Chemicals

DisC₃(5), 3, 3′-dipropylthiadicarbocyanine iodide, was purchased from Molecular Probe (Leiden, Netherlands); carbonyl cyanide mchlorophenylhydrazone (CCCP) and valinomycin were from Sigma Chemical Co. (St. Louis, MO); W-5, W-7, KN-92 and KN-93 were from Seikagakukogyo (Tokyo, Japan); tetraethylammonium chloride (TEA) from Nacalai Tesque (Kyoto, Japan); the K⁺ channel blockers, α-dendrotoxin, β-dendrotoxin, γ-dendrotoxin, δ-dendrotoxin, dendrotoxin-I, apamin and mast cell-degranulating peptide (MCD-peptide), and the Ca²⁺ channel blockers, nifedipine, nimodipine, taicatoxin, FS-2, ω-conotoxin MVIIA, PLTX-II, sFTX3.3 and FTX3.3 were from Alomone Laboratories (Jerusalem, Israel); and the cyclic AMP enzyme immunoassay (EIA) system (PRN 225) was from BIOTRAK (Amersham Pharmacia Biotech, Buckinghamshire, England). CCCP, valinomycin, DiSC₃(5) and nifedipin were dissolved in DMSO at 0.2, 0.1 and 0.1% v/v, respectively. Nimodipine was dissolved in 1% ethanol. These concentrations had no deteriorating effect on sperm motility, and the media used in the control experiment contained the same concentration of the solvents. All other reagents used were of analytical grade.

Collection of sperm

Mature males of the rainbow trout and the steelhead trout, which is the sea-run rainbow trout, Oncorhynchus mykiss were provided by the Yamanashi Prefectural Fisheries Experiment Station of the Oshino Trout Hatchery (Fujiyoshida, Japan) and kept in an indoor aquarium at 10°C. The fish were not fed during the experiments. Semen was collected directly by inserting a pipette into the sperm duct and stored on ice until use.

Assessment of sperm motility

A glass slide was coated with 1% bovine serum albumin and then dried to prevent sperm from sticking to the glass. A two-step dilution procedure for measuring sperm motility was used, as reported by Cosson et al. (1989), to achieve synchronous induction of sperm motility. The semen was diluted 10-fold in physiological saline (ASP) consisting of 130 mM NaCl, 40 mM KCl, 2.5 mM CaCl₂, 1.5 mM MgCl₂ and10 mM HEPES-NaOH at pH 7.8. The composition of the ASP was based on the ionic and osmotic composition of the seminal plasma of the rainbow trout (Morisawa, 1985). Sperm motility was completely suppressed in the ASP by the 40 mM KCl. The prediluted sperm was placed on the glass slide, which was placed on the stage of a microscope (Nikon Optiphoto, Tokyo, Japan) with a dark field condenser lens and without a cover glass. The sperm suspension was diluted 100-fold by addition of Activation Solution (AS) consisting of 150 mM NaCl and 10 mM HEPES-NaOH at pH 7.8. The second dilution step reduced the K⁺ concentration surrounding the sperm, thereby inducing motility of the sperm on the glass slide. The motility of the sperm was recorded on a video camera (Hamamatsu C2400 SIT; Hamamatsu, Japan) attached to the microscope. The percentage of motile sperm and their velocity were measured from images of the tracks of sperm using a Cellsoft semen analyzer (Cellsoft series 3000; NAC Incorporated, Tokyo, Japan).

Effects of ion channel blockers and calmodulin inhibitors on sperm motility

The effects of ion channel blockers and calmodulin inhibitors on the initiation of sperm motility were investigated by diluting the sperm in the ASP containing various concentrations of ion channel blockers and calmodulin inhibitors, and then suspending the sperm in AS containing each blocker at the same concentration.

Membrane potential measurement

The semen was diluted in the ASP at a dilution ratio of 1:10. After predilution, 20 μl of the sperm suspension was diluted in 2 ml of AS containing 1 μM DiSC₃5 in a quartz cuvette in the presence or absence of an inhibitor. Mitochondrial potential was eliminated by subsequent addition of 1 μM CCCP, followed by addition of 1 μM valinomycin to make the plasma membrane maximally permeable to K⁺. Finally, KCl was added. During the procedures, DiSC₃(5) fluorescence was monitored by a fluorescence spectrophotometer (Hitachi 650-10S; Hitachi, Tokyo, Japan) at an excitation wavelength of 620 nm and emission wavelength of 670 nm with continuous stirring at 18°C. A single loading of 1 μM DiSC₃(5) to the trout sperm did not significantly change the motility. Hyperpolarization and depolarization of the plasma membrane resulted in a decrease and an increase in fluorescence intensity, respectively, under the experimental conditions.

Assay of cyclic AMP

The semen was diluted in the ASP at a dilution ratio of 1:10, and 3 μM of the sperm suspension was suspended in 300 μM of AS alone or AS containing KCl, ion channel blockers, calmodulin inhibitors and/or valinomycin. After the necessary incubation time, 180 μl of the suspension was mixed with 20 μl of Kit buffer (lysis regent) to stop cyclic AMP synthesis and dissolve the cells. 100 μl of the mixture was then added to well of the kit to quantify cyclic AMP according to the cyclic AMP EIA system manual (BIOTRAK RPN 225; Amersham Pharmacia Biotech, England). The cyclic AMP level of each sample was calculated by measuring the 450-nm absorbance with a microplate reader (Model 550; Bio-Rad, Richmond, CA). The protein concentration was measured using a commercial reagent for protein measurement from BIO-RAD (Bio-Rad Protein Assay; Bio-Rad Laboratories, Hercules, CA).

A two-paired Student's t-test was used for the statistic analysis. Data were expressed as the means±SD.

Effects of K⁺- and Ca²⁺-channel blockers on sperm motility

Just after dilution of the ASP-pre-diluted sperm into AS, more than 95% of the sperm of the rainbow trout and steel-head trout began to move straight with an average velocity of 180 μm/sec. The percentage of motile sperm declined within 15 sec, and the swimming pattern became circular and halted by 25 sec (data not shown). The results were almost the same as those obtained previously in chum salmon (Morisawa and Suzuki, 1980), masu salmon, char (Morisawa et al., 1983) and rainbow trout (Morisawa et al., 1983; Cosson et al., 1991). Blockers effective for the voltage-dependent K⁺ channels that are mainly used in the neurons, β-dendrotoxin, δ-dendrotoxin, γ -dendrotoxin and dendrotoxin-I at the concentration of 1 μM and α-dendrotoxin at the concentration of 5 μM, effectively inhibited the sperm motility of rainbow trout (Fig. 1A). The blocker concentrations effective for inhibiting the motility of live sperm cells in the present study were higher than those used in the investigation of ion fluxes through the isolated sperm plasma membrane (Galindo et al., 2000). MCD-peptide at a concentration of 5 μM, which has been shown to inhibit the motility of Ciona sperm (Izumi et al., 1999), also inhibited trout sperm motility in the present work (Fig. 1A). Blockers of other types of K⁺ channels did not inhibit the sperm motility (data not shown). For example, apamin—a blocker of the small conductance Ca²⁺ -dependent K⁺ channel—and iberiotoxin, paxilline, penitrem and verruculogen-blockers of the large conductance Ca²⁺ -activated K⁺ channel-did not inhibit sperm motility at concentrations up to 200 μM (Fig. 1A). TEA, which is generally known to inhibit the efflux of K⁺ through the voltage-dependent K⁺ channels (Stanfield, 1983), suppressed 50% of sperm motility at concentrations lower than 25 mM in the rainbow trout (Fig. 1). The experimental media was kept at a constant osmolality of 300 mOsm/kg by replacing TEA for NaCl in AS. Sperm motility, which was blocked by 25–200 mM TEA, was reversibly recovered when the concentration of TEA was decreased by addition of AS, indicating that TEA is a useful tool for investigating transmembrane cell signaling in the trout sperm. Furthermore, sperm motility that was completely inhibited by 25 mM TEA was rescued when the plasma membrane was made maximally permeable to K⁺ by a K⁺-selective ionophore, valinomycin, at a concentration of 0.1 μM. These results suggest that K⁺ permeability that is sensitive to TEA and some other specific K⁺ channel blockers is essential for the initiation of trout sperm motility.

Fig. 1

Effects of blockers of K⁺- and Ca²⁺-channels on sperm motility. Sperm of the rainbow trout were diluted in ASP containing blockers of the voltage-dependent K⁺ channel (A): α-dendrotoxin (5 μM), β-dendrotoxin (1 μM), γ-dendrotoxin (1 μM), δ-dendrotoxin (1 μM), dendrotoxin-I (1 μM), apamin (10 μM), MCD-peptide (5 ^fi and 10 μM ^{fi fi}), TEA (25 mM); or blockers of the Ca²⁺ channel (B): nifedipine (100 μM), nimodipine (20 μM), taicatoxin (5 μM), FS-2 (20 μM), ω-conotoxin MVIIA (200 μM), PLTX-II (0.5 μM), sFTX3.3 (100 μM), FTX3.3 (10 μM) and trifluoroperazine (200 μM). After incubation for 5 minutes, aliquots of the sperm suspensions were diluted in AS containing the same concentration of blockers, and the percentage of motile sperm was measured. Valinomycin (Val) at a concentration of 0.1 μM was added to the immotile sperm in the presence of TEA or trifluoroperazine. Note that the sperm exhibited full motility in AS (control), but that some blockers known as blockers of the voltage-dependent K⁺ channels and blockers of L-/T-type Ca²⁺ channels inhibited sperm motility. Inhibition of motility by TEA or trifluoroperazine was relieved by addition of valinomycin. The data are expressed as the means±SD. Numbers of experiments are indicated in the parentheses in (A), and all experiments were performed in triplicate in (B). Data marked with asterisks were significantly different from the control data (p<0.05). TEA+Val and trifluoroperazine+Val were significantly different from +TEA (p<0.05). Double asterisks indicate an absence of significant difference.

FS-2 (20 μM) and taicatoxin (5 μM), which are considered to be blockers of L-type Ca²⁺ channels in the neuron, completely inhibited the sperm motility of the rainbow trout (Fig. 1B). Dihydropyridine, nimodipine (20 μM) and nifedipine (100 μM), which are known as blockers of T-type Ca²⁺ channels in sperm (Lievano et al., 1996) and as blockers of L-type Ca²⁺ channels in other excitable cells, inhibited sperm motility. Blockers of Ca²⁺ channels other than the L- and T- types Ca²⁺ channels such as ω-conotoxin MVIIA, PLTX-II, and FTX at the concentration of 0.5–200 μM, showed only slight inhibition. Trifluoroperazine, which can block Ca²⁺ channels as effectively as calmodulin, has been reported to inhibit the acrosome reaction in sea urchin sperm and the membrane potential of mouse sperm (Espinosa and Darszon, 1995). This blocker inhibited trout sperm motility, and this inhibition was relieved by addition of 0.1 μM valinomycin (Fig. 1B).

Effects of extracellular K⁺ concentration and K⁺ channel blocker on membrane potential

K⁺ at a concentration of 40 mM in the seminal plasma (Morisawa, 1985) completely suppresses sperm motility in the rainbow trout (Morisawa et al., 1983). A decrease in K⁺ concentration in the area surrounding the sperm causes an increase in sperm motility (Morisawa and Morisawa, 1986). In order to determine whether the initiation of sperm motility by the decrease in K⁺ concentration in the area surrounding the sperm correlates with K⁺-dependent changes in membrane potential, we directly monitored membrane potential using the fluorescent probe, DisC₃(5). Hyperpolarization of the membrane decreased DisC₃(5) fluorescence, while depolarization increased its fluorescence. As shown in Fig. 2, when rainbow trout sperm suspended in the ASP were suspended in AS containing DisC₃(5), the sperm became motile, and the dye fluorescence level became stable. After the membrane potential in AS became stable, the potential from mitochondria was cancelled by addition of CCCP, but this caused only a small increase in fluorescence (Fig. 2A), suggesting that the mitochondrial potential was negligible in the sperm cells. Therefore, the stable fluorescence level in the presence (Fig. 2A) or absence (Fig. 2B) of added CCCP is considered to represent the membrane potential of the sperm at each K⁺ concentration. The stepwise increase in external K⁺ concentration (arrows in Fig. 2) caused a stepwise decrease in the intensity of fluorescence, i.e., a stepwise depolarization of the plasma membrane both in the presence (Fig. 2A) and absence (Fig. 2B) of valinomycin. After the fluorescence intensity at the same final external concentration of K⁺ (75 or 50 mM) following stepwise addition of K⁺ was aligned (dotted line in Fig. 2), the levels of membrane potential in each experiment were compared. Consequently, the fluorescence intensity, which is comparable to the resting membrane potential at each K⁺ concentration (solid bars in Fig. 2) decreased with an increase in external K⁺ concentration. This suggests that the sperm plasma membrane behaves as a potassium electrode whose potential is defined by the Nernst equation, and that the gradual decrease in K⁺ concentration surrounding the sperm caused a gradual hyperpolarization of the plasma membrane. The decrease in fluorescence intensity was not due to quenching causing by KCl, since the fluorescence of AS containing DisC₃(5) did not change upon addition of 40 mM KCl (data not shown).

Fig. 2

Membrane potential of the sperm in the presence of various concentrations of external K⁺, ([K⁺]_ex). The sperm of rainbow trout diluted in ASP containing 40 mM [K⁺]_ex were diluted in AS containing the fluorescent dye DiSC₃(5) and 0, 10, 20 or 40 mM [K⁺]_ex. After the fluorescence level became stable, CCCP was added (filled arrowhead in A) to cancel mitochondrial membrane potential. After the fluorescence became stable, which represents the membrane potential of the sperm at each K⁺ concentration (solid bar), 0.1 μM valinomycin was added (open arrowhead) in A and then KCl was added (arrow) to the final K⁺ concentration of [K⁺]_ex (arrow with asterisk). In B, valinomycin was not added, and KCl was directly added. After the fluorescence level, namely the membrane potential, became stable in the presence of external 75 (A) or 50 mM [K⁺]_ex, the levels of 0, 10, 20 or 40 mM [K⁺]_ex were aligned (dotted line). Note that the membrane potential of the sperm (solid bar) in 40 mM [K⁺]_ex may be the membrane potential of the sperm in the seminal plasma containing 40 mM [K⁺]_ex (Morisawa, 1985), and the potential may become negative concomitantly with the decrease in K⁺ concentration from 40 mM to 0 mM [K⁺]_ex. Typical patterns of three experiments (A) and four experiments (B) are shown.

The degree of membrane hyperpolarization in the rainbow trout sperm decreased with increasing concentration of TEA, a K⁺ channel blocker (solid bars in Fig. 3A). Subsequent addition of valinomycin caused the hyperpolarization, and the membrane potentials (solid line at the bottom of Fig. 3A) were identical in the presence or absence of TEA. The difference between the fluorescence level after addition of CCCP and that after addition of valinomycin was 3- or 4-fold larger in the presence of 25 mM and 50 mM TEA, respectively, than in the absence of TEA (Fig. 3A). These results suggest that TEA inhibited not only the sperm motility (Fig. 1A) but also the membrane hyperpolarization (Fig. 3A).

Fig. 3

Effects of a K⁺- or Ca²⁺- channel blocker on membrane potential of the sperm. The sperm of the rainbow trout diluted in the ASP containing external 40 mM KCl, 40 mM [K⁺]_ex, were diluted in AS without KCl containing 0 mM, 25 mM or 50 mM TEA (A) or 100 μM nifedipine (B). After the fluorescence level, namely the membrane potential, became stable, CCCP was added (filled arrowhead). The stable level reached shows the sperm membrane potential (solid bar). Valinomycin at the concentration of 0.1 μM was then added (open arrowhead) to make the plasma membrane maximally permeable to K⁺. Subsequent addition of 30 mM KCl (arrow) depolarized the plasma membrane. The final levels after addition of 30 mM [K⁺]_ex (arrow with asterisk) were aligned (dotted line). Note that the membrane potential reached the same level (solid line in the bottom) after addition of valinomycin. The difference of membrane potential of sperm before (solid bars) and after addition of valinomycin (solid line in the bottom) became larger concomitantly with the increase in TEA concentration or presence of nifedipine. These findings suggest that a K⁺ channel blocker, TEA, and Ca²⁺ channel blocker, nifedipine, inhibited the hyperpolarization of the plasma membrane. [K⁺]_ex at the concentration of 40 mM inhibited membrane hyperpolarization (middle in B).

Nifedipine, a blocker of L-type Ca²⁺ channels in general and of T-type Ca²⁺ channels in sperm cells (Lievano et al., 1996) completely inhibited the hyperpolarization of the sperm plasma membrane (Fig. 3B) as well as the sperm motility (Fig. 1B) in rainbow trout. These results suggest that Ca²⁺ influx via the dihydropyridine-sensitive Ca²⁺ channel contributes to the membrane hyperpolarization and the initiation of sperm motility. Trifluoroperazine, which can inhibit Ca²⁺ channels (Espinosa and Darszon, 1995), suppressed the membrane hyperpolarization (data not shown).

Effects of channel blockers and valinomycin on cyclic AMP synthesis

The cyclic AMP concentration was 698 fmol/mg protein in the immotile sperm of rainbow trout in the presence of 40 mM K⁺ (40 mM [K]_ex in Fig. 4). The concentration increased significantly to 2291 fmol/mg protein (0 mM [K]_ex in Fig. 4) when sperm motility was initiated in AS without K⁺. When sperm were immobilized by 25 mM TEA, the cyclic AMP level became 774 fmol/mg protein, almost the same as that in the presence of 40 mM K⁺ (+TEA in Fig. 4). Addition of valinomycin to the sperm immobilized by TEA recovered sperm motility (Fig. 1A), and the cyclic AMP level increased to about 1588 fmol/mg protein (TEA+Val in Fig. 4), which was significantly different from that in the immotile sperm under the influence of 40 mM K⁺ (40 mM [K]_ex in Fig. 4). The cyclic AMP concentration in the immotile sperm of steelhead trout (465 fmol/mg protein) increased to 1990 fmol/mg protein in the presence of 0 mM [K]_ex, and to 545 fmol/mg protein in the presence of 200 mM TEA. Valinomycin relieved the cyclic AMP concentration up to 1071 fmol/mg protein (n=3). MCD-peptide, a blocker of the voltage-dependent K⁺ channel, inhibited the sperm motility of the rainbow trout (Fig. 1A). MCD-peptide strongly decreased the cyclic AMP, and subsequent addition of valinomycin recovered the cyclic AMP (data not shown). A Ca²⁺ channel blocker, trifluoroperazine, at a concentration of 200 μM inhibited cyclic AMP synthesis in the sperm of steelhead trout (Fig. 4B). Subsequent addition of valinomycin caused a slight increase in cyclic AMP.

Fig. 4

Effects of the K⁺ channel blocker, Ca²⁺ channel blocker and calmodulin inhibitor on cAMP synthesis. Cyclic AMP levels of sperm were measured 5 seconds after suspending the sperm of rainbow trout in the media containing 40 mM K⁺ (40mM [K⁺]_ex), 0 mM K⁺ (0 mM [K⁺]_ex), 25 mM TEA (0K+TEA) or TEA and 0.1 μM valinomycin (0K+TEA+Val) in A. In B, cyclic AMP levels of steelhead sperm were measured in the media containing 40 mM K⁺ (40 mM[K⁺]_ex), 0 mM K⁺ (0 mM[K⁺]_ex), 25 μM calmidazol (0K+Calmid), calmidazol + 0.1 μM valinomycin (0K+Calmid+Val), 100 μM trifluoroperazine (0K+TFP) or trifluoroperazine + 0.1 μM valinomycin (0K+TFP+Val). The control (0 mM[K⁺]_ex) had no K⁺ channel blocker, calmoduline inhibitors or valinomycin. Cyclic AMP level in the sperm whose motility was completely inhibited by TEA, calmidazol and trifluoroperazine was the same as that in the immotile sperm in the presence of 40 mM[K⁺]_ex. Subsequent addition of valinomycin caused the initiation of sperm motility and increase in cAMP. The data are indicated as the means±SD (n=3). An asterisk indicates a significant difference after paired t-test (p<0.05) from the data in the presence of 40 mM[K⁺]_ex.

Effects of inhibitors of calmodulin and valinomycin on sperm motility, membrane potential and cyclic AMP synthesis

Almost all rainbow trout or steelhead trout sperm became motile when suspended in AS. More than 80% of the rainbow trout sperm were motile in AS containing 0-100 μM W-5, an inactive analogue of W-7 (Fig. 5). W-7 at a concentration of 100 μM was required for 50% inhibition of sperm motility (Fig. 5). The half- maximal concentration required for inhibition by another calmodulin inhibitor, calmidazol-Cl, was 12.5 μM. Sperm whose motility was inhibited in the presence of 25 μM calmidazol-Cl or 200 μM W-7 became motile when valinomycin was added (data not shown). Sperm motility in steelhead trout was unaffected by the inhibitor of calmodulin kinase II, KN-93, or by its inactive analogue, KN-92, at concentrations up to 50 μM (data not shown).

Fig. 5

Effects of calmodulin inhibitors on sperm motility. Sperm of rainbow trout diluted in ASP containing various concentrations of the calmodulin inhibitors, W-5 (open circle), W-7 (closed circle) and calmidazol-Cl (closed triangle), were suspended in AS containing the same concentration of the inhibitors, and the percentage of motile sperm was measured. The data are expressed as the means±SD=(n=3). An asterisk indicates a significant difference from the control or +W-5 (p<0.05).

The membrane potential was slightly changed in the presence of W-5. On the other hand, W-7 (50 μM) and calmidazol (25 μM) strongly inhibited membrane hyperpolarization. However, subsequent addition of valinomycin caused only slight membrane hyperpolarization. The level of the membrane potential did not reach that seen in the controls or in the presence of W-5 (cf. solid line in the bottom of Fig. 3). In addition, stepwise addition of KCl did not cause a stepwise depolarization of the plasma membrane (data not shown). These results suggest that the calmodulin inhibitor may achieve its effect on sperm motility via some mechanism other than a direct effect on calmodulin.

Calmidazol-Cl also decreased cyclic AMP from a high level in 0 mM K⁺ to a low level comparable to that in 40 mM K⁺. Addition of valinomycin recovered the level of cyclic AMP (Fig. 4B). These results may suggest that these reagents do not specifically inhibit calmodulin. At the same time, however, they make clear that membrane hyperpolarization is prerequisite to cAMP synthesis.