernatants from the first and second centrifugations were combined and centrifuged for an additional 30 min at 30,000 × g. The final pellet was resuspended in the original homogenization buffer. For spermatozoa and GC-1spg cells, crude homogenates were prepared by disrupting the cells with a glass-glass homogenizer. Both the tissue membranes and cell homogenates were used for determination of Na,K-ATPase activity with various concentrations of ouabain.

RNA Blotting Analysis

RNA was isolated from tissue and cells using RNAzol according to the suppliers protocol (Tel-Test; Friendswood, TX). RNA content was quantified spectrophotometrically. Equal amounts of RNA (15 μg) were separated by electrophoresis in a formaldehyde gel and transferred overnight by capillary action to nitrocellulose (Micron Separations; Westboro, MA). The blots were UV-crosslinked and prehybridized at 65C according to Sambrook et al. (1989). [³²P]-dCTP-labeled probes for the Na,K-ATPase α isoforms were made from N-terminal fragments of the corresponding cDNAs, using the Rediprime random primer labeling kit from Amersham (Poole, UK). For the α1 and α3 isoforms, a cDNA segment corresponding to the 5′ end of each isoform to the AflII restriction site was used as template. For α2, a region of the isoform spanning from the 5′ end to the NheI site was used. For α4, the segment between base pairs 1314 and 1592 was utilized. Blots were washed in 0.1 × SSC (15 mM NaCl, 0.15 mM sodium citrate, pH 7.0), containing 1% SDS at 65C and exposed for autoradiography.

In Situ Hybridization

Localization of the Na,K-ATPase α4 isoform RNA was performed on 10-μm-thick sections of rat testes that had been fixed in 2% paraformaldehyde and embedded in paraffin. In situ hybridization was performed using the RNA Colour kit from Amersham. The fluorescein-11-UTP-labeled probes for the α1 and α4 isoforms were made using as template the full-length cDNA for the α1-subunit or a 301-bp cDNA fragment (bases 1314–1592 of the coding region of α4) for the α4-subunit. Transcription from both α isoform cDNAs, subcloned into pGEM-T Easy vector (Promega; Madison, WI) was performed using SP6 RNA polymerase. Sense probes using as template the same α1 and α4 cDNAs were made and used as control. Hybridization with the probes was performed for 2 hr at 55C. Samples were washed twice for 10 min each with 0.1% (v/v) of sodium dodecyl sulfate (SDS) in SSC at room temperature (RT), followed by two other washes at 55C for 10 min with 0.1% (v/v) SDS in SSC. The labeled probes were detected using an anti-fluorescein antibody conjugated to alkaline phosphatase at a dilution of 1:100.

Immunocytochemistry

Paraffin sections (10 μm) of rat testes, were treated with xylene and ethanol to remove the paraffin. After hydration, permeabilization of the sections was achieved by a 15-min incubation at 25C with 0.3% Triton X100 in HBS (25 mM Hepes, pH 7.4, 150 mM NaCl, and 1 mM EGTA). The tissue was then blocked for 2 hr at RT in 0.2% BSA, 5% normal goat serum (NGS) in HBS. The primary antibody in HBS containing 0.1% BSA, 2% NGS was added and allowed to attach overnight at 4C. After three 5-min washes in HBS, the secondary antibody in HBS containing 0.1% BSA, 2% NGS was added. After 1 hr, sections were washed three times for 5 min each with HBS and mounted. The α1 polypeptide was detected with a 1:100 dilution of an antiserum that recognizes a N-terminal peptide (DKYEPAAVSEHGD) of the isoform (Blanco et al. 1994). For the α4 isoform, a specific antiserum against a peptide (SEQKPRPTLRASNTNRQPK) corresponding to a sequence at the N-terminus of the α4 polypeptide was used at a dilution of 1:100 (Blanco et al. 1999). As secondary, an anti-rabbit antibody conjugated to fluorescein isothiocyanate (FITC) was used. Images were obtained using a Zeiss Axioskop microscope equipped with a Nikon UFX-II camera attachment.

Biochemical Assays

Protein assays were performed using the bicinchoninic acid/copper sulfate solution as described by the supplier (Pierce Chemical; Rockford, IL) after lysis of the cells in 1% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS). Na,K-ATPase activity was assayed through determination of the initial rate of release of ³²P_i from γ[³²P]-ATP as described previously (Blanco et al. 1995a). Membrane fractions from rat and mouse testes containing a total of 5 μg protein were assayed, whereas for sperm cell and GC-1spg cell homogenates, 10 μg and 20 μg total protein was used, respectively. Na,K-ATPase activity was measured in a final volume of 0.25 ml of medium containing 120 mM NaCl, 30 mM KCl, 3 mM MgCl₂, 0.2 mM EGTA, 2.5 mM sodium azide, 30 mM Tris-HCl (pH 7.4), 3 mM cold ATP, 0.2 μCi γ[³²P]-ATP ± different concentrations of ouabain. The reaction was started by the addition of ATP and incubated at 37C for 30 min. Radioactivity of 170 μl of the isobutanol-phosphomolybdate phase was measured by liquid scintillation counting. The hydrolysis of ATP in all assays was linear over the time course of the reaction. The total hydrolysis of ATP among the various preparations ranged from 30 to 50 μmoles of P_i released/mg protein/hr. Specific Na,K-ATPase activity was approximately 3 and 7 μmoles of P_i released/mg protein/hr for the Na,K-ATPase from rat and mouse testes, respectively. The germ cell line in culture and the spermatozoa showed lower specific activity, with values of approximately 1 and 2 μmoles of P_i released/mg protein/hr, respectively. Na,K-ATPase activity was determined as the hydrolysis of ATP that was dependent on Na⁺ and K⁺ and sensitive to 1 mM ouabain. Thus, total Na,K-ATPase activity was calculated by subtracting the ATPase activity obtained in the presence of 1 mM ouabain from that measured in its absence. For the ouabain dose-response curves, activity values were standardized to compare the relative contribution of each Na,K-ATPase isozyme to the total activity of each sample. For this, data were expressed as a percentage of the total Na,K-ATPase sensitive to 1 mM ouabain.

Data Analysis

Curve fitting of the experimental data was performed using a Marquardt least-squares nonlinear regression computing program (Sigma Plot; Jandel Scientific, San Rafael, CA). Dose-response relations for the ouabain inhibition of Na,K-ATPase activity showed a heterogeneous population of enzyme that was best fitted by applying an equation that assumed the existence of two (Equation 1) populations of Na,K-ATPase isozymes with different affinities for ouabain:

v = (F 1 / (1 + [I] / K_{j})) + (F 2 / (1 + [I] / K_{ii}))

where v is the Na,K-ATPase activity corresponding to a certain concentration of the inhibitor ouabain [I], expressed as a fraction of activity in the absence of ouabain. F1 and F2 are the fractional amounts of each Na pump isozyme, whereas K_i and K_ii represent the concentration of ouabain that gives the half-maximal inhibition to each of the Na,K-ATPases present in the sample. The best fit of the data to a two, rather than a one or multiple enzyme population model was confirmed by applying an F test as reported previously (Blanco et al. 1995b). This indicated that only two Na,K-ATPase isozymes, with K_is compatible with the existence of α1 and α4, were present in the samples.

Results

In the testes, the expression of the α-subunit of the Na,K-ATPase has been reported to be limited to the α1 and α4 isoforms (Shamraj and Lingrel 1994; Blanco and Mercer 1998). We confirmed this observation by Northern blotting analysis of total RNA from rat tissue. To ensure the specific identification of the α-sub-units, ³²P-labeled probes were made using as template isoform-specific cDNA segments. In addition, hybridization with the probes at 65C and stringent washing conditions using 1% SDS and 0.1% SSC were applied. As shown in Figure 1, transcripts for only the α 1- and α4-subunits of the Na,K-ATPase were detected in the testes. As control, total RNA from rat kidney, which contains only the α1 isoform, and brain, which expresses the α1, α2, and α3 isoforms, were used.

To gain insight into the distribution of the Na,K-ATPase α1 and α4 isoforms in the testes, we determined the localization of the corresponding mRNAs using in situ hybridization. Fluorescein–11-UTP-labeled RNA probes, followed by an anti-fluorescein alkaline phosphatase-conjugated antibody, were used to detect the α1 and α4 transcripts. Specific labeling of the isoforms was obtained by hybridization with the probes at 55C and stringent washes. Figure 2 shows that the α1 mRNA was found in the seminiferous tubules as well as at the interstitial cells of the gonad. In contrast, the message for α4 was localized to cells in the pseudostratified epithelium of the seminiferous tubules and mainly at the inner or luminal portions of these structures. The distribution of α4 mRNA is consistent with expression of the isoform in the germ cells. These cells also express the α1 transcript, and there appeared to be no cells that express solely the α4 mRNA. In contrast, cells at the interstitium, between the seminiferous tubules, contain only the RNA for the Na,K-ATPase α1 isoform.

Figure 1

RNA hybridization analysis of Na,K-ATPase α isoform in rat testes. Equal amounts of total RNA (15 μg) were separated on formaldehyde gels and transferred to nitrocellulose. Isoform-specific rat cDNA probes for the indicated α isoforms were used to identify the corresponding transcripts. The specificity of the probes is shown by their hybridization profile with rat kidney and rat brain RNA. Autoradiography exposure time was 14 hr for all samples except for α1 in the testes, which was developed after 25 hr. No signal was seen in testes for the α2 and α3 isoforms even after 72 hr of exposure. The mobility of the 18 S and 28 S RNA has been included for reference.

To investigate the expression of α isoforms at the protein level, sections of rat testes were subjected to immunocytochemical analysis using anti-α1 or anti-α4 antibodies, followed by secondary antibodies conjugated to FITC. As shown in Figure 3, a different pattern of distribution of the Na,K-ATPase α1 and α4 polypeptides was found in the gonad. As expected from the expression of the corresponding RNAs, the α1 antiserum evenly labeled all the cells within and between the seminiferous tubules (Figures 3A and 3D), whereas staining with the α4 antiserum showed a more restricted distribution (Figures 3B and 3E). Immunoreactivity for α4 was primarily located on the adluminal side of the seminiferous tubules, with the highest intensity of label found in the spermatozoa. This suggests that the germ cells are the main source of the Na,K-ATPase α4 isoform. However, the polypeptide is not the only Na pump isoform expressed in these cells because α1 was also detected.

To further study the pattern of expression of the Na,K-ATPase α1 and α4 isoforms in the germ cells of the testis, we determined the profiles of inhibition of Na,K-ATPase activity by ouabain. This approach has been routinely used as a tool to detect Na,K-ATPase isoform content in a sample. The method has the advantage of measuring functionally active Na,K-ATPase molecules and, in addition, it allows a more reliable quantification of the isozymes present (reviewed in Blanco and Mercer 1998). As demonstrated previously in the rat, the α1 and α4 isoforms differ drastically in their affinities for ouabain. Compared to α1, the α4 isoform is over a thousand times more sensitive to the cardiotonic steroid (Blanco et al. 1999). This enzymatic difference, which is the greatest among the Na,K-ATPase isoforms, can readily be used to determine the relative contribution of each α isoform to the total Na,K-ATPase of the sample. The functional Na,K-ATPase isoform composition of germ cells was first determined in GC-1spg cells and in spermatozoa. GC-1spg cells are a nontumorigenic murine cell line derived from germ cells of the gonad that have been immortalized by simian virus 40 large tumor antigen transformation (Hoffmann et al. 1992). As shown in Figure 4A, the dose-response curve for the inhibition of Na,K-ATPase activity by ouabain in the GC-1spg cells was heterogeneous, indicating the presence of two Na,K-ATPase isozymes. The inhibition constants for each of the isoforms, as well as their relative proportions, are shown in Table 1. The K_is in the nanomolar and millimolar range corresponded well with those reported previously for the Na,K-ATPase α1 and α4 isoforms (Blanco et al. 1999). The pattern of expression of the Na,K-ATPase α isoforms in the GC-1spg cells was corroborated by hybridization analysis of total RNA (Figure 4B). As shown, these cells do not express the α2 and α3 isoforms of the enzyme. Therefore, in agreement with the previous results, germ cells express both the α1 and α4 isoforms of the Na,K-ATPase. In addition, the level of isoform activity was unequal, with the α1 isoform making a relatively larger contribution to the total Na,K-ATPase of these cells. Because the turnover number of activity (ATP hydrolysed per min) is similar among the isoforms, their different contribution to the Na,K-ATPase hydrolysis reflect differences in expression of the polypeptides (Blanco et al. 1995a,b, 1999).

Although the GC-1spg cells have all the characteristics of germ cells, they can differentiate only to a certain extent, reaching a stage between spermatogonia Type B and spermatocytes. To investigate the level of expression of the α4 isoform in fully differentiated germ cells, we studied the profile of ouabain inhibition of the Na,K-ATPase from spermatozoa obtained from rat epididymis. As shown in Figure 5, a biphasic dose-response curve was found for the interaction of the cardiotonic steroid with the Na,K-ATPase of sperm cells. The obtained K_is for the sensitive and resistant forms of the enzyme corresponded to those of α1 and α4, confirming the expression of both Na pump isoforms in these cells. The calculated K_i and fractional amounts for each isozyme are indicated in Table 1. As shown, α4 activity constitutes the main Na,K-ATPase of spermatozoa. This contrasts with the relative distribution of the isoforms in the cultured germ cells, in which α1 activity represents the predominant Na pump present.

Figure 2

In situ hybridization of Na,K-ATPase α1 and α4 isoforms in rat testis. Testes from adult rats were fixed in 2% paraformaldehyde, embedded in paraffin, and sectioned (10 μm thick). Samples were hybridized with fluorescein-11-UTP RNA probes and detected using an antibody conjugated to alkaline phosphatase. As controls, the sense probes of the cDNA for the α1 (A,B) and α4 (C,D) isoforms were used. Antisense probes for α1 (E,F) and α4 (G,H) were used to detect the respective Na,K-ATPase isoforms. L, Leydig cells; S, Sertoli cells; G, germ cells. Bars = 30 μm.

To further confirm that the expression of the Na,K-ATPase α4 isoform originates mainly from the sperm cells, we characterized the Na,K-ATPase activity in testes from normal mice and mice made infertile through a deficiency in the production of the early growth response transcription factor, Egr4 (Tourtellotte et al. 1999). Egr4 is a member of the Egr zinc-finger transcription factors that regulate cell growth and cell differentiation (Crosby et al. 1991). Egr4 deficiency in male mice causes oligospermia due to an arrest in the maturation of germ cells. Because the Na,K-ATPase α4 isoform is mainly expressed in the mature germ cells, a reduction in the expression of the Na,K-ATPase α4 polypeptide should be expected to accompany the impaired production of spermatozoa in the transgenic mice. Figure 6 shows the ouabain inhibition profiles of the Na,K-ATPase from Egr4-deficient and normal mice testes. Reflecting the presence of both the α1 and α4 isoforms, testes from normal and Egr4-deficient mice exhibited low and high ouabainsensitive Na,K-ATPase activities. The calculated inhibition constants and relative quantity for the activity of each α isoform are summarized in Table 1. As shown, the normal mice gonad has approximately equal amounts of the α1 and α4 isoforms. This is in agreement with the results from rat testes (Blanco et al. 1999). In contrast, the Egr-4 deficient mice exhibit a significantly lower amount of the α4 isoform and, concomitantly, a proportional increase in the α1 isoform. Taken together, these results indicate that the germ cells express two functionally active isoforms of the catalytic subunit of the Na,K-ATPase, α1 and α4, and that the α4 polypeptide is the main functional form expressed in the meiotic cells of the gonad.

Figure 3

Localization of the Na,K-ATPase α1 and α4 polypeptides in rat testis. Testes were fixed in paraformaldehyde, embedded in paraffin, and sectioned. Immunocytochemistry was done as indicated in Materials and Methods using rabbit antisera that recognize the N-terminus of the α1 (anti-α1) or the α4 (anti-α4) isoform. A goat anti-rabbit antibody conjugated to FITC was used as the secondary antibody. (A,D) Tissue labeled with the anti-α1 antibody. (B,E) Labeling with the anti-α4 antibody. (C,D) Control sections treated with rabbit preimmune serum. L, Leydig cells; S, Sertoli cells; G, germ cells. Bars = 30 μm.

Discussion

The α4 polypeptide is the Na,K-ATPase isoform that exhibits the most restricted pattern of expression, having been solely identified in male gonadal tissues (Shamraj and Lingrel 1994; Blanco et al. 1999; Underhill et al. 1999; Woo et al. 1999). In the testis, the expression of the α4 isoform is not exclusive because the α1 polypeptide is present as well (Blanco et al. 1999; Woo et al. 1999). In contrast, the α2 and α3 isoforms are absent (Shamraj and Lingrel 1994; Blanco et al. 1999). We confirmed this observation by hybridization analysis of total RNA from the gonad (Figure 1). Both the α1 and α4 isoforms are catalytically functional in the gonad. Although α1 and α4 contribute approximately equally to the Na,K-ATPase activity of the tissue, the isoforms have enzymatic properties that are different (Blanco et al. 1999). The particular tissue expression and kinetics of α4 suggest that it performs a specific function. An important goal in understanding the role of α4 is to determine its pattern of expression within the cells of the testes. In the present work we have analyzed this by in situ hybridization, immunocytochemistry, and by the profile of ouabain inhibition. Our results indicate that the α4 isoform is expressed predominantly in differentiated germ cells of the testes.

Figure 4

(A) Dose-response curve for the ouabain inhibition of Na,K-ATPase from GC-1spg cells. Na,K-ATPase activity was determined as explained in Materials and Methods in medium containing 120 mM NaCl, 30 mM KCl, 3 mM MgCl₂, 0.2 mM EGTA, 2.5 mM sodium azide, 30 mM Tris-HCl (pH 7.4), 3 mM cold ATP, 0.2 μCi γ[³²P]-ATP ± different concentrations of ouabain. Na,K-ATPase activity was obtained as the difference between the Na⁺- and K⁺-dependent hydrolysis of ATP in the absence or presence of the different ouabain concentrations and the hydrolysis measured at 1 mM ouabain. Values were then expressed as percentage of the Na,K-ATPase activity in the absence of the inhibitor. The best fit of the data was obtained by assuming the presence of two enzyme populations with different affinities for ouabain. Each value is the mean and error bars represent SEM of three experiments performed in quadruplicate. (B) Northern blotting analysis of the Na,K-ATPase α isoform composition of GC-1spg cells. Total RNA (15 μg) was hybridized with isoform-specific rat cDNA probes for the indicated α isoforms. As controls, rat kidney, which contains α1, and rat brain, which expresses α1, α2, and α3 were used. Exposure time was 48 hr for α4 and 20 hr for the other isoforms. The mobility of the 18S and 28S RNA has been included for reference.

Although the polypeptide represents the majority of the Na,K-ATPase in the gametes, it is not the only α-subunit present because the cells also contain lower amounts of the ubiquitously expressed α1 isoform. The presence of the highly ouabain-sensitive and the ouabain-resistant ATPase activity typical of the α1 and α4 isoforms indicates that both polypeptides are functionally competent in the germ cells. This agrees with previous reports indicating that the meiotic cells of the testes are capable of hydrolyzing ATP in an Na⁺- and K⁺-dependent manner (Uesugi and Yamazoe 1966). The possibility that minor amounts of the α4 isoform are present in other cells of the testes can not be completely ruled out. For example, the anti-α4 antiserum appears to faintly label the Sertoli cells. However, the level of α4 expression in these cells is sparse. The finding by Muffly et al. (1985) that the Na,K-ATPase activity of rat Sertoli cells is resistant to ouabain agrees with the notion that these cells produce mainly the Na,K-ATPase α1 isoform. In addition, the analysis of the RNA and protein of the Na pump α4 isoform in the space between the seminiferous tubules suggests that the interstitial cells of the testes, such as Leydig cells, express predominantly the α1 isoform. Interestingly, we were unable to identify cells that uniquely express α4. Taken together, these results suggest that the main expression of the Na,K-ATPase α4 polypeptide occurs in the fully mature germ cells. However, the relative levels of activity of the isoform are different when the spermatozoa and the germ cell line GC-1spg are compared. In spermatozoa, the expression of α4 activity clearly predominates over that of the α1 isoform. The opposite occurs in GC-1spg cells, in which the activity of α1 prevails. This dissimilarity in isoform composition between the cultured and native cells may depend on the inability of the GC-1spg cells to fully express the Na,K-ATPase α4 isoform in vitro. This possibility seems unlikely because GC-1spg cells are able to normally express other testis-specific proteins, such as cytochrome c_t and the lactate dehydrogenase C₄ isozyme (Hoffmann et al. 1992). Alternatively, cell maturation may be involved in the regulation of expression of the isoforms. GC-1spg cells are known to differentiate only partially in culture. On the basis of their morphological and biochemical characteristics these cells have been shown to reach a stage between Type B spermatogonia and primary spermatocyte (Hoffmann et al. 1992). It is possible that upregulation of the expression of the α4 isoform is required at later stages of spermatozoa development. Supporting this notion are the results obtained from testes of Egr4-deficient mice. In these mice, maturation of the germ cells is almost completely blocked at the early-mid pachytene stage of spermatocyte development (Tourtellotte et al. 1999). Concomitant with this, the relative level of activity of the Na,K-ATPase α4 isoform is approximately a third of that found in normal testes. This difference, which suggests a developmental modulation of the expression of the Na pump α4-subunit, is not unique for the isoform, because changes in the levels of other Na,K-ATPase α isoforms have been reported during maturation (reviewed in Sweadner 1989; Lingrel et al. 1990).

Table 1

K_i values and relative amounts of the rat Na,K-ATPase isozymes α1 and α4 expressed in GC-1 cells, rat spermatozoa, and mouse testes from normal and oligospermic mice a

	α1 isoform		α4 isoform
	K_i(M)	K_i(M)	(%)
GC-1 cells	9.5 ± 1.0 × 10⁻⁴	79.7 ± 1.4	3.4 ± 3.1 × 10⁻⁹	20.3 ± 2.6
Spermatozoa	1.3 ± 0.6 × 10⁻⁴	32.6 ± 2.6	2.8 ± 2.0 × 10⁻⁹	67.4 ± 3.4
Normal mouse testes	7.5 ± 1.8 × 10⁻⁵	54.0 ± 2.6	5.2 ± 3.9 × 10⁻⁹	43.0 ± 4.4
Oligospermic mouse testes	1.1 ± 0.2 × 10⁻⁴	85.0 ± 2.8	1.7 ± 3.5 × 10⁻⁸	15.2 ± 4.0

Inhibition constants (Ki) and percentile amounts were calculated from dose-response curves for the ouabain inhibition of Na, K-ATPase activity. Values represent the mean ± SEM.

The high level of expression of the α4 isoform of the Na,K-ATPase in the mature germ cells of testes is intriguing. This occurrence may be related to the particular ionic environment of the germ cells. The medium bathing the cells inside the seminiferous tubules has a concentration of K⁺ that is higher than that of blood (Sheth and Rao 1968; Polak and Daunter 1984; Rosecrans et al. 1987). Coincidentally, when compared to the other isoforms the affinity of the α4 isoform for K⁺ is low (Blanco et al. 1999). Therefore, it is possible that the Na,K-ATPase α4 isoform function has evolved to adapt to the medium in the tubules. In addition, the increase of α4 in spermatozoa compared to the immature germ cells may be related to the fact that as the cells differentiate they come in contact with the high-K⁺ fluid in the lumen of the tubules. Another important enzymatic property of the Na,K-ATPase α4 isoform is its high affinity for ouabain. Differences in ouabain sensitivity of the Na,K-ATPase isozymes have been proposed to have a role in the regulation of Na,K-ATPase activity (reviewed in Blanco and Mercer 1998). This is supported by the identification of endogenous ouabain-like compounds in mammalian tissues (Blaustein 1993, 1994; Kelly and Smith 1994; Doris 1996). Interestingly, endogenous digoxin-like immunoreactivity has been found in the seminal fluid of humans (Vadazs et al. 1983). Ouabain has been shown to elicit various responses when applied to spermatozoa (Nelson and McGrady 1981). For example, changes in the membrane potential and in motility have been reported after exposure of the male gametes to ouabain. Therefore, the Na,K-ATPase α4 isoform in spermatozoa may be a site of regulation by endogenous ouabain-like compounds. In conclusion, our results provide evidence of a differential cell distribution for the Na,K-ATPase α4 polypeptide in testis cells. This, in addition to the fact that the isoform exhibits unique enzymatic properties, suggests that the α4-subunit may be important in the physiology of the male gonad.

Figure 5

Ouabain inhibition profile of the Na,K-ATPase from spermatozoa. Na,K-ATPase activity of sperm cells from rat epididymis was determined as described in Materials and Methods. Values for specific activity were obtained as described in Figure 4. Data are expressed as percentage of Na,K-ATPase activity determined in the absence of the inhibitor ouabain. The best fit of the experimental data was obtained using an equation describing two enzyme populations with different affinities for the cardiotonic steroid. Each value is the mean and error bars represent SEM of four experiments performed in quadruplicate.

Figure 6

Ouabain inhibition of Na,K-ATPase from normal and Egr4-deficient mice. Na,K-ATPase activity was determined as explained in Materials and Methods. Values are expressed as percentage of the activity obtained in the absence of ouabain. The biphasic curves represent the best fit of the data and indicate the presence of the highly ouabain-sensitive α4 and ouabain-resistant α1 isoforms in normal (•) and oligospermic (○) mice. Each value is the mean and error bars represent SEM of three experiments performed in quadruplicate.

Footnotes

Acknowledgments

Supported by National Institutes of Health grants GM 39746 and DK 45181, and by American Heart Association grant 96012080.

We thank Dr Jeffrey Milbrandt for providing the mice deficient in the Egr-4 transcription factor.

References

Ashraf

Peterson

Russell

(1982) Activity and location of cation-dependent ATPases on the plasma membrane of boar spermatozoa. Biochem Biophys Res Commun 107: 1273–1278

Besirli

Gong

Lomax

(1997) Novel β3 isoform of the Na,K-ATPase β subunit from mouse retina. Biochim Biophys Acta 1350: 21–26

Blanco

Koster

Mercer

(1994) The α subunit of the Na,K-ATPase specifically and stably associates into oligomers. Proc Natl Acad Sci USA 91: 8542–8546

Blanco

Koster

Sanchez

Mercer

(1995a) Kinetic properties of the α2β1 and α2β2 isozymes of the Na,K-ATPase. Biochemistry 34: 319–325

Blanco

Melton

Sanchez

Mercer

(1999) Functional characterization of a testes-specific α-subunit isoform of the Na,K-ATPase. Biochemistry 38: 13661–13669

Blanco

Mercer

(1998) Isozymes of the Na,K-ATPase: Heterogeneity in structure, diversity in function. Am J Physiol 275:F633–650

Blanco

Sanchez

Mercer

(1995b) Comparison of the enzymatic properties of the Na,K-ATPase α3β1 and α3β2 isozymes. Biochemistry 34: 9897–9903

Blaustein

(1993) Physiological effects of endogenous ouabain: control of intracellular Ca²⁺ stores and cell responsiveness. Am J Physiol 264:C1367–1387

Blaustein

(1994) Endogenous ouabain: physiological activity and pathophysiological implications. Clin Invest 72: 706–707

10.

Byers

Graham

(1990) Distribution of sodium-potassium ATPase in the rat testis and epididymis. Am J Anat 188: 31–43

11.

Crambert

Hasler

Beggah

Modyanov

Horisberger

Lelievre

Geering

(2000) Transport and pharmacological properties of nine different human Na,K-ATPase isozymes. J Biol Chem 275: 1976–1986

12.

Crosby

Puetz

Simburger

Fahrner

Milbrandt

(1991) The early response gene NGFI-C encodes a zinc finger transcriptional activator and is a member of the GCGGGGGCG (GSG) element-binding protein family. Mol Cell Biol 11: 3835–3841

13.

Doris

(1996) Endogenous inhibitors of the Na,K pump. Miner Electrolyte Metab 22: 303–310

14.

Ewart

Klip

(1995) Hormonal regulation of the Na⁺-K⁺-ATPase: mechanisms underlying rapid and sustained changes in pump activity. Am J Physiol 269:C295–311

15.

Gloor

Antonicek

Sweadner

Pagliusi

Frank

Moos

Schachner

(1990) The adhesion molecule on glia (AMOG) is a homologue of the β subunit of the Na,K-ATPase. J Cell Biol 110: 165–174

16.

Hoffmann

M-C

Narisawa

Hess

Millán

(1992) Immortalization of germ cells and somatic testicular cells using the SV40 large T antigen. Exp Cell Res 201: 417–435

17.

Jewell

Lingrel

(1991) Comparison of the substrate dependence properties of the rat Na,K-ATPase α1, α2, and α3 isoforms expressed in HeLa cells. J Biol Chem 266: 16925–16930

18.

Kelly

Smith

(1994) Endogenous cardiac glycosides. Adv Pharmacol 25: 263–288

19.

Kenneth

Hess

(1992) Localization and activity of Na,K-ATPase in the ductuli efferens of the rat. Anat Rec 234: 190–200

20.

Levenson

(1994) Isoforms of the Na,K-ATPase: family members in search of function. Rev Physiol Biochem Pharm 123: 1–45

21.

Lingrel

Kuntzweiler

(1994) Na⁺,K⁺-ATPase. J Biol Chem 269: 19659–19662

22.

Lingrel

Orlowsky

Shull

Price

(1990) Molecular genetics of Na,K-ATPase. Prog Nucleic Acids Res 38: 37–89

23.

Malik

Canfield

Beckers

Gros

Levenson

(1996) Identification of the mammalian Na,K-ATPase β3 subunit. J Biol Chem 271: 22754–22758

24.

Martin-Vasallo

Dackowski

Emanuel

Levenson

(1989) Identification of a putative isoform of the Na,K-ATPase beta subunit. Primary structure and tissue-specific expression. J Biol Chem 264: 4613–4618

25.

McGrady

(1979) The effect of ouabain on membrane potential and flagellar wave in ejaculated bull spermatozoa. J Reprod Fertil 56: 549–553

26.

Muffly

Turner

Brown

Hall

(1985) Content of K⁺ and Na⁺ in seminiferous tubule and rete testis fluids from Sertoli cell-enriched testes. Biol Reprod 33: 1245–1251

27.

Munzer

Daly

Jewell-Motz

Lingrel

Blostein

(1994) Tissue- and isoform-specific kinetic behavior of the Na,K-ATPase. J Biol Chem 269: 16668–16676

28.

Nelson

McGrady

(1981) Effects of ouabain on spermatozoan function: a review. Arch Androl 7: 169–176

29.

O'Brien

Lingrel

Wallick

(1994) Ouabain binding kinetics of the rat alpha two and alpha three isoforms of the sodium-potassium adenosine triphosphate. Arch Biochem Biophys 310: 32–39

30.

Polak

Daunter

(1984) Osmolarity of human seminal plasma. Andrologia 16: 224–227

31.

Quinn

White

(1967) Active cation transport in dog spermatozoa. Biochem J 104: 328–331

32.

Rosecrans

Jeyendran

Perez-Pelaez

Kennedy

(1987) Comparison of biochemical parameters of human blood serum and seminal plasma. Andrologia 19: 625–628

33.

Sambrook

Fritsch

Maniatis

(1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press

34.

Schneider

Mercer

Caplan

Emanuel

Sweadner

Benz

Jr Levenson

(1985) Molecular cloning of rat brain Na,K-ATPase α subunit cDNA. Proc Natl Acad Sci USA 82: 6357–6361

35.

Shamraj

Lingrel

(1994) A putative fourth Na⁺,K⁺-ATPase α subunit gene is expressed in testis. Proc Natl Acad Sci USA 91: 12952–12956

36.

Sheth

Rao

(1968) potassium levels of human semen. Ind J Med Res 56: 1806–1807

37.

Shull

Greeb

Lingrel

(1986) Molecular cloning of three distinct forms of the Na⁺,K⁺-ATPase α subunit from rat brain. Biochemistry 25: 8125–8132

38.

Sverdlov

Monastyrskaya

Broude

Ushkaryov

YuA

Allikmets

Melkov

Smirnov

YuV

Malyshev

Dulobova

Petrukhin

(1987) The family of human Na⁺,K⁺-ATPase genes. No less than five genes and/or pseudogenes related to the α subunit. FEBS Lett 217: 275–278

39.

Sweadner

(1989) Isozymes of the Na⁺/K⁺-ATPase. Biochim Biophys Acta 988: 185–220

40.

Therien

Nestor

Ball

Blostein

(1996) Tissue-specific versus isoform-specific differences in cation activation kinetics of the Na,K-ATPase. J Biol Chem 271: 7104–7112

41.

Tourtellotte

Nagarajan

Auyeung

Mueller

Milbrandt

(1999) Infertility associated with incomplete spermatogenic arrest and oligozoospermia in Egr4-deficient mice. Development 126: 5061–5071

42.

Uesugi

Yamazoe

(1966) Presence of sodium-potassium-stimulated ATPase in boar epididymal spermatozoon. Nature 5021: 403–404

43.

Underhill

Canfield

Dahl

Gros

Levenson

(1999) The Na,K-ATPase α4 gene (Atp1a4) encodes a ouabain-resistant α subunit and is tightly linked to the α2 gene (Atp1a2) on mouse chromosome 1. Biochemistry 38: 14746–14751

44.

Vadazs

Jakobi

Stoler

Makler

Krivoy

(1983) Endogenous digoxin-like immunoreactivity measured in seminal fluid. Biochem Biophys Res Commun 112: 132–138

45.

Vermouth

Carriazo

Ponce

Blanco

(1986) Lactate dehydrogenase X, malate dehydrogenase and total protein in rat spermatozoa during epididymal transit. Comp Biochem Physiol 83B:381–384

46.

Woo

James

Lingrel

(1999) Characterization of the fourth α isoform of the Na,K-ATPase. J Membr Biol 169: 39–44

The α4 Isoform of the Na,K-ATPase Is Expressed in the Germ Cells of the Testes

Abstract

Keywords

Materials and Methods

Tissue and Cells

Tissue and Cell Preparations

RNA Blotting Analysis

In Situ Hybridization

Immunocytochemistry

Biochemical Assays

Data Analysis

Results

Discussion

Footnotes

Acknowledgments

References