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Biology of Reproduction 60, 102-109 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Progesterone-Mediated Calcium Influx and Acrosome Reaction of Human Spermatozoa: Pharmacological Investigation of T-Type Calcium Channels1

Manuel A. Garcia3,a, and Stanley Meizel2,a

a Department of Cell Biology and Human Anatomy, University of California, Davis, California 95616


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mechanisms of the progesterone (P4)-activated Ca2+ influx and the relationship between the intracellular free Ca2+ concentration ([Ca2+]i) and the acrosome reaction (AR) were investigated in this study. We compared the [Ca2+]i of uncapacitated and capacitated human sperm populations in response to P4 stimulation; characterized the effects of the pharmacological agents pimozide and mibefradil, inhibitors of T-type voltage-operated calcium channels (VOCCT), on the P4-activated Ca2+ influx; and determined the effects of these drugs on the P4-initiated AR. Since pimozide can also inhibit calmodulin-dependent enzymes, we examined the effects of the calmodulin antagonist, calmidazolium, on the above-mentioned events. The basal [Ca2+]i and the amplitude of the P4-activated Ca2+ influx were significantly (p < 0.05) higher in capacitated sperm populations. Also, in capacitated sperm populations, all three pharmacological agents significantly (p < 0.05) inhibited the P4-activated Ca2+ influx (IC50): calmidazolium (0.7 µM) > pimozide (8 µM) > mibefradil (11 µM). By contrast, the effects of these drugs on the P4-initiated AR were varied: pimozide (10 and 20 µM) significantly (p < 0.05) increased the percentage of AR spermatozoa, calmidazolium was without effect, and mibefradil (20 µM) significantly (p < 0.05) inhibited the AR. These disparate results do not allow us to reach any definitive conclusion concerning the role of a sperm VOCCT in the mechanism of the P4-initiated AR. However, the differences between the [Ca2+]i and AR effects, in particular the inverse relationship in the case of pimozide, suggest a dissociation between the amplitude of the P4-stimulated Ca2+ signal and the downstream biological effect of that signal, the AR.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rapid, nongenomic biological actions mediated by cell surface receptors have been described for several steroid hormones [1]. The initiation of the acrosome reaction (AR) by progesterone (P4) is an example of a nongenomic activity that is dependent on a transient elevation of intracellular free Ca2+ concentration ([Ca2+]i) [2, 3]. Several reports have demonstrated a rapid, transient P4-activated Ca2+ influx in human spermatozoa [4–11] involving a plasma membrane P4 receptor [12–17]. While there is agreement that nearly all spermatozoa in a population respond to P4 [4, 7, 8], the pathway(s) regulating the elevation of [Ca2+]i remains unresolved. Moreover, differences in P4-activated Ca2+ influx between uncapacitated and capacitated spermatozoa [5, 18] suggest that the regulation of [Ca2+]i may vary depending on the state of sperm maturation.

Ca2+ influx, through voltage-operated calcium channels (VOCC), is required for zona pellucida-initiated acrosomal exocytosis [19–22]. The specific type of VOCC required appears to belong to the T-type (VOCCT), or the low voltage-activated calcium channel family [23]. The involvement of a similar VOCCT pathway in the P4-activated Ca2+ influx is currently unresolved; however, if such a pathway were involved, then the membrane potential would represent a key regulatory component. Studies have demonstrated cation-dependent depolarization by P4, but their relationship to [Ca2+]i was not investigated [24, 25]. Similarly, several studies support the involvement of a sperm GABAA receptor-like Cl- channel in the P4-initiated mammalian AR [26–31], and recent studies [7] suggest that this pathway may regulate the P4-activated Ca2+ influx, possibly through alterations in membrane potential.

The aim of the present study was to characterize the P4-activated Ca2+ influx pathway using the pharmacological inhibitors of VOCCT, pimozide, and mibefradil. Pimozide inhibits the zona pellucida-stimulated increase in [Ca2+]i [23], and mibefradil is a newly described Ca2+ channel antagonist with T-channel selectivity [32]. Pimozide also inhibits calmodulin-dependent enzymes [33, 34]; therefore, we investigated the effects of an unrelated calmodulin antagonist, calmidazolium, on the P4-activated Ca2+ influx. The significance of our results pertaining to the involvement of a VOCCT and calmodulin-dependent pathways in the regulation of the P4-activated Ca2+ influx and AR are discussed.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents

The following chemicals and reagents were purchased: P4 (4-pregnen-3,20-dione) and calmidazolium from Calbiochem (San Diego, CA); pimozide from RBI (Natick, MA); mibefradil dihydrochloride from Hoffmann-La Roche (Basel, Switzerland); Pentex fraction V BSA from Bayer Inc. (Kankakee, IL); Con A-fluorescein isothiocyanate (FITC) from EY Laboratories (San Mateo, CA); and fura 2-AM from Molecular Probes Inc. (Eugene, OR). All other chemicals were of reagent grade and were purchased from Sigma Chemical Company (St. Louis, MO) or Fisher Scientific Inc. (Pittsburgh, PA). Deionized water used in these experiments was purified to >= 18 M{Omega}-cm with a NANO-pure ion-exchange system (Barnstead/Thermolyne, Dubuque, IA).

Preparation of Spermatozoa

Human semen was obtained from healthy donors by masturbation with the approval of the human subjects committee at the University of California, Davis. A population of > 95% motile spermatozoa was obtained by centrifugation of semen samples through a discontinuous Percoll (Pharmacia and Upjohn, Kalamazoo, MI) gradient and subsequent washing in human sperm medium (HSM) as previously described [35]. However, the HSM was modified by increasing the NaHCO3 concentration to 34 mM. The final sperm suspensions were prepared by diluting the spermatozoa to 6 x 106/ml in HSM containing 26 mg/ml BSA (HSM-26B). These sperm suspensions were immediately used in studies involving uncapacitated spermatozoa. To promote capacitation, sperm suspensions in HSM-26B (0.5-ml aliquots/tube) were incubated at 37°C in loosely capped 15-ml polypropylene centrifuge tubes for 24 h in a humid atmosphere of 5% CO2:95% air.

Measurement of [Ca2+]i

Uncapacitated and capacitated sperm suspensions in HSM-26B (2 ml/tube) were prepared for [Ca2+]i determination by loading with the acetoxy-methyl ester of fura-2 (1 µM final extracellular concentration) for 30 min at 37°C. After dye loading, each 2-ml sample was centrifuged (300 x g for 20 min) through 750 µl of a 40% Percoll solution. This Percoll solution was prepared by diluting a stock solution of 95% Percoll, 150 mM NaCl, and 10 mM Hepes (pH 7.5) with Hepes-buffered fura medium containing 3 mg/ml BSA (FM-3B; [3]). After centrifugation, the supernatants were discarded, and each sperm pellet was washed with approximately 14 ml of FM-3B/tube and centrifuged at 300 x g for 10 min. The resulting sperm pellets were pooled, and the concentration of the pooled sperm suspension was determined. This concentrated sperm suspension was incubated for 30 min at 37°C in tightly capped centrifuge tubes and then maintained at room temperature before spectrofluorometric analysis. Aliquots of the concentrated sperm suspension were then diluted with FM-3B medium to a final sperm concentration of 6 x 106 sperm/ml. In experiments in which a high extracellular [K+] (100 mM KCl) was necessary, the FM-3B medium was modified by substituting KCl for NaCl (FMK-3B; final [KCl], 126 mM), and a combination of FMK-3B and FM-3B media was used to dilute the concentrated sperm suspension to 6 x 106 sperm/ml.

Spectrofluorometric [Ca2+]i measurements were performed using an excitation wavelength pair of 364/385 nm, an emission wavelength of 510 nm, and a 5-nm excitation/emission bandpass to abolish viscosity artifacts as previously described [36, 37]. Spectrofluorometry was performed in a methylacrylate cuvette magnetically stirred and warmed to 37°C in a heated cuvette holder. Sufficient time (2–5 min) was allowed for the temperature of the sperm suspension to reach 37°C before measuring [Ca2+]i. Pimozide (1–20 µM), mibefradil (1–20 µM), or calmidazolium (0.1–2 µM) was added to the sperm suspension approximately 100 sec after the beginning of each sample run (900 sec total) in order to facilitate the collection of basal [Ca2+]i. P4 (3.2 µM, final) was added 10 min after (t = 700 sec) the addition of the inhibitors. Sequential additions of 20 µM digitonin and 25 mM Tris-EGTA were made near the end of each sample run to facilitate determination of "calculated [Ca2+]i" as previously described [38], using a Kd of 285 nM for fura-2 at 37°C [39]. In certain experimental runs, the addition of P4, digitonin, and Tris-EGTA was omitted to determine the percentages of motile spermatozoa and fura-2-labeled spermatozoa after a 10-min treatment with calmidazolium or pimozide, by phase-contrast and fluorescence microscopy, respectively.

Background fluorescence or Ca2+-insensitive fura-2 fluorescence was determined by exposing fura-loaded spermatozoa to MnCl2 (2 mM) and ionomycin (20 µM) [6, 40]. The measured fluorescence was then subtracted from the fluorescence intensity of the corresponding experimental samples before determination of calculated [Ca2+]i. Any nonspecific effects of calmidazolium (2 µM), mibefradil (20 µM), and pimozide (20 µM) on fura-2-loaded spermatozoa were investigated using digitonin-permeabilized (10 µM) capacitated and uncapacitated spermatozoa. Moreover, any changes in autofluorescence were determined by measuring the emission fluorescence intensity (510 nm; 364/385 nm excitation) of nonloaded spermatozoa suspensions (6 x 106 spermatozoa/ml of FM-3B) before and after the addition of calmidazolium (2 µM) and pimozide (20 µM).

Determination of the AR

Capacitated human spermatozoa in HSM-26B were pretreated (10 min) with pimozide, mibefradil, and calmidazolium at concentrations used for spectrofluorometry to determine their effects on the P4-initiated AR. In other experiments, capacitated sperm suspensions in HSM-26B were pooled (2-ml/tube), centrifuged (300 x g for 10 min), and resuspended in FM-3B. The AR was initiated by adding P4 (3.2 µM) to sperm suspensions. The level of spontaneous or pimozide/calmidazolium-induced AR was determined by adding dimethyl sulfoxide (DMSO, 0.1%) in parallel with P4 treatments. Sperm suspensions were then incubated at 37°C in a humid atmosphere of 5% CO2:95% air for 5 min to allow the AR to proceed. At the end of the incubation period, 10-µl aliquots were removed from each treatment group to evaluate motility while the remaining spermatozoa were fixed for AR assay by addition of 4% formaldehyde in PBS. Fixed spermatozoa were labeled with FITC-Con A lectin [41]. A minimum number of 200 spermatozoa were scored in each sample, and identification of samples was coded to determine the percentage of AR spermatozoa in a "blind" fashion.

Measurement of Motility

Using phase-contrast microscopy, the percentage of motile spermatozoa was determined by counting 100–200 spermatozoa per treatment at x400 magnification. Additionally, a subjective score was assigned to the quality of sperm motility using a scale from 1 ("twitching," nonprogressive motion) to 4 (vigorous forward motility [35]).

Statistical Analysis

The significance of mean value differences between treatment groups was determined by ANOVA using a randomized complete block design with the donor as the blocking variable (donor effect). A Dunnett one-tailed means separation test was applied when more than two treatment groups were compared. The percentage values for motility and AR data were arcsine-transformed before analysis [42].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Motility and Fura-2 Fluorescence

At the highest concentrations used in our experiments, neither pimozide (20 µM), mibefradil (20 µM), calmidazolium (2 µM), or high extracellular [K+] (100 mM KCl) had any significant (p = 0.47) effect on the percentage of motile sperm (> 75% for all treatments). Similarly, at these or lower concentration, none of the drugs significantly (p = 0.37) decreased the percentage of spermatozoa that retained fura-2 fluorescence in the area of the sperm head. There was, however, a decrease in the subjective quality of sperm motility in capacitated spermatozoa (from grade 2–3 to 1–2) at the highest drug concentrations and in high-potassium medium (data not shown). Even higher concentrations of pimozide (50 µM), mibefradil (100 µM), or calmidazolium (5 µM) resulted in a marked decrease in the percentage of motile spermatozoa (data not shown). Therefore, our dose-response curves were limited to drug concentrations that did not significantly reduce the percentage of fura-loaded sperm or the motility of those sperm.

The fluorescence emission intensity of digitonin (10 µM)-permeabilized spermatozoa was not affected by pimozide (20 µM), mibefradil (20 µM), or calmidazolium (2 µM) (data not shown), suggesting that these compounds do not interfere with fura-2 fluorescence in a nonspecific or Ca2+-independent manner. Similarly, the autofluorescence levels of nonloaded spermatozoa at the same concentration as the fura-loaded spermatozoa suspensions (6 x 106 sperm/ml) were not increased by the addition of 2 µM calmidazolium or 20 µM pimozide (data not shown).

[Ca2+]i in Uncapacitated and Capacitated Spermatozoa

As previously reported [5, 18], the basal [Ca2+]i and the amplitude of the P4-activated Ca2+ influx, in the absence of inhibitors, were greater in capacitated than in uncapacitated spermatozoa (Fig. 1). A donor effect (p = 0.0001) was detected for basal [Ca2+]i, demonstrating the significant interdonor variation with respect to basal [Ca2+]i. The characteristics of the P4-activated Ca2+ influx were also different for capacitated and uncapacitated spermatozoa. In uncapacitated spermatozoa, the [Ca2+]i elevation was rapid (< 30 sec) and transient, returning to basal levels within a minute of P4 treatment (Fig. 2). In contrast, Ca2+ influx in capacitated spermatozoa did not return to baseline, but rather reached a plateau that was higher than basal [Ca2+]i (Fig. 2). We have previously demonstrated that the difference between plateau and basal [Ca2+]i is directly correlated with the percentage of AR spermatozoa [36].



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  		FIG. 1. The mean [Ca2+]i before P4 treatment (BASAL), and the peak [Ca2+]i immediately (< 30 sec) after the addition of P4 (PEAK) were compared in uncapacitated and capacitated human spermatozoa. The error bars represent the SEM, and the number of experimental replicates is denoted in parentheses within each bar. *Significant (p < 0.05) difference between uncapacitated and capacitated spermatozoa.



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  		FIG. 2. Dynamic differences in the P4-activated Ca2+ influx between uncapacitated and capacitated sperm populations. Uncapacitated or capacitated human sperm suspensions were treated with DMSO (0.1%) at 100 sec to represent the solvent control for pimozide and calmidazolium treatments. Ten minutes after the DMSO treatment, the sperm suspensions were treated with P4 (3.2 µM). The data points represent uncapacitated and capacitated sperm suspensions from a single donor, which were typical of the results from a total of 27 replicates (14 uncapacitated; 13 capacitated) using a total of 12 different donors.

Pharmacological Characterization of the P4-Activated Ca2+ Influx Pathway

Spermatozoa from a total of 12 different donors were used to examine the dose-dependent effect of pimozide (1–20 µM), mibefradil (1–20 µM), and calmidazolium (0.1–2 µM) on [Ca2+]i. Interestingly, the addition of any of these drugs alone resulted in a rapid (<= 60 sec) and dose-dependent increase of the [Ca2+]i in uncapacitated spermatozoa (Fig. 3). A similar increase was seen in capacitated spermatozoa for pimozide and calmidazolium, but not for mibefradil (Fig. 3). In most cases, the [Ca2+]i returned to pretreatment levels before the addition of P4; however, a small but significant (p < 0.05) elevation of the resting [Ca2+]i was caused by pimozide (>= 1 µM) and calmidazolium (>= 0.5 µM) in uncapacitated spermatozoa, and by calmidazolium (2 µM) in capacitated spermatozoa.



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  		FIG. 3. The transient stimulatory effect of pimozide (a), mibefradil (b), and calmidazolium (c) on [Ca2+]i of uncapacitated and capacitated human spermatozoa. The {partial} [Ca2+]i represents the change in [Ca2+]i immediately (<= 60 sec) after the addition of the inhibitors. The error bars represent the SEM. *Stimulatory effect significantly (p < 0.05) greater than the solvent control at the given concentration. The pimozide and calmidazolium solvent control (0.1% DMSO) values for uncapacitated and capacitated spermatozoa were 55 ± 8 nM and 76 ± 16 nM, respectively (mean ± SEM). The mibefradil solvent control (1 µl H2O/1 ml sperm suspension) values for uncapacitated and capacitated spermatozoa were 8 ± 5 nM and 12 ± 8 nM, respectively (mean ± SEM). The numbers of experimental replicates are listed in parenthesis adjacent to each data point, except for mibefradil (n = 4 at all concentrations).

In addition to their stimulatory effect, all three drugs caused a dose-dependent decrease in the P4-activated Ca2+ influx (Fig. 4). In capacitated spermatozoa, pimozide (IC50 = 8 µM) and mibefradil (IC50 = 11 µM) were equally potent at inhibiting the P4-activated Ca2+ influx. By contrast, calmidazolium was a more potent inhibitor of the P4-activated Ca2+ influx (IC50 = 0.7 µM). Additionally, except for pimozide, the potency of these inhibitors was unaffected by capacitation (uncapacitated IC50: mibefradil = 9 µM, calmidazolium = 0.7 µM). In the case of pimozide, uncapacitated sperm suspensions were less sensitive (IC50 = 16 µM) to inhibition by this drug.



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  		FIG. 4. Dose-dependent inhibition of the P4-activated Ca2+ influx in capacitated human spermatozoa by pimozide (a), mibefradil (b), and calmidazolium (c). Human sperm suspensions were treated with pimozide (n = 4–6), mibefradil (n = 5–7), or calmidazolium (n = 4–5) 10 min before the addition of P4 (3.2 µM). The data represent the percent inhibition of the solvent control treatment group (mean ± SEM). The IC50 was calculated by simple regression analysis of logit-log plots.

It has been demonstrated that the potency of pimozide as a VOCC antagonist depends on the membrane potential [43–45]. Similarly, voltage-dependent blockade by mibefradil has been observed for VOCC in somatic cells [32]. When the extracellular [K+] was increased to 100 mM to promote membrane depolarization, we saw a potentiation of the inhibitory activity of pimozide in capacitated sperm populations (Fig. 5). Mibefradil (10 µM), in contrast, did not demonstrate any significant (p > 0.05) voltage-dependent effect in capacitated spermatozoa. The change (mean value ± SEM) in [Ca2+]i for mibefradil-treated sperm suspension after a P4 stimulation in FM-3B was 954 ± 134 nM (n = 6) and in FMK-3B, 698 ± 127 nM (n = 5). The elevation of extracellular [K+] in the absence of inhibitor (solvent control group) did not affect the [Ca2+]i (data not shown) or the change in [Ca2+]i in response of to P4 stimulation (Fig. 5). However, it should be noted that a transient increase in [Ca2+]i in response to the depolarization treatment may have been missed because the [Ca2+]i of sperm suspensions was not measured until at least 30 sec after their dilution into the high-potassium medium.



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  		FIG. 5. Inhibition of the P4-activated Ca2+ influx by pimozide was potentiated in high-potassium medium (100 mM KCl). Capacitated human sperm suspensions in normal medium (FM-3B) and high-potassium medium (FMK-3B) were pretreated (10 min) with pimozide (PMO; 5 µM), or solvent control (SC; 0.1% DMSO) before the addition of P4 (3.2 µM). The {partial} [Ca2+]i represents the change in [Ca2+]i immediately (< 30 sec) after the addition of P4. The error bars represent the SEM for a total of seven experimental replicates for each treatment. Different symbols indicate that the results of corresponding treatments were significantly (p < 0.05) different from that of the 5 µM pimozide in FM-3B (Dunnett's two-tailed test).

Pharmacological Characterization of the P4-Initiated AR

Capacitated spermatozoa from a total of seven different donors were used to examine the effects of pimozide, mibefradil, and calmidazolium on the P4-initiated AR. Since pimozide or calmidazolium alone produced an elevation of the [Ca2+]i, we investigated whether these drugs could initiate the AR. The percentages of acrosome-reacted spermatozoa after a 10-min incubation with pimozide (1–20 µM) or calmidazolium (0.1–2 µM) in the absence of P4 were not significantly (p = 0.13; n = 3; data not shown) higher than those in paired solvent controls (0.1% DMSO in HSM-26B), and most likely represent spontaneous acrosome reactions that had previously occurred during capacitation.

P4 stimulation of capacitated human spermatozoa after a 10-min preincubation with pimozide, mibefradil, or calmidazolium demonstrated some interesting results. While all three of the pharmacological agents similarly inhibited the P4-activated Ca2+ influx, their effects on the P4-initiated AR were vastly different. Mibefradil (20 µM) significantly (p < 0.05) inhibited the P4-initiated AR (Fig. 6a). Since 20 µM mibefradil was higher than the level required to inhibit the P4-activated Ca2+ influx (IC50 = 11 µM), we retested this drug using FM-3B to avoid any decrease in the free drug concentration due to the higher BSA concentration of the HSM-26B medium. Under these conditions, mibefradil concentrations of 5 and 10 µM did not result in significant inhibition of the P4-initiated AR (Fig. 6b). In contrast to the inhibitory effects of mibefradil, pimozide (10 µM) produced a significant (p < 0.05) potentiation of the P4-initiated AR (Fig. 7). This effect was not due solely to an inhibition of calmodulin-dependent enzymes by pimozide, because calmidazolium (0.1–2 µM), a calmodulin antagonist and more potent inhibitor of the P4-activated Ca2+ influx, produced no significant (p = 0.98; n = 4; data not shown) effect on the P4-initiated AR.



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  		FIG. 6. Mibefradil (20 µM) inhibited the P4-initiated AR. Mibefradil (MBF; 5–20 µM) or water (SC; 1:1000 v:v dilution) was added to capacitated human spermatozoa in HSM-26B (a) or FM-3B (b) 10 min before the addition of P4 or solvent control (SC; 0.1% DMSO). Sperm motility was > 70% before the addition of P4 in all treatment groups, and did not change after the addition of P4. The data represent the mean and SEM of 4 (a) or 5 (b) experimental replicates, except P4 in HSM-26B (n = 6). *Mean value significantly (p < 0.05) lower than respective P4 treatment group.



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  		FIG. 7. The inverse relationship between pimozide's effect on the P4-activated Ca2+ influx and the P4-initiated AR in capacitated human spermatozoa. Capacitated human spermatozoa were pretreated with pimozide for 10 min before the addition of P4 (3.2 µM), and the change in [Ca2+]i and the percentage of AR sperm were determined. The data represent the mean and SEM of 5 (AR) or 4–7 ([Ca2+]i) experimental replicates. For the calcium experiments, the values within the parentheses represent the number of experimental replicates for each pimozide concentration (1–20 µM).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
P4 stimulates Ca2+ influx in both uncapacitated and capacitated spermatozoa [4–6, 10, 18, 25, 36, 46]. Previous comparisons between these two populations in the same experiment were contradictory and suggested that the amplitude of the P4-activated Ca2+ influx was similar [6, 46] or greater in the capacitated population [5, 18]. Our results are in agreement with those of the latter studies and indicate that both the amplitude and the time course of the P4-activated Ca2+ influx differ between these populations (Figs. 1 and 2). Moreover, the results of our experiments suggest that there are differences in [Ca2+]i regulation between uncapacitated and capacitated spermatozoa. A possible explanation for this difference in [Ca2+]i regulation may be that the process of capacitation results in the activation of a Ca2+ permeation pathway that is otherwise inactive in uncapacitated spermatozoa. A proposed candidate for this Ca2+ permeation pathway is the sperm VOCCT [23, 47]. Since only VOCCT are detected in spermatogenic cells, it is likely that this pathway is a key regulator of [Ca2+]i in ejaculated spermatozoa [23, 45].

The involvement of VOCC in the P4-activated Ca2+ influx has been contested. Studies using mouse and human spermatozoa have demonstrated an inhibition of the P4-activated Ca2+ influx and/or AR by VOCC antagonists such as verapamil [48] and nifedipine [29, 49]. However, other reports found no effect of these inhibitors on the P4-activated Ca2+ influx [4, 25]. These contradictory results highlight the difficulty of characterizing the P4-activated Ca2+ influx pathway in ejaculated spermatozoa. Unlike spermatogenic cells, spermatozoa are not amenable to electrophysiological experiments, thus precluding characterization of the Ca2+ influx pathway on the basis of its biophysical properties. An added complication of trying to identify the involvement of VOCC in the P4-activated Ca2+ influx is the absence of pharmacological tools with sufficient selectivity and potency. Recently, however, a new pharmacological inhibitor, mibefradil, with VOCCT selectivity has been described [32, 50]. Using mibefradil and a different class of VOCCT inhibitor, pimozide [23], we sought to characterize the P4-activated Ca2+ influx pathway. Our results demonstrated a dose-dependent inhibition of the P4-activated Ca2+ influx by both mibefradil and pimozide (Fig. 4) but opposing effects on the P4-initiated AR (Figs. 6 and 7). Moreover, a third class of pharmacological agent (a calmodulin antagonist), calmidazolium, produced a more potent inhibition of the P4-activated Ca2+ influx (Fig. 4) but was without effect on the P4-initiated AR. Therefore, while these three structurally dissimilar drugs are potent inhibitors of the P4-activated Ca2+ influx, we cannot conclude from these data whether a T-type channel is involved in this Ca2+ influx pathway or the P4-initiated AR. Interpretations of the data that support or refute the involvement of VOCCT are discussed below.

Is a VOCCT Involved in the P4-Activated Ca2+ Influx?

T-type Ca2+ currents in mouse spermatogenic cells have a low voltage activation threshold, exhibit rapid inactivation, and demonstrate steady-state inactivation in the -90 to -40 mV range [23, 47, 51]. Therefore, at the resting membrane potential of uncapacitated mouse spermatozoa (~-40 mV) [52, 53], a large fraction of the T-type channels should be inactivated. Mammalian spermatozoa must therefore undergo a capacitation-associated hyperpolarization to remove this inactivation [23, 53]. Alternatively, capacitation-associated protein tyrosine phosphorylation or P4-activated membrane depolarization may modulate the conductance state of the sperm VOCCT [54].

The higher-amplitude P4-activated Ca2+ influx detected in capacitated sperm populations (Fig. 1) supports the proposed model—reactivation of VOCCT via capacitation-associated hyperpolarization. If capacitated human spermatozoa had escaped the membrane potential-dependent inactivation of the uncapacitated state, then a P4-activated depolarization [24, 25] should have resulted in a greater Ca2+ influx. Indeed, the amplitude of the P4-activated Ca2+ elevation was greater in capacitated spermatozoa (Fig. 1).

Differences in membrane potential between uncapacitated and capacitated human spermatozoa should also affect the potency of pimozide, which demonstrates a voltage-dependent inhibition in somatic [43, 44] and spermatogenic cells [45]. When capacitated spermatozoa were subjected to conditions that promote depolarization (high extracellular [K+]), the inhibitory effect of pimozide was enhanced (Fig. 5). However, uncapacitated spermatozoa, which, according to spectrofluorometric studies with mouse and bovine sperm [52, 53], have a more depolarized resting membrane potential than capacitated spermatozoa, were less sensitive to pimozide than capacitated spermatozoa. Therefore, the effects of pimozide on the P4-activated Ca2+ influx results do not completely agree with the voltage-dependent effects described in somatic and spermatogenic cells.

It has been proposed that the inhibitory actions of pimozide on the zona pellucida-initiated [Ca2+]i elevation reflect the preferential blockade of T-type currents in mouse sperm, because spermatogenic cells demonstrate T-type currents that are inhibited by pimozide at similar concentrations (IC50 = 0.5 µM) and because they lack L-type currents [23]. However, in other cell types, pimozide inhibits both T-type (IC50 = 0.5 µM) [55] and L-type channel currents (IC50 = 0.2–5 µM) [43, 44]. Moreover, the pimozide concentration required to inhibit Ca2+ influx in mature mouse sperm (1 µM) [23, 43] is less than one order of magnitude lower than that required to block calmodulin-activated enzymes (7 µM) [34, 56]. Therefore, the insufficient selectivity of this pharmacological tool makes it difficult to distinguish the mechanism of action of this inhibitor in studies in which the target cannot be isolated. In that regard, our results, which indicate that the half-maximal inhibitory concentration for pimozide is 8 µM (Fig. 4), do not permit us to distinguish between the two possible mechanisms of action—inhibition due to VOCCT blockade or antagonism of a calmodulin-dependent pathway.

To further isolate our target molecule, a VOCCT, we tested an inhibitor of T-type channels, mibefradil, which has no reported effects on calmodulin-dependent pathways. Mibefradil demonstrates a use-dependent inhibition (IC50 = 5 µM) of T-type currents in mouse spermatogenic cells [45]; and in somatic cells, mibefradil selectively inhibits T-type channels over high voltage-activated (e.g., L-type) Ca2+ channels (e.g., IC50 1.6 µM and 28 µM, respectively) in a voltage-dependent and use-dependent manner [32]. In human spermatozoa, however, there was no difference in the potency of mibefradil between uncapacitated and capacitated spermatozoa (IC50 = 11 µM). Examination of the inhibitory effects of mibefradil on the P4-activated Ca2+ influx, under depolarizing conditions (100 mM KCl), also failed to elicit a significant effect. Therefore, the inhibition of the P4-activated Ca2+ influx by mibefradil is not dependent on the resting membrane potential of spermatozoa.

In conclusion, if mature spermatozoa possess T-type channels and no L-type channels, as has been suggested from experiments with spermatogenic cells [23, 47], then the mibefradil results suggest that at least one component of the P4-activated Ca2+ influx pathway is mediated by a VOCCT. However, recent results suggest that spermatozoa possess a unique isoform of an L-type VOCC [57]; thus the specific target through which mibefradil inhibits the P4-activated Ca2+ influx remains to be resolved.

Is a VOCCT Involved in the P4-Initiated AR?

An elevation of [Ca2+]i is required for the P4-initiated AR [2]. The amplitude of this Ca2+ signal or the spatial/temporal characteristics required to trigger the AR are not yet clearly defined. Studies that have investigated the relationship between the Ca2+ signal and the P4-initiated AR have suggested the involvement of tyrosine kinase pathways [58, 59] and gamma aminobutyric acid (GABA)A receptor-like Cl- channels [7] as potential modulators of the Ca2+ signal that are required to produce the AR. Here we report that pimozide, mibefradil, and calmidazolium—drugs that inhibit the P4-activated Ca2+ influx to the same magnitude—potentiate, inhibit, and have no effect on the P4-initiated AR, respectively. The inhibition of the P4-initiated AR by mibefradil may suggest the involvement of a VOCCT; however, the concentration of mibefradil (20 µM) required to inhibit the P4-initiated AR is higher than that needed to inhibit Ca2+ influx in spermatozoa (IC50 = 11 µM) and spermatogenic cells (5 µM). Furthermore, this concentration of mibefradil is sufficiently high that it does not facilitate distinction between T-type and L-type channels. Therefore, the disparate results obtained with the T-channel blockers, mibefradil and pimozide, and the high levels of mibefradil required to inhibit the AR do not allow us to conclude whether or not a VOCCT is involved in the P4-initiated AR.

Pimozide at a 1-µM concentration inhibits the zona pellucida-induced Ca2+ influx and AR of mouse spermatozoa [23]. In contrast, our result indicated that pimozide at a 10-fold higher concentration inhibited the P4-activated Ca2+ influx (Fig. 4) but potentiated the P4-initiated AR (Fig. 7). The mechanism of this stimulatory activity is unknown and apparently not mediated by a calmodulin-dependent pathway, given the lack of any significant (p = 0.98, n = 5) effect by calmidazolium (0.1–2 µM) on the P4-initiated AR. More importantly, though, the inverse relationship between the actions of pimozide on the P4-activated Ca2+ influx and the P4-initiated AR (Fig. 7) suggests that the ability of P4 to produce a biological effect (i.e., AR) is not dependent on the amplitude of the [Ca2+]i elevation produced by this steroid hormone.


   ACKNOWLEDGMENTS
 
We thank Harvey Florman for sharing in-press results and for providing valuable insights.


   FOOTNOTES
 
1 National Institutes of Health (NIH) grant HD-23098 to S.M. and a fellowship from NIH training grant RR-07038 to M.A.G. supported this research. Back

2 Correspondence: Stanley Meizel, Department of Cell Biology and Human Anatomy, University of California, One Shields Avenue, Davis, CA 95616-8643. FAX: 530 752 8520; smeizel{at}ucdavis.edu Back

3 Current address: Department Animal Resources (MB-18), Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037 Back

Accepted: August 28, 1998.

Received: May 26, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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