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J. Biol. Chem., Vol. 275, Issue 27, 20693-20699, July 7, 2000

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Sperm Motility Is Dependent on a Unique Isoform of the Na,K-ATPase^*

Alison L. Woo, Paul F. James, and Jerry B Lingrel

From the Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524

Received for publication, March 20, 2000, and in revised form, April 6, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The Na,K-ATPase, a member of the P-type ATPases, is composed of two subunits,  and , and is responsible for translocating Na⁺ out of the cell and K⁺ into the cell using the energy of hydrolysis of one molecule of ATP. The electrochemical gradient it generates is necessary for many cellular functions, including establishment of the plasma membrane potential and transport of sugars and ions in and out of the cell. Families of isoforms for both the  and  subunits have been identified, and specific functional roles for individual isoforms are just beginning to emerge. The 4 isoform is the most recently identified Na,K-ATPase  isoform, and its expression has been found only in testis. Here we show that expression of the 4 isoform in testis is localized to spermatozoa and that inhibition of this isoform alone eliminates sperm motility. These data describe for the first time a biological function for the 4 isoform of the Na,K-ATPase, revealing a critical role for this isoform in sperm motility.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The Na,K-ATPase is a heteromeric, integral membrane protein that is responsible for the electrogenic translocation of three sodium ions out of the cell and two potassium ions into the cell using the energy of hydrolysis of one molecule of ATP (1-3). This enzymatic activity results in the production of an electrochemical gradient that is required for many cellular processes, including establishment of the resting membrane potential, regulation of the osmotic balance, and generation of the Na⁺ gradient necessary for the transport of many ions and other substrates across the plasma membrane (1-3). Structurally, the Na,K-ATPase consists of two subunits, the  subunit with a molecular mass of 112 kDa and the glycosylated  subunit with a protein molecular mass of 35 kDa (1-3). The  subunit is the catalytic subunit of the enzyme, containing the cation-binding sites, the cardiac glycoside-binding site, and the ATP-binding site (2), whereas the  subunit is necessary for maturation of the enzyme, localization to the plasma membrane (4-7), and stabilization of the K⁺-occluded intermediate form of the protein (8, 9). An additional protein, , has been recently described to be associated with the Na,K-ATPase in some tissues and appears to modulate the enzyme's affinity for cations (10-12).

Isoforms for the  and  subunits have been identified, all exhibiting unique tissue and developmental expression patterns (3, 13-16), and specific functional roles for each are now beginning to be defined (17). Studies focusing on the biochemical properties of the Na,K-ATPase carrying different  isoforms have revealed modest differences in enzymatic activity (18); however, it is uncertain whether these differences have physiological significance. Therefore, it is important to consider other properties of these isoforms to understand the reason for the existence of multiple Na,K-ATPase  isoforms. Recently, this laboratory has reported for the first time a unique functional role for the 2 isoform in Ca²⁺ handling in cardiac myocytes (17), highlighting the importance for examination of the biological function(s) performed by other tissue-specific  isoforms.

The tissue expression pattern of the Na,K-ATPase 4 isoform is one of the most restricted, having been identified only in mouse, rat, and human testes and at lower levels in mouse epididymis (13, 17, 19). Biochemical characteristics of the 4 isoform have been recently reported, revealing it to be a high affinity ouabain receptor that also has a high affinity for both Na⁺ and K⁺ and that exhibits Na⁺- and K⁺-stimulated, ouabain-inhibitable ATPase activity (20, 21). To better understand the unique functional role of the 4 isoform, we report here an in-depth analysis of this Na,K-ATPase isoform in the rat. Our experiments show that it is expressed specifically in the mid-piece of the flagellum of mature sperm cells and that inhibition of the 4 isoform alone eliminates sperm motility, demonstrating for the first time a critical role for this isoform in normal sperm function.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

RNA Isolation and Northern Blot Analysis-- Total cellular RNA was isolated by the guanidine thiocyanate method (Tri-Reagent, Molecular Research Center, Inc., Cincinnati, OH). Total RNA samples (10 µg/sample) were denatured in 1 M glyoxal, 54% Me₂SO, and 0.01 M sodium phosphate buffer (pH 6.8); separated using a 1% agarose gel in 0.01 M sodium phosphate buffer; and then transferred to Sure Blot^® nylon membrane (Intergen Co., Purchase, NY). Northern blots were screened for expression of  isoforms of the Na,K-ATPase using isoform-specific probes (17). Quantitation of mRNA levels was determined by exposing ³²P-labeled blots to a phosphor screen. Phosphor screens were scanned using a Storm 840 scanner and analyzed using ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA). Densitometry results were corrected to levels of glyceraldehyde-3-phosphate dehydrogenase and are reported as mean volume integrated values ± S.E. between samples from three animals.

Microsome Preparation, SDS-Polyacrylamide Gel Electrophoresis, and Western Blot Analysis-- Microsome preparation, SDS-polyacrylamide gel electrophoresis, and Western blotting were performed as described previously (17, 20) using the following dilutions of primary antibody: 0.5 µg/ml b4 (20), 1:1000 of 1 isoform-specific monoclonal antibody 6F (University of Iowa Developmental Hybridoma Bank, Iowa City, IA), 1:500 of 2 isoform-specific monoclonal antibody McB2 (generous gift from K. Sweadner), and 1:1000 of 3 isoform-specific monoclonal antibody (Affinity Bioreagents, Inc., Golden, CO). Quantitation of protein expression levels was performed using scanned Western blots and ImageQuant software. Densitometry results are reported as mean volume integrated values ± S.E. between samples from three animals.

Immunohistochemical Analysis of Testis-- Testes were harvested from Harlan Sprague-Dawley rats (Harlan Sprague Dawley, Inc., Indianapolis, IN) and immediately placed in HistoPrep^TM-buffered 10% Formalin (Fisher) overnight at 4 °C. Tissues were washed three times in PBS,¹ dehydrated in ethanol, and stored in 80% ethanol until paraffin embedding. Tissue sections were cut at a thickness of 8-10 µm, fixed to glass slides (Fisher Premium Brand glass slides), and stored at 4 °C until further use.

Immmunohistochemical staining of testis sections was performed as follows. Tissue sections were deparaffinized by washing in HemoD (Fisher) three times, rehydrated in a series of ethanol washes from 100 to 70%, and placed in PBS. Slides were then incubated in methanol containing 0.5% hydrogen peroxide for removal of endogenous peroxidase activity. Tissue sections were blocked for nonspecific binding by incubation in PBS (pH 7.4) containing 0.2% Triton X-100 and 2% normal goat serum for 1 h and then placed in a solution containing b4 (5.0 µg/ml) overnight at 4 °C. To observe secondary antibody interactions with tissue sections, additional slides were incubated overnight in the absence of primary antibody. The next day, sections were washed (0.1 M PBS containing 0.2% Triton X-100) and then exposed to biotinylated goat anti-rabbit IgG (Vector Labs, Inc., Burlingame, CA) for 1 h at room temperature. At the end of the hour, sections were washed before incubation with an avidin-biotin complex (Vector Labs, Inc.) for 30 min at room temperature. Slides were washed one final time, rinsed briefly in 0.1 M acetate buffer (pH 6.0), incubated with diaminobenzidine for 4 min, rinsed briefly in Tris buffer (pH 7.6), incubated in a Tris cobalt solution (pH 7.2) for 4 min, and then placed in deionized water. Stained tissue sections were dehydrated in a series of ethanol washes from 70 to 100%, and coverslips were mounted using Permount (Fisher) diluted 1:1 with xylene.

Immunocytochemical Analysis of Sperm-- The whole epididymis was removed from adult (8-12 weeks of age) Harlan Sprague-Dawley rats and placed in PBS. Epididymis tubules were carefully minced, and sperm were allowed to swim out freely into the buffer for 15 min at room temperature. The tissue was then removed, and sperm were either pelleted by centrifugation and stored at 80 °C or fixed to glass slides for immunocytochemical analysis. Sperm microsomes were prepared from frozen cell pellets as described for testis microsomes.

Aliquots of sperm were fixed to glass slides overnight in HistoPrep^TM-buffered 10% Formalin at 4 °C. The next morning, slides were washed in PBS and then used for immunocytochemical analysis. Diaminobenzidine detection of the 4 isoform in isolated sperm cells was performed from this point on as described above for the immunohistochemical analysis of testis sections. For immunofluorescent detection of the 4 isoform, slides were blocked for nonspecific binding by incubation in PBS containing 0.2% Triton X-100 and 2% normal donkey serum and then placed in a solution containing b4 (5.0 µg/ml). Immunofluorescent detection of the 1 isoform required exposure of cells to PBS containing 2% SDS for 15 min, several washes in PBS, and then incubation in a solution containing 6F (1:100). On the third day, slides were washed (0.1 M PBS containing 0.2% Triton X-100) before incubation with the appropriate secondary antibody, either fluorescein isothiocyanate-conjugated donkey anti-rabbit or Texas Red^® dye-conjugated donkey anti-mouse secondary antibody (both from Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) diluted 1:100 for at least 1 h. Slides were washed a final time, and coverslips were mounted using Vectashield mounting medium for fluorescence with 4,6-diamidino-2-phenylindole (Vector Labs, Inc.) for visualization of nuclei. Fluorescently labeled sperm cells were examined and photographed using an Axioplan 2 and Axiophot 2 light microscope equipped for fluorescence visualization (Carl Zeiss, Inc., Thornwood, NY).

Ouabain Binding Competition Assays-- All ouabain binding competition assays were performed as described previously (20, 22). Three different sets of sperm microsomes from individual animals were analyzed to characterize each protein-ligand interaction. Purified sheep kidney enzyme was used as a positive control for individual assays. The K_D for ouabain binding and IC₅₀ values for Na⁺ and K⁺ competition were determined as described previously (20, 22), and errors reported are the S.E. values from the mean for three samples.

Sperm Motility Assays-- Sperm from whole epididymis were obtained as described above from Harlan Sprague-Dawley rats (23). A drop of the sperm suspension was then diluted in warmed, modified Tyrode's albumin/lactate/pyruvate (TALP) solution (24) (114 mM NaCl, 3.2 mM KCl, 2 mM NaHCO₃, 0.4 mM NaH₂PO₄H₂O, 10 mM sodium lactate, 2 mM CaCl₂·2H₂O, 0.5 mM MgCl₂·6H₂O, 10 mM HEPES, 100 IU/ml penicillin, 3 mg/ml bovine serum albumin (fraction V), and 0.2 mM pyruvate). Ouabain solutions were prepared in TALP solution not more than 15 min before use and kept at 35 °C. The percentage of motile sperm in the presence and absence of ouabain was determined using a hemocytometer to count duplicate samples in varied order. Motility was assessed every hour by counting at least 200 sperm/sample, and sperm with any flagellar movement were scored as motile (25, 26). The assay was performed on five different animals on separate days. The results from 1 day's experiment, in duplicate, are shown and are representative of data collected in every experiment. Results are expressed as means ± S.E. for duplicate samples from one animal on 1 day's experiment. Statistical analyses of differences in sperm motility in ouabain compared with sperm motility in buffer alone were performed using unpaired Student's t test with equal variance.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Analysis of the 4 and 1 Isoforms in Maturing Testes-- A variety of changes occur in the mammalian testis as it matures into a sexually active adult, including initiation of testosterone production, establishment of the blood-testis barrier, and spermatogenesis (27, 28). One of the first indications of sexual maturity is the production of mature sperm, called spermatozoa, in testis, an event that occurs in the rat at the average age of 33-35 days (29). Determination of the stage of sexual maturity during which the 4 isoform is produced will provide useful information concerning its functional role in testis. Therefore, we began by examining its expression in both immature and mature animals, ages 2-12 weeks, at both the RNA and protein levels. Examination of total RNA isolated from testes from these animals revealed that the 4 isoform is not expressed until 4 weeks and reaches a maximum level, approximately three times the original amount, at 6 weeks (Fig. 1, A and B). The ubiquitous 1 isoform, on the other hand, is expressed at a constant level throughout the life of the animal (Fig. 1A). Western analysis of the 4 isoform protein levels in these testes revealed that it is not present in testis until after 4 weeks, subsequent to which it increases almost 3-fold, whereas the 1 isoform is constantly present (Fig. 1, C and D). The 4 isoform is therefore not omnipresent in testis; rather, its expression is regulated in parallel to the onset of sexual maturity.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   The Na,K-ATPase 4 and 1 isoforms exhibit different expression patterns in testis throughout sexual maturation. Shown are the results from Northern blot and Western blot analyses of the expression of the 4 and 1 isoforms in testes from rats between the ages of 2 and 12 weeks. A, Northern blots probed sequentially for the expression of the 4 and 1 isoforms showed that the 4 isoform is not expressed until 4 weeks, whereas the 1 isoform is expressed constantly throughout the life of the animal. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used as a loading control. B, quantitation of the levels of 4 isoform expression, normalized to glyceraldehyde-3-phosphate dehydrogenase expression, revealed an ~3-fold increase in 4 isoform expression after 4 weeks. C, Western blots showed that the 4 isoform protein is not present in testis until after 4 weeks, whereas the 1 isoform protein is present at constant levels throughout sexual maturation. 20 µg of protein was loaded in each lane. D, quantitation of the 4 isoform protein levels revealed an ~3-fold increase in 4 isoform expression after 6 weeks. kb, kilobases.

Localization of the 4 Isoform in Testes-- Immunohistochemistry was next used to define the localization of expression of the 4 isoform in rat testis. Structurally, the testis divides into three regions, the membranous tunica albuginea that envelops the testis, the coiled seminiferous tubules, and the interstitium (28). The seminiferous tubules contain populations of both somatic cells, called Sertoli cells, and germ cells, which, beginning at puberty in mammals, develop and mature through the process of spermatogenesis (27, 28). Spermatogenesis begins with round, immature germ cells and results in the production of elongated, mature sperm, which are released into the luminal space (30). Intermediate forms of germ cells undergoing spermatogenesis include both round spermatocytes and elongating spermatids (30). Immunohistochemical examination of adult testis sections identified the 4 isoform in mature sperm, and no staining that indicates expression of the 4 isoform in any other cell type, including Sertoli and Leydig cells, was observed (Fig. 2A). In addition, immunoreactivity of the secondary antibody alone in testis was nonexistent (Fig. 2B).

View larger version (81K): [in this window] [in a new window]   Fig. 2.   Immunohistochemical analyses revealed that the 4 isoform is localized to spermatozoa. A, an adult testis section incubated with the 4 isoform-specific antibody showed the presence of the 4 isoform in mature sperm localized to the center of the seminiferous tubule, whereas sections incubated with the secondary antibody alone showed no cross-reactivity. Bar = 65 µm. B, testis sections from animals 3-12 weeks old were probed for expression of the 4 isoform protein, exposed to the secondary antibody alone, and stained with hematoxylin and eosin (H&E). Only testis sections containing mature sperm cells showed specific staining for the 4 isoform protein. Testis sections incubated with the secondary antibody alone showed no cross-reactivity. Hematoxylin and eosin staining revealed mature sperm in the lumen of seminiferous tubules from animals ages 6, 8, and 12 weeks old and not in the seminiferous tubules of younger animals 3 and 4 weeks old. Bar = 75 µm.

The expression of the 4 isoform in mature sperm suggests that it must be present in germ cells in earlier stages of spermatogenesis since mature sperm synthesize little to no new protein. Because spermatozoa represent a large population of cells in these adult testis sections, the strong 4 isoform-specific staining in these cells may mask the expression of this isoform in smaller populations of germ cells such as spermatocytes and spermatids. Before the onset of spermatogenesis, spermatogonia are the only germ cells present in the mammalian testis (29, 30). Testes of animals in which spermatogenesis has recently begun contain only early to intermediate developmental stages of germ cells, with little to no spermatozoa. Investigation of testes from these animals therefore allows detection of the 4 isoform in developing germ cells in the relative absence of mature sperm. Toward this end, testis sections from animals 3-12 weeks old were examined for expression of the 4 isoform protein, and the presence of mature sperm was determined by hematoxylin and eosin staining. Testis sections from animals 3 and 4 weeks old do not express the 4 isoform protein (Fig. 2B), nor do they contain mature, elongating spermatozoa (Fig. 2B). Examination of testis sections from 6-week-old animals, however, revealed some 4 isoform protein expression in intermediate stages of developing sperm, possibly spermatids, and little to no spermatozoa present (Fig. 2B). Finally, testis sections from animals 8 and 12 weeks old express the 4 isoform protein in spermatozoa (Fig. 2B), similar to the pattern described above for adult testes (Fig. 2A), and hematoxylin/eosin-stained tissues confirmed the abundance of mature sperm in the seminiferous tubule lumen (Fig. 2B). Again, testis sections incubated with the secondary antibody alone did not show any immunoreactivity (Fig. 2B). The expression of the 4 isoform protein therefore immediately follows the onset of spermatogenesis, and its expression is localized to mature spermatozoa and some intermediate stages of developing sperm.

Identification of Na,K-ATPase  Isoforms in Sperm-- Microsomes of isolated sperm collected from the epididymis of sexually mature rats were subsequently examined for expression of each of the four  isoforms of the Na,K-ATPase. Western blots containing microsome samples from testis, sperm, red blood cells, and brain were probed individually using  isoform-specific antibodies. These Western blots revealed that sperm express only the 4 and 1 isoforms; and compared with testis, the level of expression of the 4 isoform is very high, whereas that of the 1 isoform is low (Fig. 3). As expected, whole testis expresses the 4 and 1 isoforms; red blood cells, included since they are the only cell contaminant in sperm preparations, express only the 1 isoform; and brain expresses the 1, 2, and 3 isoforms (Fig. 3).

View larger version (57K): [in this window] [in a new window]   Fig. 3.   The 4 and 1 isoforms of the Na,K-ATPase are expressed in rat epididymal sperm. The expression of each of the  isoforms of the Na,K-ATPase was examined in microsomes from testis, sperm, red blood cells (RBC), and brain by Western blot analysis. 10 µg of protein was loaded in each lane, except the lane for the brain sample, which contained 1 µg of protein. The 4 isoform is only in testis and sperm microsomes; the 1 isoform is in all samples; and the 2 and 3 isoforms are only in brain microsomes.

Localization of the 4 and 1 Isoforms in Sperm-- The distributions of the 4 and 1 isoforms of the Na,K-ATPase were next examined in isolated sperm cells. Immunocytochemical localization of the 4 isoform by diaminobenzidine staining identified the 4 isoform specifically in the flagellum of the sperm, most heavily perceived in the mid-piece region of the tail (Fig. 4A). Sperm incubated without primary antibody did not show any staining (Fig. 4B). Immunofluorescent labeling of the 4 isoform in sperm confirmed this pattern of expression (Fig. 4, C and D). The entire sperm flagellum was examined for 4 isoform expression by sequentially photographing sperm with first the head and then the tail in the focal plane (Fig. 4, C and D). This paired series of photographs showed that regardless of the focal plane, the expression pattern of the 4 isoform protein is localized to the mid-piece of the flagellum, and there is no detectable secondary antibody immunoreactivity (Fig. 4, C, D, F, and G). Visualization of the 1 isoform revealed its location in the same region of the sperm where the 4 isoform was found, whereas nonspecific secondary antibody binding to sperm was undetectable (Fig. 4, E and H). Therefore, both of the  isoforms of the Na,K-ATPase present in sperm are localized to the mid-piece of the flagellum and do not have distinct patterns of localization that are detectable at this level of resolution.

View larger version (45K): [in this window] [in a new window]   Fig. 4.   The 4 and 1 isoforms of the Na,K-ATPase are localized to the mid-piece region of sperm. Diaminobenzidine staining of isolated sperm cells incubated with an 4 isoform-specific antibody revealed high expression of this isoform in the mid-piece of the flagellum (A), whereas sperm cells incubated with the secondary antibody alone revealed no cross-reactivity (B). Black bars = 25 µm. A sperm cell incubated with both an 4 isoform-specific antibody and a fluorescein isothiocyanate-conjugated secondary antibody, taken with the head of the sperm cell in the focal plane, revealed intense 4 isoform protein expression in the mid-piece of the flagellum (C). The same sperm cell was photographed with the end of the flagellum in the focal plane, again revealing intense 4 isoform protein expression only in the mid-piece of the flagellum (D). A sperm cell incubated with both an 1 isoform-specific antibody and a Texas Red-conjugated secondary antibody, taken with the head of the sperm in the focal plane, revealed 1 isoform protein expression in the head and mid-piece of the flagellum (E). Isolated sperm cells incubated with either the fluorescein isothiocyanate- or Texas Red-conjugated secondary antibody alone did not show any cross-reactivity (F-H). White bars = 20 µm.

Biochemical Analysis of the 4 Isoform in Sperm-- The biochemical characteristics of the 4 isoform in sperm were next examined to define any distinguishing characteristics between it and the 1 isoform. Our laboratory (20) and others (21) have previously measured the biochemical characteristics of the 4 isoform using tissue culture cell expression systems, but it is important to examine this isoform in endogenous cells, as differences in the kinetics of the Na,K-ATPase are not only isoform-specific, but also tissue-specific (31, 32). Using ouabain binding competition assays (20, 22), the ligand binding affinities for the 4 isoform in sperm microsomes were found to be similar to those previously reported (20) and are listed here as means ± S.E. between three unique samples: the K_D for ouabain binding is 148.50 ± 23.44 nM, the IC₅₀ for Na⁺ is 6.13 ± 0.62 mM, and the IC₅₀ for K⁺ is 3.49 ± 0.35 mM.

Ouabain Inhibition of Sperm Motility and Fertilization-- The results presented in this paper thus far have defined the 4 isoform of the Na,K-ATPase to be specific to sperm. The identification of the 4 isoform in spermatozoa and not in spermatogonia suggests that its biological role must be related to a specialized function of mature sperm, e.g. flagellar movement for sperm motility. The Na,K-ATPase is the molecular receptor for cardiac glycosides such as ouabain, which bind to and inhibit the activity of the enzyme (17). The effects of ouabain on sperm motility have been previously examined in other species, but the results were interpreted without considering the existence of Na,K-ATPase molecules carrying different  isoforms (33). Two  isoforms of the Na,K-ATPase have now been identified in rat sperm: the high affinity ouabain receptor, 4, and the low affinity ouabain receptor, 1. Because of their different pharmacological properties, the effects of ouabain inhibition of the 4 isoform alone on sperm motility were examined. Freshly isolated epididymal rat sperm were incubated in buffer containing either 1 × 10⁵ M ouabain, which will inhibit only the 4 isoform, or 1 × 10² M ouabain, which will inhibit both the 4 and 1 isoforms. The percentage of motile sperm in each ouabain solution was counted each hour and compared with the percentage of motile sperm in buffer alone (Fig. 5). The results from these motility assays revealed that ouabain inhibition of the 4 isoform alone is sufficient to reduce sperm motility to the same level as ouabain inhibition of all of the Na,K-ATPase, whereas the motility of sperm in control buffer is constant and sometimes even increases over the time span of each experiment, up to 18 h (Fig. 5 and data not shown). Motility was assessed by scoring sperm with any flagellar movement as being motile. The residual motile sperm observed in ouabain solutions exhibited little to no forward movement and overall less activity compared with the motile sperm in control buffer, indicating that sperm movement is essentially abolished by inhibiting 4. These data clearly show the dependence of sperm motility on the 4 isoform, leading to the question of the consequences of 4 inhibition on fertilization. Interestingly, the effect of ouabain inhibition of the Na,K-ATPase on in vitro fertilization has been previously examined in the mouse, but again these results were interpreted without considering the presence of multiple  isoforms in sperm (34). In that study, acrosome-reacted spermatozoa were exposed to different concentrations of ouabain, and their ability to successfully fertilize zona-free mouse oocytes was examined (34). Compared with control studies in the absence of ouabain, the number of oocyte fertilizations by sperm exposed to low concentrations of ouabain (1 × 10⁵ M) was maximally reduced to 0-5% (34). Ongoing studies from our laboratory have defined the  isoforms of the Na,K-ATPase in mouse testis to be identical to those in rat (17).² Therefore, the inhibition of fertilization events observed at low concentrations of ouabain in the mouse can only be attributed to specific inhibition of the 4 isoform in sperm, revealing a critical role for this isoform in both sperm motility and fertilization.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Inhibition of sperm motility by low doses of ouabain. The percentage of motile sperm in TALP solution containing 0, 1 × 105, or 1 × 102 M ouabain was reported every hour and revealed a significant inhibition of motility for sperm incubated in ouabain at both concentrations. The percentage of motile sperm incubated in 1 × 102 M ouabain was never statistically less than that of sperm in 1 × 105 M ouabain, showing that inhibition of the 4 isoform alone is sufficient to eliminate sperm motility. In addition, motile sperm in ouabain solutions exhibited little to no forward movement compared with motile sperm in TALP solution alone. *, p <0.05; **, p <0.001.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

One of the major objectives of our laboratory is to identify and define specific functional roles for  isoforms of the Na,K-ATPase. Until now, a unique role has been demonstrated only for the 2 isoform as a Ca²⁺ regulator in cardiac myocytes (17). The data presented here constitute the first description of a biological function for the most recently characterized Na,K-ATPase  isoform, 4. Expression of the 4 isoform has now been identified in spermatozoa, specifically in the mid-piece of the flagellum, suggesting a functional role related to the specialized activity of these cells. In fact, this novel Na,K-ATPase isoform does play a critical role in sperm function since selective inhibition of the 4 isoform alone is sufficient to eliminate sperm motility, providing new perspectives in the studies of both biological functions of Na,K-ATPase  isoforms and general mechanisms of sperm motility.

Sperm motility is dependent on a number of different parameters, one of which is the cytosolic pH (35, 36). Environmental conditions that inhibit sperm motility such as the absence of Na⁺ also decrease the intracellular pH, resulting in a more acidic cytoplasm in these immobile sperm than in mobile sperm (35, 36). The reinitiation of motility of these sperm, by resuspension in Na⁺-containing medium, is immediately preceded by their release of H⁺ (35). In fact, the addition of NH₄Cl (35) or bicarbonate (36) alone to the external medium, both of which stimulate H⁺ release, is also sufficient to induce sperm motility. These findings highlight the importance of H⁺ extrusion and the regulation of intracellular pH for the initiation and maintenance of sperm motility.

The Na⁺/H⁺ exchangers are a family of proteins involved in intracellular pH regulation in many epithelial tissues (37), and recently, the NHE-1 (Na⁺/H⁺ exchanger-1) protein has been detected in porcine spermatozoa (36). The Na,K-ATPase establishes the Na⁺ gradient across the membrane that the Na⁺/H⁺ exchanger uses to remove H⁺ from the cell (37). The functional role of the Na⁺/H⁺ exchanger in regulating the internal pH of sperm has been investigated using ouabain (35), a specific inhibitor of the Na,K-ATPase; amiloride (35), an inhibitor of the Na⁺/H⁺ exchanger; and the amiloride analog 5-(N-ethyl-N-isopropyl) amiloride (36). These drugs were shown to inhibit acid release, internal pH recovery, and motility initiation and, together with the identification of NHE-1 in sperm, suggest that proper functioning of the Na⁺/H⁺ exchanger is essential for regulating intracellular pH, and therefore motility of sperm (35, 36). Specific inhibition of the Na,K-ATPase carrying the 4 isoform likely induces intracellular acidification of sperm by eliminating Na⁺ gradients necessary for the Na⁺/H⁺ exchanger to remove excess H⁺, resulting in the loss of motility (Fig. 6). The localization of the Na,K-ATPase to the mid-piece region of the sperm where the mitochondria are found (38) is therefore not surprising since sperm mitochondria are responsible for producing the ATP necessary for flagellar movement, and during this metabolic activity, large amounts of H⁺ leak from the mitochondrial inner membrane space into the cytoplasm (39). In the sperm mid-piece, the mitochondria lie directly below and possibly in contact with the plasma membrane (38), providing the likelihood for the existence of a restricted-volume space in these cells into which H⁺ leaks from the mitochondria. The presence of a unique isoform of the Na,K-ATPase, working in concert with the Na⁺/H⁺ exchanger in the mid-piece, would thereby provide a mechanism for the tight control of H⁺ concentration in this region of the sperm, allowing for normal sperm motility (Fig. 6).

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Model for the functional role of the Na,K-ATPase 4 isoform in sperm motility. The H+ leak from the mitochondria during chemiosmotic synthesis of ATP is regulated through the enzymatic activity of the Na,K-ATPase working in concert with the Na+/H+ exchanger (NHE).

The acidic internal environment in sperm may directly affect flagellar movement through inhibition of dynein activity. Structurally, the sperm tail is a flagellum, a cellular component composed of microtubules whose movement is powered by the ATPase motor, dynein (30). In flagella, the outer and inner dynein arms are the axonemal structures involved in the production of the stroke necessary for the sliding of adjacent microtubules (40). Recently, one study demonstrated that sperm lacking outer dynein arms do not increase motility in response to an increase in pH, which stimulates normal sperm, but produce a low level of constant motility at an acidic pH where normal sperm are relatively inactive. These data suggest that the outer dynein arms contain a pH-sensitive regulatory mechanism (40). Therefore, in an acidic environment, dynein outer arms may inhibit flagellar activity, whereas in more alkaline conditions, dynein outer arms activate flagellar activity and motility.

The mechanism of reducing sperm motility by specifically inhibiting the 4 isoform may also involve disruption of the plasma membrane potential. The plasma membrane potential of a spermatozoon undergoes many changes throughout maturation that are critical for its ability to fertilize the ovum; therefore, precise regulation of the plasma membrane potential is necessary for normal sperm function (41). Previous studies using bull sperm have shown that ouabain decreases the progressive motility and flagellar wave of sperm and that the membrane potential of these sperm is dramatically more positive, providing a connection between the regulation of membrane potential and sperm motility (42). The contribution of individual Na,K-ATPase  isoforms to this phenomenon was not considered. The inhibition of sperm motility by the loss of the 4 isoform alone may therefore be a result of disturbing the regulation of the membrane potential.

Interestingly, a recent study of immotile sperm collected from asthenozoospermic, infertile humans demonstrated that they exhibit both decreased motility and dramatically less negative plasma membrane potentials compared with sperm from healthy, normal, fertile men (41). Again, the contribution of individual Na,K-ATPase  isoforms was not considered, but future studies of these infertile patients may reveal the absence of, or dysfunctional, Na,K-ATPase carrying the 4 isoform. Once this has been established, agents that increase Na,K-ATPase activity or gene therapy techniques that introduce a functional 4 isoform gene into sperm could be used to restore motility and fertility in these patients.

Another application suggested by the data presented here on the 4 isoform of the Na,K-ATPase involves the design of unique pharmacological agents that exclusively inhibit the 4 isoform for use as male birth control agents. The protein sequence of the 4 isoform is the least similar of the four  isoforms of the Na,K-ATPase (13), which should facilitate the design of specific inhibitors. In addition, the limited expression pattern of the 4 isoform suggests that patients treated with these inhibitors would see effects only on sperm without consequence to other organ functions.

The localization of the 4 isoform in sperm cells and the identification of its critical role in sperm motility and fertilization now necessitate further study of its contribution to other sperm-specific biochemical processes, including capacitation and the acrosome reaction (43-46). These studies will provide a better understanding of the spectrum of biological functions connected to this novel isoform of the Na,K-ATPase and the potential use of this protein as a specific target for male fertility/contraception treatments.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants RO1HL28573 and PO1HL41496 (to J. B L.) and Training Grant T32HL07382 (to A. L. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence and reprint requests should be addressed: Dept. of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, 231 Bethesda Ave., Cincinnati, OH 45267-0524. E-mail: Jerry.Lingrel@uc.edu.

Published, JBC Papers in Press, April 10, 2000, DOI 10.1074/jbc.M002323200

² A. L. Woo, P. F. James, and J. B Lingrel, manuscript in preparation.

	ABBREVIATIONS

The abbreviation used is: PBS, phosphate-buffered saline.

	REFERENCES

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