Molecular Human Reproduction, Vol. 7, No. 9, 819-828,
September 2001
© 2001 European Society of Human Reproduction and Embryology
Testis and spermatogenesis |
Effects of the ion-channel blocker quinine on human sperm volume, kinematics and mucus penetration, and the involvement of potassium channels
Institute of Reproductive Medicine of the University, Domagkstrasse 11, D-48129 Münster, Germany
Abstract
Sperm defects in the infertile c-ros knockout mouse model have recently highlighted the importance of volume regulation in sperm function. In this study, washed human spermatozoa were shown to change size and shape, as detected by flow cytometry and light microscopy, in response to the ion-channel blocker quinine (minimum effective doses at 20 and 125 µmol/l respectively). The increase in sperm volume was accompanied by reduced straight-line velocity (VSL) and linearity (LIN) of the swim-path but increased lateral head displacement and curvilinear velocity, while percentage motility was unaffected. Spermatozoa in semen and in artificial cervical mucus were similarly affected at 0.2 and 0.5 mmol/l quinine, resulting in marked reduction of mucus penetration and migration. The effects of quinine on sperm volume and kinematics were reduced or abolished by the K+-ionophores valinomycin (1 and 5 µmol/l) and gramicidin (0.5 and 1 µmol/l). In Ca2+-free medium; however, the quinine effects largely persisted. The K+-channel blocker, 4-aminopyridine (1 and 4 mmol/l), mimicked the quinine effects in the reduction of VSL and LIN, while the K+-channel blocker, tetraethylammonium chloride (TEA, 2.5–10 mmol/l), did not affect kinematics. The K+-channel (Kv1.3)-specific inhibitor, margatoxin, and the Ca2+-dependent K+-channel blocker, charybdotoxin, also had no effects. This study suggests that volume regulation in human spermatozoa and the linear trajectory of their motion may rely on quinine-sensitive and TEA-insensitive, largely calcium-independent, potassium channels, and possibly volume-sensitive organic anion channels. These channels could be targets for contraception.
infertility/K+ channel/regulatory volume decrease/sperm function/sperm motility
Introduction
Defective sperm volume regulation has recently been shown to be the major cause of infertility in the c-ros knockout mouse model where the swelling of spermatozoa leads to angulation of the tail and results in the failure of spermatozoa to pass through the utero-tubal junction (Yeung et al., 1998
, 2000). Somatic cells can counteract swelling initiated by osmotic challenge by activating mechanisms of regulatory volume decrease (RVD). However, the physiological significance of RVD in sperm function has scarcely been studied.
In contrast to rodents, where sperm transport in the female tract demands vigorous forward progressive motility for penetrating the tightly folded utero-tubal junction (Gaddum-Rosse, 1981), the main obstacle to sperm transport in the human female tract is less clear. Limited studies in few subjects, including fertile women, have indicated the presence of barriers since less than one in a million spermatozoa deposited in the vagina can be recovered from the oviduct (Barratt and Cooke, 1991). There is a correlation between sperm velocity measured in vitro and intrauterine insemination success rates (Holt et al., 1989), and the morphology of spermatozoa, along with motility characteristics, are known to be important factors for penetration of cervical mucus (Aitken et al., 1985; Mortimer et al., 1986; Katz et al., 1989, 1990). However, observations of labelled microspheres used as surrogate spermatozoa led to the controversial interpretation that transport from the cervix into and through the uterus does not involve sperm motility but is effected by uterine peristalsis aided by local factors (Kunz et al., 1996). Nevertheless, these authors did not rule out the significance of mucus penetration, such as in the creation of sperm reservoirs in the cervical crypts (Kunz et al., 1997).
In somatic cells, RVD involves effluxes of K+, Cl– and organic osmolytes through separate K+-channels and anion channels (Lang et al., 1998; O'Neill 1999). Quinine is a wide spectrum channel blocker affecting voltage-sensitive K+-channels as well as Ca2+-activated ones (Grinstein and Foskett, 1990; Kuriyama et al., 1995). Angulated sperm flagella, as observed in the infertile c-ros knockout mouse, can be created in wild type spermatozoa by treatment with known blockers of cell volume regulation, quinine, NPPB [5-nitro-2-(3-phenylpropylamino)-benzoic acid] or BaCl2, with the best response being obtained with quinine (Yeung et al., 1999). In this study, the effects of quinine on human sperm shape and size, and the consequences of these physiological effects on sperm transport in the female tract, namely kinematics and mucus penetration, were investigated. The nature of the K+-channel(s) involved in the mechanism of the quinine action was investigated using various channel blockers and openers to mimic or counteract the effects of quinine.
Materials and methods
Chemicals and media
Unless stated otherwise, all chemicals, including the potassium channel blockers 4-aminopyridine (4-AP) and tetraethylammomium chloride (TEA), were from Sigma (Taufkirchen, Germany), except charybdotoxin and margatoxin and the potassium ionophore valinomycin which were from Calbiochem (Bad Soden, Germany) and gramicidin which was from Molecular Probes (Leiden, The Netherlands). The medium used for sperm incubation was Biggers–Whitten–Whittingham medium (BWW) (Biggers et al., 1971) containing 4 mg bovine serum albumin/ml (BWW/BSA). The osmolality of the medium was adjusted before use to around 310 or increased to 330 mmol/kg using NaCl; the former value is close to osmolality of serum and the latter to that of semen (see Results). For Ca2+-free BWW, CaCl2 was replaced by NaCl and 0.5 mmol/l EGTA was added.
Ejaculates
Normozoospermic ejaculates were obtained by masturbation from 72 men who attended the andrology clinic of the institute and who had provided written consent for the use of their semen for research purposes. Ejaculates were analysed by the routine procedure according to published guidelines (World Health Organization, 1999) after liquefaction at 37°C for 30 min. The ejaculates used in this study had sperm concentrations of 89 ± 68x106/ml, motility of 63 ± 8% and normal morphology of 24 ± 7% (mean ± SD). Semen samples were used directly for the mucus penetration test or washed to remove the seminal plasma (see below).
Treatment of washed spermatozoa with quinine, channel openers and other channel blockers
One to 2 ml of liquefied semen was layered on a gradient consisting of 3 ml 60% (v/v) and 3 ml 30% (v/v) Percoll made up in BWW identical in composition to the control BWW used throughout the same experiment, and centrifuged at 500 g for 10 min. The sperm pellet was washed again for 5 min in BWW/BSA. Washed spermatozoa were resuspended to a concentration of 20x106/ml and incubated at 37°C in 5% CO2 in air. Quinine was added at final concentrations of 2–300 µmol/l from secondary stocks in BWW diluted from a primary aqueous 100 mmol/l stock solution. Washed spermatozoa incubated with and without quinine were examined at 5, 20 and 45 min. For the study of channel blockers, spermatozoa were analysed as described below after 20 min incubation with the channel blocker, since the effects of quinine had stabilized at this time (see Results). All drugs tested were added to the sperm suspension from secondary stock solutions made by diluting primary stock solutions with BWW. For each set of drugs tested in each experiment, one aliquot without any drug (serving as the non-treatment control) and another sperm aliquot containing 125 µmol/l quinine alone (as positive control) were also incubated and analysed for comparison. This quinine dose was chosen since it was well above the minimum effective dose (20 µmol/l) and well below 1 mmol/l which inhibited the percentage motility in some sperm samples. For the study of valinomycin (1 or 5 µmol/l) and gramicidin (0.5 or 1 µmol/l) as channel openers, spermatozoa were incubated with or without 125 µmol/l quinine for a total of 20 min, and the ionophore was then added during this incubation for the last 6 min (for valinomycin) or 3 min (for gramicidin) before sperm analysis. Experiments where 4-AP and TEA were tested were carried out using BWW with an osmolality of ~310 mmol/kg, the rest were done with 330 mmol/kg as this is closer to the osmolality of semen. Effects of quinine on sperm kinematics and volume were shown to be similar at the two osmolalities (see Results).
Light microscopic observation of sperm swelling
A 2 µl aliquot of spermatozoa was placed on a pre-warmed 20 µm deep slide chamber (2X-CEL; Hamilton-Thorne Research, Beverly, MA, USA) with a 22x22 mm coverslip, on a heated microscope stage (37°C). The motility percentage and swollen status (Jeyendran et al., 1984) of 100 spermatozoa were assessed using positive phase contrast optics and x40 objective.
Computer-assisted analysis of sperm kinematics
Video-recording of 5–15 microscopic fields of the sperm sample in the 20 µm chamber at 37°C, as described above, was made over 1 min using a negative phase contrast x10 objective and x3.3 photo-eyepiece. For each sample, ~200 motile spermatozoa were tracked for 1 s and analysed with the Hamilton–Thorne CASA system (Beverly, MA, USA). Kinematic parameters measured included curvilinear (VCL), straight-line (VSL) and averaged path (VAP) velocities, amplitude of lateral head displacement (ALH), beat cross frequency (BCF) and the derived parameters of linearity (LIN = VSL/VCL x100%) and straightness (STR = VAP/VCL x100%). Washed spermatozoa were analysed with the HT-master C (version 10.6H) at 25 Hz frame rate for 25 frames with minimum contrast of 60, minimum size of 3, minimum of 13 track points and minimum VAP of 5 µm/s. For spermatozoa in semen and mucus, a new version (IVOS version 10.8) was used to enable analysis at 50 Hz frame rate, with the relevant software settings adjusted accordingly (50 frames, minimum of 30 track points and minimum VAP of 10 µm/s). For each kinematic parameter, the median value in each sample was used to represent the sample.
Detection of changes in sperm volume by flow cytometry
In flow cytometry, cell volume is reflected in the forward scatter of the laser by the cell. 30 µl of the incubated sperm sample was diluted in 300 µl of the same incubation medium containing 5 µg/ml propidium iodide (PI) as a vitality stain. After 3 min staining at room temperature, forward scatter and PI fluorescence signal intensities (channel nos.) from ~5000 cells were collected for each sample using a flow-cytometer with laser excitation at 488 nm (Coulter Epics XL, version 3.0, Krefeld, Germany). Viable (PI negative) and dead (PI positive) spermatozoa were gated and analysed separately for their mean channel no. of forward scatter and compared with the values from the parallel control samples.
Treatment of semen with quinine and the mucus penetration test
Fifteen µl BWW medium containing 0, 2 or 5 mmol/l quinine were mixed with 135 µl liquefied ejaculate in a 1.5 ml Eppendorf tube (without lid). While 100 µl of this was reserved for the penetration test, a 50 µl aliquot was transferred to another tube for sperm motility assessment at 20 and 60 min, and both tubes were incubated at 37°C in 5% CO2. At 15 min, 5 µl from these semen aliquots was added to 25 µl mucus containing the corresponding concentration of quinine (see below). For the mucus penetration test, hyaluronic acid (from rooster comb; Sigma H5388) was dissolved in BWW/BSA at 6 mg/ml to serve as cervical mucus surrogate (Neuwinger et al., 1991; Tang et al., 1999). Quinine in BWW/BSA or medium alone was added to the mucus at 1:10 to give final concentrations of 0, 0.2 or 0.5 mmol/l. After standing at 37°C in 5% CO2 to expel any air bubbles created by the mixing, each mucus was carefully drawn into a 10 cm flat capillary tube (0.3 mm deep; Camlab, Cambridge UK) to form an 8 cm mucus column. The capillary was sealed at the air-column end with plasticine and kept at 37°C in 5% CO2 until use, with the mucus end inserted in a corresponding tube of 300 µl 0, 0.2 or 0.5 mmol/l quinine medium to prevent drying out. After 15 min of incubation of the above 100 µl semen samples with or without quinine, the mucus column was inserted into the tube of semen and held horizontally in a 9 cm Petri dish padded with moistened filter paper. The test was carried out at 37°C and scored at 0.5, 1 and 3 h. The number of spermatozoa observed in the field of a 10x objective and 10x eyepiece at distances of 1, 4 and 6 cm along the mucus column were counted (up to 200 at each site).
Measurement of semen osmolality
A 10 µl aliquot of liquefied semen (mostly after 60–90 min of ejaculation) was loaded onto a filter paper disc in the sample holder of a vapour pressure osmometer to measure osmolality by dew point depression (Vapro, model 5520; Wescor, Kreienbaum Wissenschaftliche Mess-systeme, Langenfeld, Germany). The osmometer was calibrated each day using a 290 mmol/kg standard supplied by the manufacturer.
Statistics
Data groups in each experiment were analysed using the statistical software SigmaStat (version 2.03; SPSS Inc., Erkrath, Germany). When the whole set of doses or treatments in the experiment were tested on spermatozoa from the same source (i.e. same controls for different treatments), mean values and standard error of mean (SEM) of the primary data were presented in the results, and analysed for statistical differences (P < 0.05) by one-way repeated measures analysis of variance (ANOVA) followed by Dunnett's test. When different doses had different control samples, primary data were standardized against the relevant control values before statistical analysis using one-way ANOVA on ranks followed by Dunnett's test.
Results
Effect of low concentrations of channel blockers and openers on percentage motility of spermatozoa
The numbers of motile and immotile spermatozoa were counted for every sample, and there were no significant changes in the percentage motility at any of the doses of drugs added to washed spermatozoa, semen or artificial mucus in this study (e.g. Figures 1 and 2, other data not shown). In preliminary experiments, some washed sperm samples showed a rapid decline of motility to total arrest during incubation with 1 mmol/l quinine. This high dose was subsequently abandoned to avoid non-specific toxic effects and complications in the interpretation of results.
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), the extent of swelling of all sperm (•) and the percentage of swollen motile spermatozoa (
) observed in medium of 310 mmol/kg osmolarity, and on (b) sperm volume (measured as laser light forward scatter signal by flow cytometry) after 20 min incubation in media of 310 (•) and 330 mmol/kg (
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Effects of quinine on sperm morphology
Increasing the concentration of quinine in medium of 310 mmol/kg increased the total percentage of swollen cells as judged by subjective visual assessment (Figure 1
). Spermatozoa were mainly swollen at the tip of the tail and only few cells responded. Nevertheless this reached statistical significance at 125 and 300 µmol/l quinine (Figure 1).
Osmolality of semen
Measurement of seminal plasma osmolality after 60–90 min of liquefaction showed a mean ± SD value of 342 ± 21 mmol/kg (n = 66).
Effect of quinine on sperm volume
After incubating washed spermatozoa with or without quinine, sperm volume was assessed by monitoring forward scatter of a laser beam by individual cells using flow cytometry, where dead spermatozoa were distinguished from live ones by their propidium iodide fluorescence. There was a dose-dependent increase in forward scatter signals from viable (PI negative) sperm cells incubated in quinine, compared with viable cells incubated in its absence, regardless of the osmolality of the medium (Figure 2). A statistically significant effect was obtained at concentrations as low as 20 µmol/l. The percentage of viable spermatozoa (averaged 76–84%) was not significantly affected by any of the five quinine doses, and non-viable spermatozoa did not show dose-dependent changes in size upon incubation with quinine (data not shown).
Effect of quinine on kinematics of washed motile spermatozoa
The effect of quinine was rapid, already detectable at 5 min, stabilized at maximal levels at 20 min, and maintained over 45 min of incubation (data not shown). Subsequent experiments utilized 20 min incubation. Whereas percentage motility was unaffected, kinematic parameters were affected by concentrations as low as 20 µmol/l quinine in media of both 310 and 330 mmol/kg osmolality (Figure 2). ALH was increased and both LIN and VSL were decreased (Figure 2) by quinine. Increases in VCL of 10–30% were obtained with 20 µmol/l or higher concentrations of quinine (data not shown). As found for LIN, STR was also suppressed, whereas BCF was not significantly altered (data not shown).
Motility of human spermatozoa in quinine-containing semen and artificial mucus
When added to seminal plasma, both 0.2 and 0.5 mmol/l quinine led to decreased VSL and LIN at 20 min, whereas VCL and the ALH were increased (Figure 3). Most kinematic parameters were lower in mucus than in seminal plasma, reflecting the greater viscosity of the former. Spermatozoa within the artificial mucus containing quinine showed little change in either VCL or ALH compared to the control, but displayed decreased VSL and LIN (Figure 3). Similar effects were maintained when examined at 60 min of incubation in semen and in mucus (data not shown).
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Sperm migration through artificial mucus
The number of spermatozoa found at 1 cm into the mucus column was taken to represent the extent of sperm entry from semen into mucus. In the presence of 0.5 mmol/l quinine, sperm number was markedly lower than in the control, even up to 3 h of contact with the mucus (Figure 4
). Migration through the mucus was even more drastically inhibited, as indicated by the significantly reduced sperm numbers at 4 cm (Figure 4) and 6 cm (data not shown) over 3 h of incubation. The effect of a lower dose of 0.2 mmol/l was variable among the ejaculates tested and did not reach statistical significance. When the same experiment was performed but with 0.5 mmol/l quinine present only in semen and not in the artificial mucus, the effects of quinine were largely abolished. The penetration scores were similar to the non-treated spermatozoa, with the only statistically significant decline revealed in their migration to 4 cm after 3 h of incubation (Figure 4).
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) or presence of quinine 0.2 mmol/l (
) or 0.5 mmol/l (
) in both semen and mucus or in semen alone (0.5 mmol/l, Quinine action and the effect of K+ ionophores
In the absence of valinomycin, quinine (125 µmol/l) changed the kinematics of washed human spermatozoa from a forward progressive into a hyperactivation-like pattern after a 20 min incubation, by significantly reducing VSL and LIN while increasing VCL and ALH; cell volume was also increased (Figure 5
). These stimulatory effects on VCL and ALH were reduced in a dose-dependent manner to statistically non-significant levels (when compared to treatments with valinomycin alone) by treating the spermatozoa with 1 or 5 µmol/l valinomycin for 6 min before the end of the incubation. In the presence of valinomycin, the reduction in VSL and LIN by quinine no longer reached statistically significant levels, although the valinomycin-induced changes were minimal. The increase in cell volume by quinine was also reduced by valinomycin at both concentrations (Figure 5).
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A similar but more marked interference with quinine action was observed with gramicidin (Figure 6
). Both the increases in VCL and ALH brought about by quinine were abolished by 1 µmol/l gramicidin. There was even a tendency for the quinine effects to be reversed (suppression instead of stimulation) in the presence of 5 µmol/l gramicidin. The suppression of VSL and LIN, and the increase in cell volume, by quinine were also reduced or abolished by a 3 min exposure to gramicidin at both concentrations (Figure 6).
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Effect of calcium ions on quinine action
When Ca2+ ions were removed from the medium, sperm flagellation was less vigorous and the swim-paths appeared more straight than those exhibited in the presence of Ca2+, as indicated by lowered VCL and ALH with higher LIN. The quinine-induced decrease in LIN and increase in ALH observed in the presence of Ca2+ still occurred in the absence of Ca2+, although the extent of such effects was muted (Figure 7
). The decrease in VSL and increase in VCL by quinine, on the other hand, were both dampened in Ca2+-free medium. The quinine effect on cell size in Ca2+-containing medium, as reflected in the increase in forward scatter (increases of 8.1 ± 1.4 channel no. versus control, n = 9), remained significant in Ca2+-free medium (increases of 6.0 ± 1.2 versus Ca2+-free control, n = 9).
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Effects of other potassium channel blockers on sperm kinematics
The potassium channel blockers, TEA and 4-AP, were checked for their ability to mimic quinine in altering human sperm kinematics. In these experiments, spermatozoa were examined at 20 or 45 min after drug treatment. Since the effect, or the lack of it, appeared to be similar at the two time points, as illustrated also by the quinine effects, pooled data are presented here. 4-AP in the millimolar range significantly decreased VSL but the increase in VCL was not significant (Figure 8
). LIN values, as a ratio of control values, were also reduced to 0.70 ± 0.04 (mean ± SEM; n = 14). On the other hand, TEA did not have any effect on any kinematic parameters except at a high dose of 20 mmol/l where both VCL and VSL were decreased (Figure 8).
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) and TEA (tetra-ethylammonium; Neither charybdotoxin (seven doses from 5–100 nmol/l) nor margatoxin (eight doses from 0.1–200 nmol/l) had any influence on VSL or VCL of the motile spermatozoa, or any other kinematic parameter (n = 4–10, data not shown), although spermatozoa from the same ejaculate, prepared by the same procedure, responded to 125 µmol/l quinine with decreases in VSL and LIN (to 64 ± 5 and 52 ± 5% control values) and increases in VCL and ALH (to 133 ± 7 and 159 ± 13% control values).
Effects of ouabain on sperm kinematics
The Na+ and K+ adenosine triphosphatase inhibitor ouabain, at either 1 or 2 mmol/l, had little effect on the kinematic parameters (Figure 9). In contrast to the response of spermatozoa from the same source to the presence of 125 µmol/l quinine, VSL was only slightly, though significantly, reduced by ouabain, whereas no changes in VCL or ALH were consistently observed. A slight decrease in LIN at 1 mmol/l was not confirmed at 2 mmol/l (Figure 9). Ouabain also had no effect on sperm cell size (increase in forward scatter signals was 1 ± 2 channel number versus control, compared to 11 ± 1 for quinine).
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) and ouabain at 1 mmol/l (
) and 2 mmol/l (Discussion
The present study has shown that human spermatozoa are susceptible to the effects of the wide spectrum channel blocker quinine at concentrations which did not affect percentage motility. It causes a change in sperm cell shape and size, and alterations in kinematic parameters underlying the effectiveness of forward progression. The response of human spermatozoa to quinine in flagellar shape differed from that of murine spermatozoa where it is more prominent (Yeung et al., 1998, 1999), presumably reflecting their prominence and location of the droplet which is different in human spermatozoa. Murine epididymal spermatozoa, swollen hypotonically or upon quinine treatment, bend at the site of the cytoplasmic droplet at the end of the mid-piece. Human ejaculated spermatozoa do not have an obvious droplet and swell in grossly hypotonic solutions to form a variety of patterns of coiled tails, as described in the swelling test (Jeyendran et al., 1984; Hossain et al., 1998; World Health Organization, 1999). The present effect of quinine on washed human spermatozoa was most marked in kinematics, amounting to 40–50% inhibition of VSL and LIN of swim path, and 20–40% stimulation of VCL and ALH at 125 µmol/l. On the other hand, the effect on cell shape was only detectable in a maximum of 15% coiled tails at 300 µmol/l. The latter is a subjective method to detect sperm swelling. Whereas measurement of cell size using laser light scattering (Sucic et al., 1989) and the monitoring of volume changes by flow cytometry (Freedman and Novak, 1997) are well established for somatic cells, the present application to sperm cells yielded very consistent results, although the increases in forward scatter signals only amounted to a small percentage of control values. That such small increases reflected volume increases in response to quinine by viable spermatozoa was substantiated by the dose-dependence and the lack of changes in dead spermatozoa.
In the present study, the lowest effective concentration of quinine affecting the volume of washed spermatozoa, as detected objectively by laser light scattering, was 20 µmol/l. A higher concentration of 125 µmol/l was required for the induction of coiled tails, as observed in microscopy. Tail coiling is probably triggered when sperm volume increase exceeds a certain threshold. Kinematic changes, which were also detectable at a low dose of 20 µmol/l, could be a hydrodynamic consequence of the volume increase. On the other hand, they could also be a consequence of intracellular ionic changes, e.g. concentration decrease due to volume increase. Since the quinine effects on kinematics as well as volume increase persisted in the absence of extracellular Ca2+, the involvement of any Ca2+ influx, if at all, is probably not obligatory. The relationship between kinematics and cell volume effects was also borne out by the simultaneous abolition of both by K+ ionophores.
There is abundant information that quinine and its analogue, quinidine, can prevent RVD and influence the size of many somatic cell types. The drug can be effective at doses as low as 20 and 50 µmol/l for the volume-sensitive Cl– channel (Voets et al., 1996; Schmid et al., 1998) and the voltage-sensitive Kv1.5 and 1.3 channels (Felipe et al., 1993; Attali et al., 1997). The effect of such low doses of quinine on the volume of human spermatozoa has not been investigated before. Bovine spermatozoa are susceptible to the effects of 1 mmol/l quinine and swell in response, although the rapid response of epididymal spermatozoa demonstrated (Kulkarni et al., 1997) could not be confirmed for bovine ejaculated spermatozoa (Petroukina et al., 2000). Effects on other sperm functions are limited to those employing high doses of quinine that are spermicidal (Brown-Woodman and White, 1977) or which suppress motility and metabolism [1% w/v (~28 mmol/l) (Chow et al., 1980)]. In a transmembrane migration assay of sperm motility, the ED50 for the related compound quinidine, which was the most effective of several anti-arrhythmic drugs tested, was 0.5 mmol/l (Hong and Chiang, 1984).
The involvement of K+-channels in quinine action was confirmed in the present study by the abolition of its effects on both sperm volume and kinematics by the K+-ionophores valinomycin and gramicidin, with the latter being more effective. Although gramicidin is a monovalent cationic ionophore, it has a selectivity of K+ over Na+ of ~3:1 (Wallace, 2000). Whereas gramicidin used at 5 µmol/l can eliminate the membrane potential of mammalian spermatozoa (Zeng et al., 1995; Rockwell and Storey, 1999), it abolished the quinine effect at 0.5 µmol/l without affecting percentage motility. The same concentration is effective in opening the charybdotoxin-insensitive K+-channel responsible for RVD (MacLeod and Hamilton, 1999). Valinomycin, though a specific K+-ionophore, is less potent, achieving a <50% effect in opening K+-channels, including Kv1.5 (Holmes et al., 1997; Murayama et al., 1997). On the other hand, ouabain, an inhibitor of the Mg2+-activated Na/K-ATPase, exhibited no effect on sperm volume and failed to mimic the quinine influence on kinematics after 20 min incubation. The slight suppression of VSL by 1 and 2 mmol/l ouabain was not unexpected, as inhibition of sperm motility at longer incubation times or higher doses is well established (Nelson and McGrady, 1981). The present findings indicate that disruption of K+-channel(s) function was responsible for both the volume increase and the kinematic alterations imposed on human spermatozoa by quinine.
Depending on cell types, RVD can be Ca2+-dependent via the Ca2+-activated K+-channels (McCarty and O'Neil, 1992), or Ca2+-independent K+-channels may be involved (Moran et al., 1997). Human spermatozoa still responded to quinine in the absence of Ca2+ with increased ALH and reduced LIN, although significant changes in VSL and VCL similar to those obtained in the presence of Ca2+ were not attained. This indicates that Ca2+ is not directly involved in the quinine action on human spermatozoa. Omission of Ca2+ also attenuated but did not abolish swelling of mouse spermatozoa induced by quinine (Yeung et al., 1999). These results are consistent with the summary view that Ca2+-independent K+-channels for RVD are operational in non-epithelial cells and the Ca2+-dependent ones in epithelial cells (O'Neill, 1999), whereas Cl– and organic osmolyte efflux pathways are Ca2+-independent (Pasantes-Morales and Morales Mulia, 2000).
Features of the apparently Ca2+-insensitive K+-channel involved in the quinine action on spermatozoa described here were probed using other well-studied or specific blocking agents to see if they could mimic the quinine action. Kinematic effects were particularly examined as they were more sensitive than changes in sperm size. The most frequently used K+-channel blocker, TEA, was ineffective up to 10 mmol/l and suppressed both VSL and VCL at 20 mmol/l, which could mark the beginning of non-specific effects. TEA has well-known effects on the maxi-K channels and the shaker family members including Kv1.3 (at 10–5 to 10–3 mol/l) (Kuriyama et al., 1995; Coetzee et al., 1999), but not Kv1.5 which is one of the two members involved in RVD. That Kv1.3 is not involved in the quinine action on spermatozoa was also suggested by the failure of its specific inhibitor, margatoxin (Knaus et al., 1995), to affect sperm kinematics over a wide dose range. The other scorpion toxin, charybdotoxin, was also ineffective at doses up to 100 nmol/l. This is known to inhibit the Ca2+-dependent maxi-K channels as well as the Ca2+-independent Kv channels (see Kuriyama et al., 1995) at 1–10 nmol/l, but not affect Kv1.5 channels (Coetzee et al., 1999). 4-AP, which is more selective and potent than TEA, was the only blocker shown to mimic the quinine effect partially. This inhibitor has effects on different types of outwardly rectifying K+-channels, with IC50 for Kv channels at 10–4 to 10–3 mol/l (Kuriyama et al., 1995; Coetzee et al., 1999). Therefore, the present findings suggest that quinine acts on spermatozoa through certain TEA-insensitive, Ca2+- independent and 4-AP-sensitive K+-channel(s) excluding Kv1.3.
Mammalian spermatozoa have high intracellular K+ concentrations, reported to be 90–122 mmol/l in bulls and mice (Babcock, 1983; Chou et al., 1989; Zeng et al., 1995), and this could serve as an osmolyte for RVD. The presence of sperm K+-channels has been indicated in lipid bilayer (Cox et al., 1991; Chan et al., 1997) and germ cell mRNA heterologous expression studies (Chan et al., 1998), and suggested by charybdotoxin binding to spermatozoa (Jacob et al., 2000). However, the only fully identified germ cell K+-channel is the Ca2+-insensitive, voltage-sensitive and pH-dependent slo3 (Schreiber et al., 1998). The sperm functions regulated by such K+-channels are not known. Besides K+-channels, quinine can also affect the volume-sensitive organic anion channels (VSOAC) (Strange and Jackson, 1995; Okada, 1997). These can be voltage-gated chloride channels (ClC-2), large conductance channels or outwardly rectifying intermediate conductance channels that permit passage of organic osmolytes (e.g. taurine, inositol, sorbitol, betaine, glycerophosphocholine) (Strange et al., 1996). Although the presence or concentrations of such osmolytes in spermatozoa is not known, the epididymis is known to synthesize, secrete and transport some of these low molecular weight organic solutes in high amounts (Cooper, 1998).
Somatic cells have intracellular osmolality similar to that of blood serum (~300 mmol/kg) and to study their RVD, somatic cells are exposed to hypotonic conditions (usually ~190 mmol/kg) where swelling caused by water influx activates the RVD mechanism involving efflux of K+, Cl– or organic osmolytes. The cells then rapidly regain their original volume, except when the osmolyte channels are blocked by pre-treatment with channel blockers. In the present study, quinine was effective in media of both 310 and 330 mmol/kg, as also shown for the induction of tail angulation in mouse spermatozoa (Yeung et al., 1999). This suggests that the intracellular osmolality of spermatozoa is higher than in somatic cells and may reflect their normal environment in the epididymis. Data for human epididymal fluid are not available but the vas deferens contains fluid of 342 mmol/kg (Hinton et al., 1981) and epididymal fluid from many mammalian species is of high osmolality (up to 430 mmol/kg) (Cooper 1986; Yeung et al., 1999). Therefore it is likely that mammalian sperm cells have a high intracellular concentration of osmolytes which are yet to be identified.
Upon ejaculation, spermatozoa experience a decrease in extracellular osmolality as they leave the seminal plasma (~340 mmol/kg, present data) to ~290 mmol/kg as they enter cervical mucus and are eventually bathed in follicular fluid (Edwards, 1974; Casslén and Nilsson, 1984; Rossato et al., 1996). Therefore, RVD is required to counteract cell swelling. This RVD mechanism appears to be developed during sperm maturation in the epididymis (Yeung et al., 1998, 1999). The present findings showed that human sperm kinematics in seminal plasma and artificial mucus are affected by the RVD inhibitor quinine in the same way as washed spermatozoa, although the effects occurred to a lesser extent since the viscosity of the semen and mucus restricted the motility of even the control spermatozoa. One consequence of the quinine-induced decrease in effectiveness of forward propulsion was revealed in the curtailment of sperm cell penetration into, and migration through, a column of surrogate cervical mucus. Interestingly, when the mucus column did not contain quinine, migration was little different from that of control spermatozoa migrating from quinine-free semen, attesting to the reversibility of the quinine action.
The role of ion channels in sperm function, especially those involved in volume regulation, as well as the identities of osmolytes in spermatozoa, has hardly been studied. The infertile c-ros knockout mouse model and the hindrance of human sperm penetration of mucus by quinine have highlighted the physiological importance of a new aspect of sperm function regulation which warrants further investigation, both for the understanding of male infertility and the possible development of male contraceptives.
Acknowledgements
The authors are grateful to Barbara Hellenkemper and Raphaele Kürten for their competent technical help and Heidi Kersebom, Diane Küsters, Sebine Rehr and Daniela Schmidt for the routine semen analysis. The work was supported by the Deutsche Forschungsgemeinschaft Confocal Grant no. Ni130/15: `The Male Gamete: Production, Maturation, Function'. We thank Professor Eberhard Nieschlag for his support and encouragement.
Notes
1To whom correspondence should be addressed. E-mail: yeung{at}uni-muenster.de
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