Mol. Hum. Reprod. Advance Access originally published online on January 18, 2006
Molecular Human Reproduction 2005 11(12):891-897; doi:10.1093/molehr/gah208
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Characterization of potassium channels involved in volume regulation of human spermatozoa
1Institute of Reproductive Medicine, University of Münster, Münster, Germany and 2Department of Biological Sciences, University of New Orleans, New Orleans, LA, USA
3 To whom correspondence should be addressed at: Institute of Reproductive Medicine, Domagkstr. 11, D-48129 Münster, Germany. E-mail: trevorg.cooper{at}ukmuenster.de
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Abstract |
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Fertility depends in part on the ability of the spermatozoon to respond to osmotic challenges by regulating its volume, which may rely on the movement of K+. These experiments were designed to characterize the K+ channels possibly involved in volume regulation of human ejaculated spermatozoa by simultaneously exposing them to a physiological hypo-osmotic challenge and a wide range of K+ channel inhibitors. Regulation of cellular volume, as measured by flow cytometry, was inhibited when spermatozoa were exposed to quinine (QUI; 0.3 mM), 4-aminopyridine (4AP; 4 mM) and clofilium (CLO; 10 µM) which suggests the involvement of voltage-gated K+ channels Kv1.4, Kv1.5 and Kv1.7, acid-sensitive channel TASK2 and the ß-subunit minK (IsK) in regulatory volume decrease (RVD). QUI and 4AP and, to some extent, CLO also induced hyper activation-like motility. A sensitivity of RVD to pH could not be demonstrated in spermatozoa to support the involvement of TASK2 channels. Western blotting indicated the presence of Kv1.5, TASK2, TASK3 and minK channel proteins, but not Kv1.4. Furthermore, Kv1.5, minK and TASK2 were localized to various regions of the spermatozoa. Although Kv1.4, Kv1.7, TASK2 and TASK3 channels may have important roles in human spermatozoa, Kv1.5 and minK appear to be the most likely candidates for human sperm RVD, serving as targets for non-hormonal contraception.
Key words: contraception/human spermatozoa/inhibitors/potassium channels/volume regulation
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Introduction |
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Ion channels have several important functions in sperm physiology, as they serve as a means of communication between the spermatozoon and its environment. They play vital roles in the regulation of sperm motility, chemotaxis and the acrosome reaction (Darszon et al., 1999
). Recently, the physiological significance for spermatozoa of volume regulation, a phenomenon dependent on ion channels, has been realised by studies on c-ros tyrosine kinase knockout mice. Spermatozoa from these mice are unable to regulate their volume and as a result are unable to pass through the utero-tubal junction (Yeung et al., 1999, 2000). When c-ros knockout males are mated, the spermatozoa found in the uterus are formed into hairpin bends or angulated at the cytoplasmic droplet—morphologies associated with excessive swelling. Human spermatozoa in which volume regulation is blocked by quinine (QUI) are unable to penetrate viscous mucus (Yeung and Cooper, 2001). This infertility not only emphasises the physiological importance of volume regulation, but also creates opportunities to manipulate this phenomenon for purposes of post-testicular male contraception. However, the specific mechanisms of sperm volume regulation must be determined before this process can become a valid target for contraceptive inhibitors.
A thorough knowledge of volume regulation has been established for somatic cells where the typical response of a cell to swelling is the release of osmolytes, which most often involves separate K+ and anion channels (Lang et al., 1998). The role of K+ in volume regulation of somatic cells has been widely confirmed (Hoffmann and Dunham, 1995). It is known that luminal concentrations of K+ increase along the length of the epididymis, giving spermatozoa the opportunity to accumulate this osmolyte (Turner, 2002), and the concentration of intracellular K+ in spermatozoa is high (bulls, Babcock, 1983; Chou et al., 1989; mice, Zeng et al., 1995). From these observations, a role for K+ in regulatory volume decrease (RVD) of sperm cells is likely.
Studies in bulls (Kulkarni et al., 1997; Petrunkina et al., 2001), boars (Petrunkina et al., 2001), dogs (Petrunkina et al., 2004), monkeys (Yeung et al., 2004), humans (Yeung and Cooper, 2001; Yeung et al., 2003) and mice (Yeung et al., 1999; Barfield et al., 2005) have demonstrated an increase in sperm size when exposed to hypotonic media in the presence of QUI, a broad spectrum K+ channel blocker. The reversal of the effects of QUI by the K+ ionophore valinomycin on spermatozoa from bulls (Kulkarni et al., 1997; Petrunkina et al., 2001), mice (Yeung et al., 2005) and humans (Yeung and Cooper, 2001) further supports the involvement of K+.
Potassium channels have been previously studied in spermatozoa as they are believed to play a role in the events that prepare a spermatozoon for fertilization, and several have been identified in spermatozoa, spermatogenic cells and testicular tissue. Voltage-gated channels Kv1.1 and Kv1.2 have been localized to the principal piece and head, Kv3.1 to the annulus and Girk1 to the connecting piece of murine epididymal spermatozoa, and the presence of the mRNA of Kv1.1, 1.2 and 3.1 confirmed (Felix et al., 2002). Slo3, a potassium channel regulated by pH and membrane voltage, is expressed in seminiferous tubules by developing spermatocytes in humans and mice (Schreiber et al., 1998). Large-conductance Ca2+-activated K+ channels are expressed in germ cells but undergo a down-regulation in post-meiotic germ cells that coincides with an up-regulation of voltage-gated potassium channels (Gong et al., 2002). Voltage-gated delayed outwardly rectifying K+ (probably Kv1.3) channels are also expressed in the cytoplasm of primary spermatocytes and post-meiotic elongating spermatids in rats (Jacob et al., 2000) and the inwardly rectifying (Kir) channel on spermatozoa from mice (Munoz-Garay et al., 2001). Complete inhibition of these channels by barium (Ba) prevents membrane hyperpolarization which may indicate a role for the Kir channels in capacitation by enhancing K+ permeability (Munoz-Garay et al., 2001). Specifically, Kir5.1 is expressed in the rat testis, and the protein has been detected in the seminiferous tubules, on spermatogonia, primary and secondary spermatocytes, spermatids, and the head and tail of spermatozoa from rats (Salvatore et al., 1999).
One strategy employed to characterize channels involved in volume regulation of somatic cells is to assess the cell’s ability to regulate volume in the face of a hypo-osmotic challenge and in the presence of compounds known to block specific channels (Coetzee et al., 1999; Cho, 2002; Wehner et al., 2003). Although some valuable studies have been conducted on volume regulation by spermatozoa, the specific mechanisms, including the channels employed, remain elusive. In an attempt to understand volume regulation by human ejaculated spermatozoa better, the effect of specific and non-specific channel inhibitors on the ability of sperm to regulate their volume was investigated.
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Materials and methods |
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Ejaculates
Twenty-six ejaculates were obtained by masturbation at the institute after mean ± SEM (range) abstinence periods of 3.8 ± 0.5 (2–10) days from 20 volunteers age 29.6 ± 1.7 years (24–55) who had provided written consent for the use of their semen for research purposes. Ejaculates were analysed by routine procedures according to published guidelines (WHO, 1999
) after liquefaction for 30 min at 37°C. Ejaculates were characterized by semen volume (4.0 ± 0.4 ml, 1.3–7.8), osmolality (316.8 ± 5.0 mmol/kg, 278–383), sperm concentration (41.2 ± 6.8 million/ml, 3.8–127), sperm count (173.3 ± 36.1 million/ejaculate, 24.7–571.5), percentage motility grades a (35.0 ± 2.1, 21–55), b (14.2 ± 1.6, 4–28), c (8.5 ± 0.9, 1–15), d (42.6 ± 1.1, 35–53) and percentage normal morphology (15.0 ± 1.0, 5–22). Fifteen of these ejaculates were normozoospermic.
Preparation of sperm suspensions
Upon liquefaction, the osmolality of 10 µl semen was measured in a vapour pressure osmometer (Wescor Vapro model 5520, Kreienbaum Messsystem, Langenfeld, Germany), with a 2 min delay to ensure saturation of the chamber. Spermatozoa were washed through an 80% (v/v)/40% (v/v) Percoll gradient (Biosciences, Freiburg, Germany) adjusted to the individual seminal osmolality by combining high and low osmolality Percoll solutions in the relevant proportions and centrifuged at 500 g for 20 min The pellet was isolated by removing all but 100 µl of the supernatant. The pellet was resuspended in this and replaced in an incubator with 5% (v/v) CO2 in air at 37°C until use.
The ability of the Percoll washing step to remove non-sperm cells was evaluated on 23 ejaculates collected from 17 men by expressing the number of non-sperm cells as a percentage of the total number of sperm and non-sperm cells. The percentage of spermatozoa with ‘true’ cytoplasmic droplets (Cooper et al., 2004) before and after the Percoll wash was determined after fixation in the WHO diluent used for assessing concentration (WHO, 1999) and examined in a wet preparation by phase contrast microscopy at 40x magnification. The same observer performed all evaluations.
Incubation media and inhibitors
All spermatozoa were incubated in warmed (37°C) Biggers–Whitten–Whittingham medium (BWW; Biggers et al., 1971) containing 20 mM Hepes or Mopso in addition to NaHCO3 and 4 mg/ml bovine serum albumin (BSA) at pH 7.4. All media were adjusted to an osmolality of 290 mmol/kg (BWW290) by the addition of deionized water or 1 M NaCl. Aliquots of the washed sperm suspension were then dispensed into incubation media with or without one of the inhibitors listed in Table I and incubated for 30 min. All chemicals were from Sigma (Taufkirchen, Germany) except for clofilium tosylate (CLO; Alexis Biochemicals, Grünberg, Germany) and phrixotoxin (PTX; Alomone Labs, Jerusalem, Israel). Ten-times concentrated phosphate-buffered saline (PBS) was obtained from Gibco (Invitrogen, Karlsruhe, Germany). This stock was diluted with deionized water, and the 1 x PBS had an osmolality of 290 mmol/kg. In certain cases, its osmolality was raised to 320 mmol/mg (PBS320) by adding NaCl.
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Measurement of cell size by flow cytometry
Changes in sperm cell volume were measured by a flow cytometer (Coulter Epics XL, version 3.0, Krefeld, Germany) according to the method established by Yeung et al. (2003). After 30 min incubation, approximately 40 µl sperm suspension were added to 200 µl of the same medium lacking BSA and containing 3 µl propidium iodide (PI, final concentration 6 µg/ml). Upon gentle agitation to mix the sample, flow cytometric measurements were immediately recorded by monitoring forward and side scatter signals from laser excitation at 488 nm. Data were collected from 6000 particles excluding cellular debris, determined by their forward and side scatter signals, and spermatozoa with ruptured membranes, gated out by the detection of PI fluorescence (Em 605–635 nm). Mean values of forward scatter of the PI-negative cells were used for subsequent analysis, and the drug-treated sperm were compared with the control from the same ejaculate. The same flow cytometer and settings were maintained throughout the study and controlled by standard beads (Flow-check fluorospheres, Beckman Coulter, Fullerton, CA, USA) before each experiment.
Measurement of kinematics using computer-assisted semen analysis
A 1.5 µl aliquot of sperm suspension was taken after 30 min incubation and viewed on a pre-warmed (37°C) dual-sided sperm analysis chamber (2X-Cel chamber, 20 µm depth, Hamilton Thorne, Beverly, MA, USA) using a negative phase contrast 10x objective and 3.3x photo-ocular. Several fields of view were video-recorded over approximately 1.5 min for later analysis using the Hamilton Thorne CASA system (Animal-version 10.9i). For each sample, approximately 200 spermatozoa were tracked and measured for curvilinear velocity (VCL), straight-line velocity (VSL), averaged path velocity (VAP), amplitude of lateral head displacement (ALH), beat-cross frequency (BCF), linearity (LIN; VSL/VCL x 100) and straightness (STR; VAP/VCL x 100). Measurements were made on 50 frames at a frame rate of 50 Hz, minimum contrast 60, minimum size 3, minimum of 25 track points and minimum VAP of 10 µm/s.
Effects of pH on RVD and action of inhibitors
To determine the effect of pH on the RVD response, spermatozoa were incubated in BWW290 at pH 6.3, 7.4 or 8.4. For pH 6.3, Hepes was replaced by Mopso (final concentration 20 mM). As pH 8.4 falls just within the buffering capabilities of Hepes, spermatozoa were also incubated at pH 8.4 buffered with 20 mM Tris to determine if the buffering system influenced RVD. All media were adjusted to the desired pH with HCl or NaOH. Flow cytometric measurements were taken as indicated above.
Western blot analysis
Spermatozoa were lysed in a 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-X100), supplemented with protease inhibitor cocktail and phosphatase inhibitor Na3VO4 (Sigma). Fifty microliters of buffer were added to the pellet of a Percoll-washed sperm sample and intensely vortexed for 3 min The samples were kept on ice and incubated in the lysis buffer for 1 h and vortexed intermittently. The samples were then centrifuged at 20 000 g for 20 min at 4°C (Heraeus Biofuge Stratus, Kendro Lab Products, Langendselbold, Germany). The supernatant was collected, and protein was measured using a bicinchoninic acid protein assay using BSA as standard (Bio-Rad, Munich, Germany). Aliquots of 50–60 µg protein were stored at –80°C. Although no sonication was performed, the presence of nuclear, mitochondrial and cytosolic proteins cannot be excluded. However, functional potassium channels are membrane proteins that contain transmembrane domains.
To prepare the pre-adsorbed control, primary antibody was added to the control peptide antigen (1 µg peptide per 1 µg antibody for TASK2, TASK3, Kv4.3, Kv4.2; 3 µg peptide per 1 µg antibody for Kv1.5, Kv1.4, as recommended by the antibody supplier) and incubated overnight at 4°C on a rotating plate. Unadsorbed primary antibody was also rotated overnight at 4°C. Aliquots of the resulting adsorbed solution and primary antibody were frozen and kept at –20°C until use. Westerns for Kv1.7 were not performed because of the unavailability of antibodies.
One aliquot of frozen protein was thawed and prepared for each lane of the gel. Lysates were heated at 65°C for 10–15 min and resolved on denaturing 4–12% (w/v) NuPAGE Novex Bis-Tris gels (Invitrogen, Carlsbad, CA, USA) and transferred to nitrocellulose membranes (Hybond ECL, Amersham Pharmacia, Uppsala, Sweden). Membranes were probed with primary affinity-purified rabbit antibodies against Kv1.4, Kv1.5, Kv4.2, Kv4.3, TASK2 and TASK3 diluted 1:2500 (Alomone Labs) followed by a secondary goat anti-rabbit horseradish peroxidase-conjugated antibody diluted 1:250,000 (Pierce, Bonn, Germany). The blots were developed with Super Signal West Femto maximum sensitivity substrate (Pierce). Molecular weights were estimated using line densitometer software (ChemImage System, 15440, version 5.5, Alpha Innotech, San Leandro, CA, USA).
Immunolocalization
Spermatozoa were washed through Percoll adjusted to 320 mmol/kg, as described above, and resuspended in 3 ml PBS320. They were centrifuged for 5 min at 500 g and resuspended in 0.5 or 1 ml of PBS depending on the concentration. Five microliters of the highly concentrated sperm suspension were smeared across a Polysine slide (Menzel GmbH and Co KG, Braunschweig, Germany) and air-dried at room temperature for at least 15 min. Slides were placed in a Wheaton staining jar and submerged in 4% paraformaldehyde in PBS for 30 min. The paraformaldehyde was poured off and replaced with PBS for washing. The jar was then placed on a rotating plate for 5 min, and the PBS was changed twice during this time. A circle with an inner diameter of approximately 1 cm was drawn in the centre of the slide with a grease pen (Dako, Glostrup, Denmark). In some instances, the cells were permeabilized by placing a drop of 0.1% (v/v) Triton X-100 in PBS over the cells within the circle for 5 min followed by washing in PBS. Cells were then blocked by covering the spermatozoa with PBS containing 3% (w/v) BSA (approximately 30 µl) and incubated for 30 min. The slides were washed, and a drop of primary antibody (1:60) or adsorbed antibody (adsorbed with 10x more antigen than recommended) was placed over the sperm and incubated overnight at room temperature in a humid chamber. For detection of minK, incubation with secondary antibody only was used as a negative control rather than an adsorbed antibody preparation. Slides were washed in PBS for 20 min in the dark, and a drop of secondary antibody (1:200, anti-rabbit IgG tetramethylrhodamine isothiocyanate (TRITC) conjugate, Sigma) was incubated on the spermatozoa for 1 h. Slides were washed in PBS for 20 min, mounted with Vectashield anti-fade medium (Vector Laboratories, Burlingame, CA, USA) and observed with a Zeiss Axiovert 200 fluorescence microscope, Göttingen, Germany. Immunolocalizations of TASK3 and Kv1.7 were not performed because of lack of appropriate antibodies.
Statistical analysis
Comparisons between the forward scatter measurements from spermatozoa incubated in different inhibitors were performed using one-way repeated measures analysis of variance (ANOVA) followed by Dunnett’s post hoc test against the control. For data that were not normally distributed, one-way ANOVA on ranks was followed by Dunnett’s post hoc test against the control. Comparisons of the non-sperm cells and spermatozoa with cytoplasmic droplets before and after Percoll centrifugation were made using the Mann–Whitney Rank sum test. Values were considered significantly different with P < 0.05.
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Results |
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Purification of sperm samples by Percoll gradient centrifugation
All semen samples washed through an 80/40 Percoll gradient adjusted to seminal osmolality resulted in a statistically significant decrease of non-sperm particles from the pre-washed semen (before, 15.0 ± 2.1%; after, 1.4 ± 0.2%). In these same samples, there was also a significant reduction in the percentage of spermatozoa bearing a cytoplasmic droplet before and after Percoll gradient centrifugation (before, 34 ± 3; after 7 ± 1).
Measurement of size by flow cytometry
BWW290 containing 0.3 mM QUI was used as the positive control, since previous studies showed that human spermatozoa swell when exposed simultaneously to QUI and hypo-osmotic challenge (Yeung and Cooper, 2001; Yeung et al., 2003). Spermatozoa from all men in these experiments were capable of volume regulation as indicated by the statistically significant increase in their forward scatter in the presence of QUI compared with the control (mean ± SEM difference in channel number from the control, 16 ± 2.8).
Human spermatozoa had significantly higher forward scatter values when incubated in 4-aminopyridine (4AP, 4 mM), CLO (10 µM) and QUI (0.3 mM) for 30 min (Figure 1). There were no significant differences in the percentage of spermatozoa with intact plasma membranes in the presence of the different inhibitors, as indicated by the exclusion of PI.
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Computer-assisted semen analysis
There were no significant differences in the percentage of motile spermatozoa incubated in the various inhibitors with the exception of 2 mM cadmium (Cd, 37.1 ± 8.1; control, 85.9 ± 1.4). Incubation in QUI (0.3 mM) or 4AP (4 mM) induced a hyperactivation-like motility in the spermatozoa. Both drugs increased ALH and VCL, but significantly decreased VSL, STR (straightness) and LIN (linearity) (Table I). In addition, Cd (2 mM) decreased VSL, VAP, ALH and VCL, and Ba significantly decreased BCF without affecting the percentage of cells impermeable to PI.
Western blotting
Specific protein bands were defined as those not present on blotted membranes incubated with the antibody pre-adsorbed with that particular channel antigen (Figure 2). One specific band detected by an antibody against Kv1.5 protein was estimated to be of size 76 kDa. Previous reports have indicated two bands corresponding to Kv1.5 at 65 and 90 kDa in Schwann cells (Sobko et al., 1998) and 60 and 75 kDa in rat atrium (Yamashita et al., 2000). Antibodies against TASK2 also revealed specific bands of 55 and 72 kDa. This is in contrast to the reported value of 45 kDa in rat kidney membranes (Alomone Labs) and rat taste receptor cells (Lin et al., 2004). Antibodies to minK revealed a specific band at 14 kDa. Incubation of sperm membrane proteins with TASK3 antibodies yielded two specific bands of 75 and 49 kDa. The value of 49 kDa is identical to the value reported by the supplier for rat cerebellum lysates, but a band at 75 kDa has not been reported. No specific bands were observed when membranes were incubated with antibodies against Kv1.4, Kv4.2 or Kv4.3 (data not shown).
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Immunolocalization
Kv1.5 was localized to the midpiece and distal post-acrosomal region of the sperm head (Figure 3). In the case that a cytoplasmic droplet was still present, there was an intense fluorescence at that site (inset in Figure 3A and B). The protein minK was localized to the distal post-acrosomal region of the head and also on the neck of the spermatozoon (Figure 4). However, staining was weaker along the midpiece and became more intense but patchy along the principal piece of the flagellum. TASK2 appeared to be localized on the neck and midpiece but staining on the tail was weak (Figure 5). Some non-specific staining on the sperm head was still detected on sperm incubated with the adsorbed preparation.
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Effects of pH on RVD
After 5 and 30 min of incubation, spermatozoa incubated in BWW290 at acidic or alkaline pH without inhibitors were not significantly different in size from the control (cells in BWW290 pH 7.4 without inhibitors), although cells in pH 8.4 with Tris tended to be larger after 30 min (Table II).
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Discussion |
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Volume regulation is a vital process for spermatozoa. A surge of research during the last decade is beginning to uncover how spermatozoa achieve a stable volume and what implications this may have for sperm function in both the male and female tracts. Notably, K+ has been implicated as important in the RVD process, and these studies were designed to identify specific K+ channels that may be involved in this process in human spermatozoa.
Volume regulation is a complicated process, and it is likely that RVD involves more than one channel working in concert to reduce volume. Owing to the lack of absolutely specific inhibitors, swelling due to blockage of a single channel could not be distinguished from that caused by blockade of multiple channels. To screen for possible channels involved, inhibitors and doses were chosen for their reported ability to block K+ currents in somatic cells. The channels listed in Table III are those that can be inhibited by at least one of the tested blockers. The profiles of all channels were then compared against the blockers that did and did not cause swelling of spermatozoa. Candidates were accepted from their being inhibited by 4AP and/or CLO, which inhibited RVD in sperm, but not inhibited by TEA, PTX, FLC, Ba2+ or Cd2+ as these inhibitors did not prevent RVD. Channels were not excluded if there was no information about the effects of an inhibitor on that channel (empty box in Table III), or if the channel was insensitive to a tested inhibitor (x in box). By this reasoning, from the inhibitors used in this study, it is likely that Kv1.4, Kv1.5, Kv1.7, minK and TASK2 are involved in volume regulation of human spermatozoa. Of these channels, Kv1.5, minK and TASK2 have been implicated in RVD of somatic cells.
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Correlating changes in cell volume and kinematic parameters may be a more sensitive assay for detecting failure of RVD in cells, the response of which is very small, such as human spermatozoa (Yeung et al., 2003). In these experiments, spermatozoa swollen by incubation in QUI and 4AP exhibited a hyperactivation-like motility. This corroborates evidence from other studies that have also found higher incidences of hyperactivated motility of human and monkey spermatozoa exposed to QUI or 4AP (Yeung and Cooper, 2001; Yeung et al., 2003, 2004; Gu et al., 2004). CLO caused a less marked change in motility, perhaps indicating an incomplete transition towards hyperactivated motility. Although Cd and Ba caused changes in sperm motility, the flagellar movements induced did not resemble the hyperactivated-like motility that was associated with cell swelling. This analysis of motility supports the conclusions from measurement of size with the flow cytometer—that the channels sensitive to 4AP and CLO are candidates for RVD.
The possible involvement of an acid-sensitive channel (TASK2) prompted the investigation of the effect of pH on RVD. TASK channels (TWIK (Tandem of P-domains in a Weakly Inward rectifying K+ channel)-related acid-sensitive K+ channels) are thought to be involved in the leak or background conductance of K+ in somatic cells and are involved in the RVD process (Niemeyer et al., 2001). TASK2, when expressed in HEK-293 cells, is effectively inhibited by CLO with an IC50 of 25 µM (Niemeyer et al., 2001). TASK2 has also been isolated in Ehrlich ascites tumour cells (Niemeyer et al., 2001) which have RVD responses that are accelerated by alkaline pH and decelerated by acidic pH (Hougaard et al., 2001). Our results did not indicate acid-sensitivity of sperm volume regulation. In fact, there was a tendency for the cells incubated at pH 8.4 with Tris to be larger than those at other pH. However, because the cells incubated at pH 8.4 with Hepes were not larger than those at 7.4 and 6.3, this may indicate a pH-independent effect of Tris that hinders RVD.
Although this weakens the argument for the involvement of TASK2 in sperm RVD, western blot experiments revealed two specific bands, one of which was of the size previously reported for TASK2 proteins, and the immunolocalization revealed a distribution over the principal piece of the flagellum. In addition, the presence of TASK2 mRNA has previously been detected in post-meiotic germ cells of the mouse (Schultz et al., 2003; Shima et al., 2004). TASK3 was also detected in western blots in human sperm, but immunolocalization was not possible owing to lack of appropriate antibodies. TASK3 is sensitive to Ba in somatic cells (Kim et al., 2000), and although Ba did have a slight effect on motility, there was not enough evidence to support a significant role for this channel in volume regulation of human spermatozoa.
MinK is a 130 amino acid protein that, when associated with KvLQT1, forms the potassium channel complex responsible for the underlying slow (IKs) cardiac current (Barhanin et al., 1996; Sanguinetti et al., 1996). This protein has been implicated in RVD of some populations of renal proximal tubule cells (Millar et al., 2004), and murine tracheal epithelial cells from IsK (minK) knockout mice are unable to recover normal size when exposed to hypotonic solutions (Lock and Valverde, 2000). Vestibular dark cells of the inner ear (Wangemann et al., 1995) and Xenopus oocytes (Busch et al., 1992) also have minK-mediated currents that are activated by cell swelling. In this study, specific bands for the minK protein were detected on western blots, and immunolocalization revealed a distribution along the principal piece of the flagellum with more intense staining over the distal post-acrosomal region and neck of the spermatozoa. This channel appears to be a good candidate for participation in the RVD process.
Kv1.5 was one of the first potassium channels implicated in RVD of somatic cells, and there continues to be evidence from electrically excitable and non-excitable cells that this channel is involved in RVD (Wehner et al., 2003). Immunolocalization of Kv1.5 resulted in the most intense staining of the proteins tested and was concentrated at the neck of the spermatozoa. This corresponds to the site of the cytoplasmic droplet and where there was an intact droplet (inset of Figure 3A and B) fluorescence was especially strong. The cytoplasmic droplet is the sperm’s largest cytoplasmic reservoir and possibly the site of the most fluid exchange (Cooper and Yeung, 2003). When murine sperm are exposed to hypotonic conditions, there is an obvious swelling at the cytoplasmic droplet, and the sperm accommodates the change in volume and avoids excessive increase in surface area by angulating at the site of the droplet or assuming a hairpin-like morphology. A cytoplasmic droplet has recently been demonstrated to be a normal component of functioning human ejaculated spermatozoa (Cooper et al., 2004). The localization of all three potential proteins to the neck region supports the hypothesis that most RVD is mediated through the membrane at the cytoplasmic droplet.
In conclusion, several K+ channels may be involved in the volume regulation of human spermatozoa, including those in the voltage-gated and acid-sensitive K+ channel families. These channels, which may have overlapping ranges of activation, could work in concert to produce successful volume regulation. Whereas K+ channels appear to have a prominent role in RVD of human spermatozoa, it is possible that other channels are involved. These experiments do not rule out the participation of other types of channels blocked by the inhibitors tested here, thus future research should determine if various anion and organic osmolyte channels contribute to the volume regulatory responses of human spermatozoa. Targeting these channels may have contraceptive potential by preventing volume regulation of sperm in the female tract and hindering their migration to the site of fertilization.
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Acknowledgements |
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We thank Barbara Hellenkemper for preparation of human sperm plasma membrane proteins for the western blotting experiments and Raphaele Kürten, Daniela Hanke, Sabine Rehr and Margret Kloth for performing the semen analyses. J.P.B. was 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.
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Submitted on May 6, 2005; resubmitted on June 29, 2005; accepted on July 12, 2005
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