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BOR - Papers in Press, published online ahead of print January 26, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.038448
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BIOLOGY OF REPRODUCTION 72, 1275–1281 (2005)
DOI: 10.1095/biolreprod.104.038448
© 2005 by the Society for the Study of Reproduction, Inc.

The Effects of Putative K+ Channel Blockers on Volume Regulation of Murine Spermatozoa1

J.P. Barfield 3,4 , C.H. Yeung 3, and T.G. Cooper 2 3

Institute of Reproductive Medicine of the University,3 D-48129 Münster, Germany Department of Biological Sciences,4 University of New Orleans, New Orleans, Louisiana 70148


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Volume regulation is a necessary task for spermatozoa as the osmolarity of female tract fluids is lower than that in the epididymis and because the disruption of it in transgenic mice results in infertility. As the specific mechanisms behind this phenomenon are unknown, spermatozoa from mice were screened for sensitivities to inhibitors known to affect specific channels involved in volume regulation of somatic cells. Spermatozoa from the cauda epididymidis were exposed to physiological hypotonic conditions with and without inhibitor. Flow cytometric forward scatter measurements were taken to indicate relative sperm size at 5 and 75 min of incubation. The presence of quinine (0.8 mM), cadmium (0.2 mM), flecainide (100 µM), 4-aminopyridine (4 mM), barium (1 mM), clofilium (10 µM), and phrixotoxin (100 nM) for 75 min resulted in significantly higher forward scatter values than sperm incubated in medium without an inhibitor. These results imply that channels potentially involved in volume regulation of murine spermatozoa include the voltage-dependent Kv1.4 (also known as KCNA1), Kv1.5 (KCNA5), Kv4.1 (KCND1), Kv4.2 (KCND2), Kv4.3 (KCND3), mink (KCNE1), and acid-sensitive TASK2 (KCNK5) and TASK3 (KCNK9). Western blots confirmed the presence of Kv1.5 and TASK2 proteins in sperm plasma membranes at similar (Kv1.5) or higher (TASK2) molecular weight than in somatic cells. Incubation in a different pH did not reveal acid sensitivity of volume regulation. Volume regulation of spermatozoa may involve novel voltage-gated and pH-sensitive potassium channels, which could be valuable targets for the development of a posttesticular male contraceptive.

epididymis, fertilization, gamete biology, male reproductive tract, sperm


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Regulation of cellular volume is vital for a wide range of physiological functions. While it has been generally accepted that spermatozoa have mechanisms for adjusting their volume at times of osmotic stress, the physiological significance of volume regulation for sperm function has only recently been appreciated. This avenue of research was stimulated by studies on c-ros (ROS1) tyrosine kinase knockout mice, which produce spermatozoa unable to regulate their volume [1]. These mice lack the initial segment of the epididymis, and their spermatozoa do not acquire the ability to regulate volume. On exposure to female tract fluids, these spermatozoa swell, form hairpins or angulate at the cytoplasmic droplet, and are unable to migrate to the site of fertilization [2]. By contrast, spermatozoa from wild-type mice under such a hypo-osmotic challenge perform regulatory volume decrease (RVD) to maintain their volume [3]. This loss of fertility in the knockout mice not only highlights the physiological significance of sperm volume regulation but also opens up the possibility of manipulating this phenomenon for the purpose of posttesticular male contraception. However, before this application can be realized, the specific mechanisms of volume regulation must be determined.

The ability of spermatozoa to undergo RVD is acquired in the epididymis [1, 4, 5]. It has been suggested that this maturation occurs through isovolumetric regulation, by which the cells gradually take up osmolytes available to them in the epididymal lumen when they encounter the increasing osmolarity generated by the secretion of osmolytes by the epididymis [6]. This can be a lengthy process taking up to 5 days in the mouse [7]. In this situation, changes in cell volume would be undetectable, yet there would be a continual increase in intracellular concentrations of osmolytes. The osmolytes acquired in the epididymis could then be used in the subsequent volume regulation required by the spermatozoa during their transition from the epididymis to the site of fertilization, as the difference in osmolarity of the male tract is approximately 80 mmol/kg higher than that measured in the murine female tract [1, 2].

Although the nature of the osmolytes is becoming clearer [8], more thorough knowledge of volume regulation has been established for somatic cells. The typical response of a cell to swelling is the release of osmolytes, which in somatic cells [9] and porcine spermatozoa [10] most often involves separate K+ and anion channels, although tissue-specific differences exist [9]. The role of K+ in volume regulation of various somatic cells has been widely confirmed [11]. Several studies have found high concentrations of intracellular K+ in spermatozoa (bulls [12], mice [13, 14]), and it is known that concentrations of K+ increase in luminal fluid along the length of the epididymis, giving spermatozoa the opportunity to accumulate this osmolyte [15].

Judged by the significant role of potassium in volume regulation by somatic cells and the high amounts of K+ in spermatozoa, a role for K+ in regulatory volume decrease of sperm cells is likely. Studies in mice [1], bulls [16, 17], boars [17], humans [18, 19], dogs [20], and monkeys [5] have demonstrated a sustained increase in sperm size when exposed to hypotonic media in the presence of quinine, a broad-spectrum K+ channel blocker. The reversal of the effects of quinine by the K+ ionophore valinomycin demonstrated in bovine [16, 17] and human [18] spermatozoa further supports the involvement of K+.

It is common practice in electrophysiology to characterize channels by their pharmacological sensitivities. The channels possibly involved in the volume regulatory response can be narrowed down by systematically testing the cell's response to various compounds known to block specific channels [21, 22]. This strategy has been employed to determine the channels involved in volume regulation of somatic cells [23]. In an effort to understand volume regulation of spermatozoa better, in this study the effects of specific and nonspecific channel inhibitors, as well as the effects of extracellular pH, on the ability of murine sperm to regulate their volume were investigated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Incubation Media and Inhibitors

All spermatozoa were incubated in warmed (37°C) Biggers-Whittingham-Whitten medium [24] containing 20 mM Hepes and 4 mg/ml bovine serum albumin (BSA) at pH 7.4. All media were adjusted to an osmolality of 330 mmol/kg (BWW330), the osmolarity of postcopulatory uterine contents [2], as measured by a vapor pressure osmometer (Wescor Vapro model 5520; Kreienbaum Messsysteme, Langenfeld, Germany) by addition of deionized water or 1M NaCl. The osmometer was calibrated each day using a 290-mmol/kg standard supplied by the manufacturer. Inhibitors and the concentrations tested are listed in Table 1. All chemicals were from Sigma (Taufkirchen, Germany) except for clofilium tosylate (Alexis Biochemicals, Grünberg, Germany) and phrixotoxin (Alomone Labs, Jerusalem, Israel).


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  		TABLE 1. Characteristics of channel blockers used

Animals

All 33 mice used in this study were at least 70 days old, were of the C57BL6 strain (Charles River, Sulzfeld, Germany), and were used for the collection of epididymal spermatozoa. Animals were killed by asphyxiation with CO2 followed by cervical dislocation. The experiments using these animals were conducted according to the German Federal Law on the Care and Use of Laboratory Animals (license no. G67/01).

Collection of Epididymal Spermatozoa

The testis-epididymis complex was dissected out and the cauda epididymidis isolated. The capsule of the cauda epididymidis was gently torn open with fine forceps to expose the tubule. The tubule was then carefully uncoiled so that the entire length of the mid- and distal cauda tubule was exposed. Beginning from the distal end, sections of the tubule (about 10) were cut out and placed in separate 10-µl drops of BWW medium ± inhibitor on a plastic spatula. The order of inhibitors tested during the experiment was rotated to ensure that the location of the spermatozoa within the cauda did not confound the results. The spermatozoa were then gently teased from the tubule, and the tubule was removed from the drop. The drop was then dispersed in 200 µl of the same medium, and the sperm suspensions were placed in an incubator (37°C, 5% [v/v] CO2 in air). After approximately 2 min, sperm suspensions were gently agitated to ensure dispersal of the spermatozoa and the incubation continued for 5–75 min.

Measurement of Cell Size by Flow Cytometry

Changes in sperm cell volume were measured in a flow cytometer (Coulter Epics XL, version 3.0, Krefeld, Germany) according to the method established by Yeung et al. [3]. After 5 and 75 min of incubation, approximately 80 µl sperm suspension (approximately 2–10 x 106 sperm/ ml) were added to 200 µl of the same medium lacking BSA and containing 3 µl of propidium iodide (PI: 6 µg/ml). On gentle agitation to mix the sample, flow cytometric measurements were recorded immediately. Forward and side scatter signals were recorded under laser excitation at 488 nm. Data were collected from 5000 particles excluding cellular debris gated out by forward and side scatter signals and spermatozoa with ruptured membranes gated out by detection of PI fluorescence (emission 605– 635 nm). Mean values of forward side scatter of the PI-negative cells were used for subsequent analysis. Relative cell sizes were calculated by dividing the mean forward scatter of cells incubated in inhibitor by the mean forward scatter of cells from the same epididymis incubated without inhibitor at each time point. The same flow cytometer and settings were maintained throughout the study.

Effects of pH on Volume Regulation and Action of Inhibitors

To determine the effect of pH on the volume regulation, spermatozoa were incubated in BWW330 with pH 6.3, 7.4, and 8.4. BWW330 pH 7.4 was prepared as described previously. For BWW330 pH 6.3, Hepes was replaced by 20 mM Mopso, and BWW330 pH 8.4 was buffered with either 20 mM Tris or 20 mM Hepes. All media were adjusted to the desired pH with HCl or NaOH.

Western Blotting

For membrane protein extraction, thawed pellets from 6 million spermatozoa were taken up in 50 µl lysis buffer (125 mM NaCl, 25 mM Hepes, 10 mM EDTA, 10 mM Na-pyrophosphate, 10 mM NaF, 0.1% [w/ v] SDS, 0.5% [w/v] deoxycholate, 1% [v/v] Triton X-100 at pH 7.3) containing 10 µl protease inhibitor cocktail (Sigma) and phosphatase inhibitor 1 mM Na3VO4 and intensely vortexed for 3 min. The samples were kept on ice for 1 h with frequent vortexing. The suspension was centrifuged for 20 min at 20 000 x g at 4°C (Heraeus Biofuge Stratus; Kendro Lab Products, Langendselbold, Germany). The supernatant membrane extract was stored at –80°C.

Polyacrylamide gel electrophoretic separation of proteins was carried out using 4–12% (w/v) NuPAGE Novex Bis-Tris precast gels (8 x 8 cm, 1 mm thick; NuPage, Invitrogen, Carlsbad, CA) according to the manufacturer's instructions, with 50 µg protein sample in each lane after heating at 65°C for 10 min in the absence of DTT. Separated proteins were transferred onto ECL-Hybond membranes (Hybond ECL; Amersham Pharmacia, Uppsala, Sweden) using 150 mA at 35 V for 3 h and stained with Ponceau to check protein loading and visualize the molecular weight markers. After blocking with StartingBlock (Pierce, Perbio Science, Bonn, Germany) for 1 h at room temperature, the membrane was incubated with primary affinity-purified rabbit antibodies against Kv1.4, Kv1.5, Kv4.2, Kv4.3, TASK2, and TASK3 diluted 1:2500 (Alomone Labs, Jerusalem, Israel) followed by a secondary goat anti-rabbit horseradish peroxidase-conjugated antibody diluted 1:250 000 (Pierce). Antigen-adsorbed primary antibody was prepared by adding 10 µg to the control peptide antigen (10 µg: TASK2, TASK3, Kv4.3, Kv4.2; 30 µg Kv1.5, Kv1.4) and incubated overnight at 4°C on a rotating plate. The remaining primary antibody was also incubated overnight at 4°C on the same rotating plate. Aliquots of the resulting adsorbed solution and primary antibody were frozen and kept at –20°C until exposed to blotted membranes. Peroxidase-bound protein bands were visualized using the ECL-Plus method (Femto-signal; Pierce). The molecular weights of the signal bands were analyzed using the line densitometer software (ChemImage System, IS4400; Alpha Innotech Corp., San Laendro, CA).

Statistics

All comparisons were made using one-way repeated measures ANOVA followed by the Dunnett post hoc test against the control. In the event that the data failed the normality or equal variance test, one-way ANOVA on ranks was used followed by the Dunnett or Dunn post hoc test against the control, respectively. Values were considered significantly different with P < 0.05. Graphs indicate the mean + SEM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
As spermatozoa do not respond to quinine under isotonic conditions [16, 17], cell swelling in response to quinine was taken as evidence of volume regulation under the hypotonic conditions employed. Thus, BWW330 with 0.8 mM quinine was used as the positive control based on previous studies showing that murine sperm swell when exposed simultaneously to quinine and a hypo-osmotic challenge [3]. Spermatozoa from all mice used in these experiments were undergoing volume regulation in the inhibitor-free hypotonic medium as indicated by the statistically significant increase in their forward scatter in the presence of quinine (at 5 min, mean difference in channel number from the control ± SEM, 109.9 ± 3; at 75 min, mean 85.2 ± 4.75).

Measurement of Cell Size by Flow Cytometry

After 5 min incubation, two inhibitors caused a significant difference in forward scatter. Spermatozoa incubated in quinine (0.8 mM) and 4AP (4 mM) were significantly larger than the control (Fig. 1). Preliminary time course experiments indicated that the maximum difference in size between control and quinine-treated spermatozoa occurred after 75 min (data not shown). Thus, the secondary incubation time point of 75 min was chosen to maximize the sensitivity of the screening test despite possible confounding effects of capacitation [16, 25]. This different time course from that obtained before [3] may reflect the addition of Hepes to the medium to confer greater pH control of samples held outside the incubator.



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  		FIG. 1. The effect of channel inhibitors on the size (expressed as ratio of non-drug-treated control value, mean + SEM, ordinate) of murine epididymal spermatozoa incubated for 5 min in quinine (QUI), 4-aminopyridine (4AP), flecainide (FLE), cadmium (Cd), barium (Ba), clofilium (CLO), phrixotoxin (PTX), threo-ß-hydroxyaspartate (HOA), gadolinium (Gd), margatoxin (MTX), charybdotoxin (CTX), apamin (AP), phlorizin (PHZ), glybenclamide (GlY), and tetraethylammonium (TEA). Concentrations given on abscissa. N = 56 for quinine, 4–10 for other inhibitors. *, significantly different from inhibitor-free control (P < 0.05)

Several inhibitors induced swelling of murine spermatozoa at 75 min, including cadmium (0.2 mM), flecainide (100 µM), 4-aminopyridine (4 mM), barium (1 mM), clofilium (10 µM), phrixotoxin (100 nM), and quinine (0.8 mM). Spermatozoa incubated in phlorizin (500 µM) produced significantly lower forward scatter values than those of the control (Fig. 2), whereas other inhibitors showed no effect.



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  		FIG. 2. The effect of channel inhibitors on the size (expressed as ratio of non-drug-treated control, mean + SEM, ordinate) of murine epididymal spermatozoa incubated for 75 min. See Figure 1 for abbreviations of inhibitors. Concentrations given on abscissa. N = 56 for quinine, 4–10 for other inhibitors. *, significantly different from inhibitor-free control (P < 0.05)

Cell Viability

At 5 min, there were no significant differences in the percentage of membrane-intact sperm between any of the inhibitors tested as indicated by PI staining. Only flecainide (100 µM) and cadmium (0.2 mM) had significantly lower percentages of PI-negative cells after 75 min incubation (control, 63.5% ± 1.8; flecainide, 45.5% ± 3.6; cadmium, 48.0% ± 4.0).

Effects of pH on Volume Regulation

After 5 min incubation, spermatozoa incubated in drug-free BWW330 at pH 6.3 and pH 8.4 in Hepes buffer were the same size as those at pH 7.4, but those incubated at pH 8.4 in Tris were significantly larger. The same phenomenon was found after 75 min incubation (Table 2). The viability of spermatozoa was not significantly different at different pH values at either time point.


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  		TABLE 2. Relative volumes of murine spermatozoa at 330 mmol/kg at different pH (expressed as ratio of pH 7.4 control) after 5 and 75 min incubation (values are mean ± SEM)

Western Blotting

Two specific bands detected by an antibody against Kv1.5 protein were visible and estimated to be of size 84.8 and 76.4 kDa but were not present when the antibody was adsorbed with antigen. Incubation of sperm membrane proteins with a TASK2 antibody also yielded two specific bands at 113 and 99 kDa that were not present on membranes incubated with the preadsorbed antibody (Fig. 3). No specific bands were observed when membranes were incubated with antibodies against Kv1.4, Kv4.2, Kv4.3, and TASK3 (data not shown).



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  		FIG. 3. Identification of Kv1.5 and TASK2 channels in mouse epididymal spermatozoa by Western blotting. Lanes 1 and 2 were incubated with anti-Kv1.5, lanes 3 and 4 with adsorbed anti-Kv1.5 (preincubated with the control peptide antigen), lanes 5 and 6 with anti-TASK2, and lanes 7 and 8 with adsorbed anti-TASK2. Specific bands are indicated by arrows and the determined molecular weight (kDa)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Volume regulation is an extremely important process for spermatozoa that, if disrupted, leads to infertility. The mechanisms underlying volume regulatory responses of sperm from various species are just beginning to be revealed. The goal of these experiments was to characterize the channels, in particular the K+ channels, involved in the volume regulation of murine spermatozoa. Candidate channels were identified by comparing responses of spermatozoa to concentrations of channel blockers that prevent volume regulation of somatic cells. The potential channels identified here on murine spermatozoa have pharmacological sensitivities that correspond to those found in somatic cells.

Four-aminopyridine (4AP) is a quaternary ammonium blocker routinely used in electrophysiological studies to identify voltage-gated (Kv) channels [26]. The significant increase in sperm size after 5 and 75 min incubation in hypotonic medium with 4AP suggests that voltage-gated potassium channels are involved in volume regulation. The insignificant results from incubation in TEA, charybdotoxin, and margatoxin excluded the involvement of the majority of Kv channels except Kv1.4 and Kv1.5 (see Table 1). Kv1.5 has previously been implicated in volume regulation of electrically unexcitable cells [27], but there is currently no evidence for the involvement of Kv1.4 in volume regulation of other cell types. Recently, a high-conductance, 4AP-sensitive channel has been identified in human spermatozoa using patch clamping [28]. In addition, 4AP also inhibits volume regulation of human [18] and cynomolgus monkey spermatozoa [5].

The sensitivity of murine spermatozoa to flecainide, cadmium, phrixotoxin, and 4AP suggests that the Kv4 potassium channels may have a role in volume regulation of murine spermatozoa. These channels (Kv4.1, Kv4.2, and Kv4.3) are involved in A-type currents (rapidly inactivating, 4AP-sensitive, K+ currents [29]). Murine colonic myocytes contain all three Kv4 channels, but Kv4.3 transcripts are relatively more abundant and extremely sensitive to micromolar concentrations of flecainide [29]. COS7 cells transfected with wild-type Kv4.2 channels show a concentration-dependent inhibition by flecainide [30]. When transfected into HEK293 cells, Kv4.3 channels, which mediate the transient outward potassium current in the human heart, are inhibited by cadmium chloride with an EC50 of 0.110 µM [31]. Phrixotoxins specifically inhibit Kv4.2 and Kv4.3 by altering the gating properties [32]. Collectively, these results suggest the involvement of Kv4.1, Kv4.2, and Kv4.3 in volume regulation by murine spermatozoa.

Spermatozoa were significantly larger when incubated in hypotonic medium containing clofilium, another quaternary ammonium derivative, and barium chloride. The family of potassium channels known to have two pore regions and four transmembrane helices (2P-4TM) are blocked by clofilium. These proteins are thought to channel the leak or background conductances that maintain the passive properties of the cell [33], including volume regulation. The TWIK-related, acid-sensitive potassium channel 2 (TASK2) in Ehrlich cells is blocked by clofilium with an IC50 of 25 µM and may participate in volume regulation of these cells [33]. The 2P-4TM channels are also characterized by an insensitivity or low sensitivity to a range of conventional K+ channel blockers such as 4AP, TEA, and barium chloride [33]. However, barium was found to inhibit TASK3 transfected into COS-7 cells [34].

The present results also suggest channels that are unlikely to be involved in volume regulatory responses of murine sperm. That glybenclamide did not affect volume regulation argues against inwardly rectifying channels Kir6.1 (KCNJ8) or Kir6.2 (KCNJ11) being involved. By similar reasoning the Ca2+-activated K+ channels Slo1 (KCNMA1) and SK4 (KCNN2) (CTX-sensitive) and SK2 (KCNN2) and SK3 (KCNN3) (apamin-sensitive) are unlikely to mediate sperm volume regulation [21, 35]. Of the Kv channels, Kv1.2 (KCNA2) and Kv1.3 (KCNA3) are inhibited by CTX; Kv1.3, Kv1.6 (KCNA6) and Kv1.7 (KCNA7) are inhibited by MTX and Kv1.1 (KCNA1), Kv1.2 (KCNA2), Kv1.3 (KCNA3), Kv1.6 (KCNA6), Kv2.1 (KCNB1), Kv2.2 (KCNB2), Kv3.1–3.4 (KCNC1-4), Kv7.1 (KCNQ1), Kv7.2 (KCNQ2), Kv7.4 (KCNQ4), EAG1 (KCNH1), EAG2 (KCNH5), ERG1 (KCNH2), ERG2 (KCNH6), and ERG3 (KCNH7) are inhibited by TEA [21, 22] thus ruling out their operation in sperm volume regulation as the process was not affected by these inhibitors.

Phlorizin, 4-aminobutyrate, and threo-ß-hydroxyaspartate are inhibitors of channels used by organic osmolytes (myo-inositol [36], carnitine [37], and glutamate [38], respectively). Judging by lack of swelling by incubation in these inhibitors, there was no indication of significant involvement of such channels in the volume regulation of spermatozoa at this osmolality during this time period. Other inhibitors are known to block channels used by organic osmolytes that may inhibit volume regulation of sperm cells. Phlorizin is a sodium-dependent hexose transport inhibitor that blocks myo-inositol transport in murine oocytes [36]. However, this nonspecific blocker also inhibits water transport through the sodium-glucose cotransporter (SGLT1) expressed in Xenopus laevis [39] and fluid absorption by renal proximal tubules of the teleost Carassius auratus [40]. Thus, the finding that murine spermatozoa incubated in phlorizin were significantly smaller than the control may be due to the inability of water to enter the treated cell.

Western blotting of proteins from several potential osmolyte channels (Kv1.5, Kv1.4, Kv4.2, Kv4.3, TASK2, and TASK3) revealed specific bands for Kv1.5 and TASK2. The specific bands found for Kv1.5 (85 and 76 kDa) were similar but not identical to those reported for Schwann cells (90 and 65 kDa [41]) and rat atrium (75 and 60 kDa [42]). The TASK2-specific bands at 113 and 99 kDa were different from the reported value of 45 kDa found in rat kidney by the antibody distributor (Alomone Labs). The differences in the values found here and those reported in the literature may suggest the presence of sperm-specific isoforms of these channels on murine spermatozoa.

Results from the pH experiments indicate that regulation of volume may be influenced by the extracellular pH of the cell although not in the manner expected. Clofilium-sensitive TASK2 seems to be involved in volume regulation of murine spermatozoa as indicated by the channel inhibitor experiments. When expressed in Ehrlich ascites tumor cells, the conductance of K+ through this acid-sensitive channel is strongly inhibited at an acidic pH (6.3) and enhanced at an alkaline pH (8.4) [33]. The voltage-gated K+ channel Kv1.5, which is also implicated in volume regulation of murine sperm cells in these experiments, is similarly sensitive to changes in pH. Kv1.5 channels expressed in Xenopus oocytes are blocked at pH 6.3 and insensitive to alkaline pH [43]. One would expect that by incubating a cell in a hypotonic medium of alkaline pH, the return to normal volume would be faster than a cell incubated in a hypotonic medium of an acidic pH as reported for Ehrlich cells [44]. However, in the current experiments, only spermatozoa in Tris-buffered medium of an alkaline pH were significantly larger than the control (BWW330 pH 7.4) rather than sperm incubated in acidic pH. Because spermatozoa exposed to an alkaline medium buffered with Hepes (although not an optimal buffer at this pH range) were not significantly different than the control, it cannot be excluded that this increase in volume is an effect of the buffer reagent rather than the pH. On the other hand, this variation in response to pH and the differences found for the size of TASK2 proteins in these experiments compared to those previously reported may be indicating that an unknown acid-insensitive channel exists in the murine sperm membrane that is inhibited by Tris.

In conclusion, several potassium channels seem to be involved in volume regulation of murine spermatozoa as judged from the effects of specific channel blockers on this process. Quinine is a broad-spectrum K+ channel inhibitor, and 4AP affects members of the voltage-dependent (Kv) family [22]. After excluding K+ channels not likely to be involved in volume regulation, Kv1.4, Kv1.5, Kv4.2, and Kv4.3 remain as putative channels, suggestions consistent with the inhibitory effects of phrixotoxin (which inhibits Kv4.2 and Kv4.3), flecainide (inhibits Kv4.2 and Kv4.3), and Cd2+ (inhibits Kv4.3). The sensitivity to clofilium also suggests an involvement of the TASK2 and mink channels. Western blots confirmed the presence of Kv1.5 and TASK2 protein in sperm membranes but did not provide evidence for Kv4.2, Kv4.3, or Kv1.4. These channels, which may have overlapping ranges of activation, could work in concert to produce successful volume regulation. While K+ channels appear to have a prominent role in volume regulation of murine spermatozoa, it is possible that other channels are also involved. Future research should determine if various anion and organic osmolyte channels contribute to the volume regulatory responses of murine spermatozoa. Targeting these channels may have contraceptive potential by preventing volume regulation of sperm in the female tract and preventing their migration to the site of fertilization.


   FOOTNOTES
 
1 J.P.B. supported by a Crescent City doctoral scholarship from the University of New Orleans. The research was supported by the Schering Research Foundation—CONRAD AMPPAII Research Network. Back

2 Correspondence: T.G. Cooper, Institute of Reproductive Medicine of the University, Domagkstrasse 11, D-48129 Münster, Germany. FAX: 49 251 835 6093; TrevorG.Cooper{at}ukmuenster.de Back

Received: 24 November 2004.

First decision: 15 December 2004.

Accepted: 18 January 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Yeung CH, Sonnenberg-Riethmacher E, Cooper TG. Infertile spermatozoa of c-ros tyrosine kinase receptor knockout mice show flagellar angulation and maturational defects in cell volume regulatory mechanisms. Biol Reprod 1999 61:1062-1069[Abstract/Free Full Text]
  2. Yeung CH, Wagenfeld A, Nieschlag E, Cooper TG. The cause of infertility of male c-ros tyrosine kinase receptor knockout mice. Biol Reprod 2000 63:612-618[Abstract/Free Full Text]
  3. Yeung CH, Anapolski M, Cooper TG. Measurement of volume changes in mouse spermatozoa using an electronic sizing analyzer and a flow cytometer: validation and application to an infertile mouse model. J Androl 2002 23:522-528[Abstract/Free Full Text]
  4. Yeung CH, Anapolski M, Sipila P, Wagenfeld A, Poutanen M, Huhtaniemi I, Nieschlag E, Cooper TG. Sperm volume regulation: maturational changes in fertile and infertile transgenic mice and association with kinematics and tail angulation. Biol Reprod 2002 67:269-275[Abstract/Free Full Text]
  5. Yeung CH, Barfield JP, Anapolski M, Cooper TG. Volume regulation of mature and immature spermatozoa in a primate model, and possible ion channels involved. Hum Reprod 2004 19:2587-2593[Abstract/Free Full Text]
  6. Cooper TG, Yeung CH. Acquisition of volume regulatory response of sperm upon maturation in the epididymis and the role of the cytoplasmic droplet. Microsc Res Tech 2003 61:28-38[CrossRef][Medline]
  7. Dadoune JP, Alfonsi MF. Autoradiographic investigation of sperm transit through the male mouse genital tract after tritiated thymidine incorporation. Reprod Nutr Dev 1984 24:927-935
  8. Yeung CH, Anapolski M, Setiawan I, Lang F, Cooper TG. Effects of putative epididymal osmolytes on sperm volume regulation of fertile and infertile c-ros transgenic mice. J Androl 2004 25:216-223[Abstract/Free Full Text]
  9. Lang F, Busch GL, Ritter M, Volkl H, Waldegger S, Gulbins E, Haussinger D. Functional significance of cell volume regulatory mechanisms. Physiol Rev 1998 78:247-306[Abstract/Free Full Text]
  10. Petrunkina AM, Harrison RAP, Ekhlasi-Hundrieser M, Töpfer-Petersen E. Role of volume-stimulated osmolyte and anion channels in volume regulation by mammalian sperm. Mol Hum Reprod 2004 10:815-823[Abstract/Free Full Text]
  11. Hoffmann EK, Dunham PB. Membrane mechanisms and intracellular signalling in cell volume regulation. Int Rev Cytol 1995 161:173-262[Medline]
  12. Babcock DF. Examination of the intracellular ionic environment and of ionophore action by null point measurements employing the fluorescein chromophore. J Biol Chem 1983 258:6380-6389[Abstract/Free Full Text]
  13. Chou K, Chen J, Yuan SX, Haug A. The membrane potential changes polarity during capacitation of murine epididymal sperm. Biochem Biophys Res Commun 1989 165:58-64[CrossRef][Medline]
  14. Zeng Y, Clark EN, Florman HM. Sperm membrane potential: hyperpolarization during capacitation regulates zona pellucida-dependent acrosomal secretion. Dev Biol 1995 171:554-563[CrossRef][Medline]
  15. Turner TT. Necessity's potion: inorganic ions and small organic molecules in the epididymal lumen. In: Robaire B, Hinton BT (eds.), The Epididymis: From Molecules to Clinical Practice: A Comprehensive Survey of the Efferent Ducts, the Epididymis and the Vas Deferens. New York: Kluwer Academic Press/Plenum Press; 2002:131–150
  16. Kulkarni SB, Sauna ZE, Somlata V, Sitaramam V. Volume regulation of spermatozoa by quinine-sensitive channels. Mol Reprod Dev 1997 46:535-550[CrossRef][Medline]
  17. Petrunkina AM, Harrison RA, Hebel M, Weitze KF, Töpfer-Petersen E. Role of quinine-sensitive ion channels in volume regulation in boar and bull spermatozoa. Reproduction 2001 122:327-336[Abstract]
  18. Yeung CH, Cooper TG. Effects of the ion-channel blocker quinine on human sperm volume, kinematics and mucus penetration, and the involvement of potassium channels. Mol Hum Reprod 2001 7:819-828[Abstract/Free Full Text]
  19. Yeung CH, Anapolski M, Depenbusch M, Zitzmann M, Cooper TG. Human sperm volume regulation: response to physiological changes in osmolality, channel blockers and potential sperm osmolytes. Hum Reprod 2003 18:1029-1036[Abstract/Free Full Text]
  20. Petrunkina AM, Radcke S, Gunzel-Apel AR, Harrison RA, Töpfer-Petersen E. Role of potassium channels, the sodium-potassium pump and the cytoskeleton in the control of dog sperm volume. Theriogenology 2004 61:35-54[CrossRef][Medline]
  21. Coetzee WA, Amarillo Y, Chiu J, Chow A, Lau D, McCormack T, Moreno H, Nadal MS, Ozaita A, Pountney D, Saganich M, Vega-Saenz de Miera E, Rudy B. Molecular diversity of K+ channels. Ann NY Acad Sci 1999 868:233-285[CrossRef][Medline]
  22. Wehner F, Olsen H, Tinel H, Kinne-Saffran E, Kinne RK. Cell volume regulation: osmolytes, osmolyte transport, and signal transduction. Rev Physiol Biochem Pharmacol 2003 148:1-80[Medline]
  23. Cho WK. Characterization of regulatory volume decrease in freshly isolated mouse cholangiocytes. Am J Physiol Gastrointest Liver Physiol 2002 283:G1320-G1327[Abstract/Free Full Text]
  24. Biggers JD, Whittem WK, Whittingham DG. The culture of mouse embryos in vitro. In: Daniel JC Jr (ed.), Methods in Mammalian Embryology. San Francisco: Freeman; 1971:86–116
  25. Petrunkina AM, Hebel M, Waberski D, Weitze KF, Töpfer-Petersen E. Requirement for an intact cytoskeleton for volume regulation in boar spermatozoa. Reproduction 2004 127:105-115[Abstract/Free Full Text]
  26. Mathie A, Wooltorton JR, Watkins CS. Voltage-activated potassium channels in mammalian neurons and their block by novel pharmacological agents. Gen Pharmacol 1998 30:13-24[Medline]
  27. Felipe A, Snyders DJ, Deal KK, Tamkun MM. Influence of cloned voltage-gated K+ channel expression on alanine transport, Rb+ uptake, and cell volume. Am J Physiol 1993 265:C1230-C1238
  28. Gu Y, Kirkman-Brown JC, Korchev Y, Barratt CL, Publicover SJ. Multi-state, 4-aminopyridine-sensitive ion channels in human spermatozoa. Dev Biol 2004 274:308-317[CrossRef][Medline]
  29. Amberg GC, Koh SD, Hatton WJ, Murray KJ, Monaghan K, Horowitz B, Sanders KM. Contribution of Kv4 channels toward the A-type potassium current in murine colonic myocytes. J Physiol 2002 544:403-415[Abstract/Free Full Text]
  30. Caballero R, Pourrier M, Schram G, Delpon E, Tamargo J, Nattel S. Effects of flecainide and quinidine on Kv4.2 currents: voltage dependence and role of S6 valines. Br J Pharmacol 2003 138:1475-1484[CrossRef][Medline]
  31. Calmels TP, Faivre JF, Cheval B, Javre JL, Rouanet S, Bril A. hKv4.3 channel characterization and regulation by calcium channel antagonists. Biochem Biophys Res Commun 2001 281:452-460[CrossRef][Medline]
  32. Diochot S, Drici MD, Moinier D, Fink M, Lazdunski M. Effects of phrixotoxins on the Kv4 family of potassium channels and implications for the role of Ito1 in cardiac electrogenesis. Br J Pharmacol 1999 126:251-263[CrossRef][Medline]
  33. Niemeyer MI, Cid LP, Barros LF, Sepulveda FV. Modulation of the two-pore domain acid-sensitive K+ channel TASK-2 (KCNK5) by changes in cell volume. J Biol Chem 2001 276:43166-43174[Abstract/Free Full Text]
  34. Kim Y, Bang H, Kim D. TASK-3, a new member of the tandem pore K+ channel family. J Biol Chem 2000 275:9340-9347[Abstract/Free Full Text]
  35. Vergara C, Latorre R, Marrion NV, Adelman JP. Calcium-activated potassium channels. Curr Opin Neurobiol 1998 8:321-329[CrossRef][Medline]
  36. Higgins BD, Kane MT. Inositol transport in mouse oocytes and preimplantation embryos: effects of mouse strain, embryo stage, sodium and the hexose transport inhibitor, phloridzin. Reproduction 2003 125:111-118[Abstract]
  37. Kido Y, Tamai I, Ohnari A, Sai Y, Kagami T, Nezu J, Nikaido H, Hashimoto N, Asano M, Tsuji A. Functional relevance of carnitine transporter OCTN2 to brain distribution of L-carnitine and acetyl-L-carnitine across the blood-brain barrier. J Neurochem 2001 79:959-969[CrossRef][Medline]
  38. Chen Y, Swanson RA. The glutamate transporters EAAT2 and EAAT3 mediate cysteine uptake in cortical neuron cultures. J Neurochem 2003 84:1332-1339[CrossRef][Medline]
  39. Loo DD, Hirayama BA, Meinild AK, Chandy G, Zeuthen T, Wright EM. Passive water and ion transport by cotransporters. J Physiol 1999 518:195-202[Abstract/Free Full Text]
  40. Kanli H, Terreros DA. Transepithelial transport and cell volume control in proximal renal tubules from the teleost Carassius auratus. Acta Physiol Scand 1997 160:267-276[CrossRef][Medline]
  41. Sobko A, Peretz A, Shirihai O, Etkin S, Cherepanova V, Dagan D, Attali B. Heteromultimeric delayed-rectifier K+ channels in schwann cells: developmental expression and role in cell proliferation. J Neurosci 1998 18:10398-10408[Abstract/Free Full Text]
  42. Yamashita T, Murakawa Y, Hayami N, Fukui E, Kasaoka Y, Inoue M, Omata M. Short-term effects of rapid pacing on mRNA level of voltage-dependent K+ channels in rat atrium: electrical remodeling in paroxysmal atrial tachycardia. Circulation 2000 101:2007-2014[Abstract/Free Full Text]
  43. Steidl JV, Yool AJ. Differential sensitivity of voltage-gated potassium channels Kv1.5 and Kv1.2 to acidic pH and molecular identification of pH sensor. Mol Pharmacol 1999 55:812-820[Abstract/Free Full Text]
  44. Hougaard C, Jorgensen F, Hoffmann EK. Modulation of the volume-sensitive K+ current in Ehrlich ascites tumour cells by pH. Pflugers Arch 2001 442:622-633[CrossRef][Medline]
  45. Lock H, Valverde MA. Contribution of the IsK (MinK) potassium channel subunit to regulatory volume decrease in murine tracheal epithelial cells. J Biol Chem 2000 275:34849-34852[Abstract/Free Full Text]
  46. Garcia-Calvo M, Leonard RJ, Novick J, Stevens SP, Schmalhofer W, Kaczorowski GJ, Garcia ML. Purification, characterization, and biosynthesis of margatoxin, a component of Centruroides margaritatus venom that selectively inhibits voltage-dependent potassium channels. J Biol Chem 1993 268:18866-18874[Abstract/Free Full Text]
  47. Khanna R, Chang MC, Joiner WJ, Kaczmarek LK, Schlichter LC. hSK4/hIK1, a calmodulin-binding KCa channel in human T lymphocytes: roles in proliferation and volume regulation. J Biol Chem 1999 274:14838-14849[Abstract/Free Full Text]
  48. Deutsch C, Chen LQ. Heterologous expression of specific K+ channels in T lymphocytes: functional consequences for volume regulation. Proc Natl Acad Sci U S A 1993 90:10036-10040[Abstract/Free Full Text]
  49. Krasznai Z, Morisawa M, Krasznai ZT, Morisawa S, Inaba K, Bazsane ZK, Rubovszky B, Bodnar B, Borsos A, Marian T. Gadolinium, a mechano-sensitive channel blocker, inhibits osmosis-initiated motility of sea- and freshwater fish sperm, but does not affect human or ascidian sperm motility. Cell Motil Cytoskeleton 2003 55:232-243[CrossRef][Medline]
  50. Taouil K, Hannaert P. Evidence for the involvement of K+ channels and K+-Cl- cotransport in the regulatory volume decrease of newborn rat cardiomyocytes. Pflugers Arch 1999 439:56-66[CrossRef][Medline]
  51. Thinnes FP, Walter G, Hellmann KP, Hellmann T, Merker R, Kiafard Z, Eben-Brunnen J, Schwarzer C, Gotz H, Hilschmann N. Gadolinium as an opener of the outwardly rectifying Cl– channel (ORCC): is there relevance for cystic fibrosis therapy?. Pflugers Arch 2001 443:suppl_1S111-S116
  52. Macho P, Solis E, Sanchez G, Schwarze H, Domenech R. Mitochondrial ATP dependent potassium channels mediate non-ischemic preconditioning by tachycardia in dogs. Mol Cell Biochem 2001 216:129-136[CrossRef][Medline]



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