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Biology of Reproduction 62, 1380-1386 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Article

Induction of a Sodium Ion Influx by Progesterone in Human Spermatozoa1

Catherine Patrat2,,a, Catherine Serresb, and Pierre Jouanneta

a Service d'Histologie-Embryologie-Biologie de la Reproduction, Université Paris V-Cochin, 75014 Paris, France b Université Paris XI, UFR Kremlin Bicètre, 92470 Kremlin Bicètre, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In human spermatozoa, progesterone (P4) induces a depolarization of the plasma membrane, a rapid calcium (Ca2+) influx, and a chloride efflux. The sodium ion (Na+) was partly responsible for the P4-induced depolarizing effect but was not required for calcium influx. We used fluorescent probes for spectrofluorometry to investigate whether P4 induced a Na+ influx and whether voltage-operated channels were involved in Na+ and/or Ca2+ entries. We found that 10 µM P4 significantly increased intracellular Na+ concentration from 17.8 ± 2.0 mM to 27.2 ± 1.6 mM (P < 0.001). Prior incubation of spermatozoa with 10 µM flunarizine, a Na+ and Ca2+ voltage-dependent channel blocker, inhibited the sodium influx induced by 10 µM P4 by 84.6 ± 15.4%. The Ca2+ influx induced by 10 µM P4 was also significantly inhibited in a Na+-containing medium by 10 µM flunarizine or 10 µM pimozide (P < 0.01). In contrast, flunarizine had no inhibitory effect on the Ca2+ influx induced by 10 µM P4 in spermatozoa incubated in Na+-depleted medium. The P4-promoted acrosome reaction (AR) was significantly higher when spermatozoa were incubated in Na+-containing medium as compared to Na+-depleted medium. These data demonstrate that P4 stimulates a Na+ influx that could be involved in the AR completion. They also suggest that voltage-dependent Na+ and Ca2+ channels are implicated in P4-mediated signaling pathway in human spermatozoa.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Before fertilization, capacitated mammalian spermatozoa undergo exocytosis of the acrosomal vesicle (the acrosome reaction) in response to interaction with the zona pellucida (ZP). The acrosome reaction (AR) can be promoted by the ZP3 glycoprotein [1] and also by a non-genomic effect of progesterone (P4) at the sperm plasma membrane level. In vivo, spermatozoa can be exposed to a high local level of P4 (>1 µg/ml) when they penetrate through the cumulus surrounding the ZP-oocyte complex, since this steroid is secreted by the cumulus cells [2]. In consequence, a physiological role of P4 on the fertilizing capacity of spermatozoa has been envisaged. P4 has been found to initiate the AR, to stimulate sperm movement, and, more recently, to increase the sensitivity of spermatozoa to the subsequent AR-inducing action of ZP [3].

It has been demonstrated that P4 stimulates two ionic fluxes in human spermatozoa, a biphasic calcium (Ca2+) influx characterized by a rapid transient intracellular Ca2+ peak followed by a long-lasting plateau phase [4, 5], and a chloride (Cl-) efflux [6]. Moreover, P4 induces a plasma membrane depolarization that is not carried by Ca2+ ion entry but is partly dependent on extracellular sodium (Na+), assuming the existence of a P4-induced Na+ influx [7]. Two studies have also demonstrated that extracellular Na+ ions are not required for P4-stimulated calcium influx [7, 8], arguing for an absence of link between Na+-mediated depolarization and Ca2+ influx. That seems to rule out the involvement of voltage-operated calcium channels (VOCC) in P4-mediated Ca2+ entry. However, Garcia and Meizel [9] have recently reported that one component of P4-activated Ca2+ influx pathway could be mediated by VOCC. The type of VOCC, L or T, was uncertain for these latter authors, but Blackmore and Eisoldt [10] ruled out the role of T-type channels. Moreover it is not yet known whether P4 opens either a nonspecific cation channel permeable to both Na+ and Ca2+ or two distinct channels [7]. Consequently, the P4-promoted Ca2+ influx pathway is still discussed.

The purpose of this study was to determine whether P4 actually triggers a Na+ influx into human spermatozoa and also to characterize the P4-activated Ca2+ and Na+ influx pathways. In order to investigate the physiological relevance of P4-stimulated Na+ influx, the influence of extracellular Na+ on P4-induced AR in human spermatozoa was also tested. We have decided to look at the P4 action on capacitated human spermatozoa since they exhibit a higher calcium response to P4 than uncapacitated spermatozoa [9].


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

Sigma-Aldrich (Saint-Quentin Fallavier, France) was the supplier of P4, pimozide, flunarizine, and gramicidin, all dissolved in absolute ethanol; Fura-2 AM, sodium-binding benzofuran isophtalate-acetoxymethyl ester (SBFI-AM), and Pluronic F127, all dissolved in dimethyl sulfoxide; EGTA; Triton X-100; sodium pyruvate; Hepes; and BSA (fraction V; cat. no. A2153). All other chemicals (salts for buffers) were purchased from Merck-Clévenot S.A. (Nogent-sur Marne, France).

Media

BWW+BSA consisted of 95 mM NaCl, 4.8 mM KCl, 1.7 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 5.6 mM glucose, 0.25 mM sodium pyruvate, 20 mM Na+-Hepes, 25 mM NaHCO3, 20 mM lactic acid, and 5 mg/ml BSA (pH: 7.5; osmolarity: 300–310 mosmol/L).

In some experiments, a modified BWW medium depleted of Na+ was used (mBWW+Cho): NaCl was replaced by choline chloride, NaHCO3 by KHCO3, and Na+-Hepes by acid-free Hepes. Sodium pyruvate was omitted. The corresponding control, Na+-containing medium (mBWW+Na+), was similar to the Na+-deficient medium except that NaCl replaced choline chloride. The pH was 7.5 and was adjusted with KOH in mBWW+Cho. The osmolarity was 280–290 mosmol/L (Table 1).


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  		Table 1. Main composition of BWW, mBWW + Na±, and mBWW + Cho

Preparation of Spermatozoa

Human semen with normal sperm characteristics according to World Health Organization criteria (concentration >= 20 x 106 cells/ml; motility >= 50%; normal morphology >= 30%) was collected by masturbation from healthy donors. After liquefaction at 37°C, motile spermatozoa were selected by centrifugation (20 min, 300 x g) through a two-step Percoll density gradient (95% v:v and 47.5% v:v). The pellet was washed (10 min, 600 x g) and suspended at a concentration of 107 cells/ml in BWW medium containing 5 mg/ml BSA (BWW+BSA). The sperm suspension was incubated in BWW+BSA overnight at room temperature (22°C, under air) to allow capacitation.

Determination of Intracellular Free Calcium ([Ca2+]i) and Sodium ([Na+]i) Concentrations

Dye loading protocol For [Ca2+]i determination, the selected and capacitated motile spermatozoa were incubated with 2 µM Fura-2 AM in BWW+BSA at 37°C for 45 min. They were then washed by dilution in 3 volumes of BSA-free medium (BWW medium, modified BWW Na+-containing medium [mBWW+Na+], or modified BWW Na+-depleted medium [mBWW+Cho]) and centrifugated (10 min, 600 x g). The pellets were suspended at a density of 5 x 106 cells/ml in BWW, mBWW+Na+, or mBWW+Cho, depending on the experiment. Sperm suspensions were kept at room temperature in the dark for a further 30 min before use.

For [Na+]i determination, capacitated motile spermatozoa (15 x 106 cells/ml) were incubated in BWW+BSA containing 25 µM SBFI-AM for 90 min at 37°C under an atmosphere of 5% CO2, 95% air. Loading was optimized by mixing SBFI-AM (v:v) with 25% Pluronic F127 (w:v in dimethyl sulfoxide) before use [11]. The cells were washed by dilution in BSA-free media (BWW, mBWW+Na+, or mBWW+Cho) and centrifugated (600 x g, 10 min). Pellets were suspended at a density of 107 cells/ml in the appropriate medium. Sperm suspensions were kept at room temperature in the dark for a further hour before fluorescence recording.

Fluorescence recordings Fluorescence was recorded at 37°C with a PTI M-2001 spectrofluorimeter (Kontron Instruments, Saint-Quentin en Yvelines, France). Aliquots (1 ml) of sperm suspension were placed in a glass cuvette and were gently stirred with a magnetic bar. P4 and various calcium or sodium channel blockers were added at a final dilution of 1/500 or 1/1000. In control experiments, the addition of 0.2% ethanol did not induce any modification in [Na+]i or [Ca2+]i. After each treatment, 30 µl of the sperm sample was removed from the cuvette for estimation of the percentage of motile spermatozoa by light microscopy.

For [Ca2+]i determination, fluorescence was measured at an emission wavelength of 505 nm using dual excitation of 340 and 380 nm (5 nm bandpass). [Ca2+]i levels were calculated from the 340:380 nm excitation ratio. For signal calibration, at the end of each experiment, the maximum and minimal fluorescence signals were measured after the sequential additions of 0.05% Triton X-100 and 10 mM EGTA (pH 9.5), respectively. These values were used to calculate [Ca2+]i according to Grynkiewicz et al. [12] and as previously reported [4]. The effect of P4 on [Ca2+]i was expressed as the amplitude of the Ca2+ peak and the plateau phase (3 min after P4 addition) induced over the basal level of calcium (nM).

For [Na+]i determination, fluorescence was measured at an emission wavelength of 500 nm for dual excitation wavelengths of 340 and 385 nm (19-nm bandpass). The fluorescent signal was calibrated to convert the excitation ratio (340:385 nm) for fluorescence intensity into [Na+]i as follows: loaded cells were incubated in mBWW containing various concentrations of NaCl (0–30 mM) in the presence of the Na+-ionophore gramicidin (2 µM), which equilibrated extracellular [Na+] with [Na+]i. Various concentrations of choline chloride were added in order to ensure the iso-osmolarity of the media.

AR Measurement

After capacitation, sperm cells were divided into two aliquots and washed by dilution in mBWW+Na+ or mBWW+Cho and centrifugated (600 x g, 10 min). Then, sperm samples were suspended in mBWW+Na+ or mBWW+Cho (107 cells/ml) and incubated with 10 µM P4 or ethanol (0.1%) at 37°C for 1 h. The acrosome status was determined with fluorescein isothiocyanate-Pisum sativum agglutinin according to Cross et al. [13]. The ability of P4 to induce AR was expressed as the difference between the AR rate promoted by P4 and the spontaneous AR rate observed in the control.

Statistical Analysis

The data are expressed as means ± SEM. The means were compared using paired Student's t-test and Mann and Whitney's test, and P < 0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of P4 on Intracellular Na+

The basal [Na+]i of capacitated human spermatozoa in Na+-containing medium was 17.8 ± 2.0 mM (n = 9). The addition of 10 µM P4 increased [Na+]i in each experiment. The increase was progressive, reaching a sustained maximal level after 1 min (Fig. 1A). The mean maximal value of [Na+]i after P4 treatment was 27.2 ± 1.6 mM, as a result of a mean sodium influx of 9.4 ± 1.5 mM (n = 9). The ion flux responsible for the fluorescence increase recorded after the addition of P4 was Na+ dependent, as no change in the fluorescent signal occurred in Na+-depleted medium (Fig. 1B) or after the addition of 40 mM KCl (not shown).



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  		FIG. 1. Time course of the induction of a Na+ influx in human spermatozoa by 10 µM P4. Capacitated, SBFI-loaded spermatozoa were incubated in mBWW+Na+ (A, C) or in Na+-depleted mBWW (mBWW+Cho) (B). P4 induced a Na+ influx in mBWW+Na+ (A, C) whereas it had no effect on [Na+]i in mBWW+Cho (B). The subsequent addition of 40 mM NaCl led to a Na+ entry (B). Na+ influx promoted by 10 µM P4 was inhibited when spermatozoa were preincubated for 5 min with 10 µM flunarizine (C). For each incubation, a typical experiment is shown

Influence of Na+ and Ca2+ Channel Inhibitors on the Na+ and Ca2+ Influx Promoted by P4

The incubation of spermatozoa for 5 min in Na+-containing media with 10 µM flunarizine, a Ca2+- and Na+-channel inhibitor [14], inhibited [Na+]i increase promoted by 10 µM P4 (typical experiment shown in Fig. 1C). The sodium influx induced by P4 was significantly inhibited by 84.6 ± 15.4% in four different sperm samples (P < 0.01; Student's t-test; Fig. 2).



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  		FIG. 2. Effect of flunarizine on the Na+ influx stimulated by P4. Capacitated, SBFI-loaded spermatozoa were incubated in mBWW+Na+ medium for 5 min with 10 µM flunarizine before the addition of 10 µM P4. Results are expressed as the Na+ influx (mean ± SEM) induced by P4 for four sperm samples treated with flunarizine (solid bars) compared to control (open bars). *P < 0.01; Student's t-test

The prior incubation of spermatozoa for 5 min in BWW with 10 µM flunarizine or 10 µM pimozide inhibited [Ca2+]i increase stimulated by 10 µM P4 (typical experiments shown in Fig. 3). The mean calcium peak and calcium plateau phase induced by P4 were significantly decreased in seven different sperm samples (P < 0.05; Student's t-test; Fig. 4).



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  		FIG. 3. Time course of the induction of a Ca2+ influx in human spermatozoa by 10 µM P4. Capacitated, Fura-2-loaded spermatozoa were incubated in BWW with 10 µM P4 alone or after a previous exposure for 5 min with 10 µM flunarizine or 10 µM pimozide. P4-Induced calcium was inhibited when spermatozoa were preincubated with 10 µM flunarizine or 10 µM pimozide. For each incubation, a typical experiment is shown



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  		FIG. 4. Effects of flunarizine and pimozide on the Ca2+ influx stimulated by P4. Capacitated, Fura-2-loaded spermatozoa were incubated in BWW medium with 10 µM flunarizine or 10 µM pimozide or without Ca2+ and Na+ channel blockers for 5 min before 10 µM P4 addition. Results are expressed as the Ca2+ peak (mean ± SEM; top) and Ca2+ plateau phase (mean ± SEM; bottom) induced by P4 for seven sperm samples treated with 10 µM flunarizine (solid bars) and seven sperm samples treated with 10 µM pimozide (solid bars) compared to respective controls (open bars). *P < 0.05; Student's t-test

The prior incubation of three sperm samples for 5 min in BWW medium with 0.1, 1, or 10 µM flunarizine inhibited the calcium peak induced by 10 µM P4 in a dose-dependent manner by 21 ± 23%, 48 ± 25%, and 72 ± 9.8%, respectively (Fig. 5). An inhibitory effect on the calcium peak induced by 10 µM P4 was also observed with 0.1, 1, or 10 µM pimozide, giving 35.1 ± 11.8%, 40.3 ± 14.3%, and 79.8 ± 5.3% inhibition, respectively (Fig. 5).



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  		FIG. 5. Dose dependence of effects of Ca2+ and Na+ channel inhibitors (flunarizine and pimozide) on the ability of P4 to increase intracellular Ca2+. Capacitated, Fura-2-loaded spermatozoa were incubated in BWW medium for 5 min with various doses of flunarizine (n = 3) or pimozide (n = 5) before 10 µM P4 addition. The results (means ± SEM) are expressed as a percentage of the control Ca2+ peak measured in spermatozoa treated with 10 µM P4 alone

Consequences of Extracellular Na+ Depletionon the Calcium Influx Induced by P4

A calcium influx was also induced by 10 µM P4 in Na+-depleted medium. The mean amplitude of the calcium peak was lower but not statistically different (P = 0.09) in mBWW+Cho as compared to mBWW+Na+ (n = 5, Fig. 6). The mean amplitude of the calcium plateau phase was not statistically different between the two media (n = 5; Fig. 6).



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  		FIG. 6. The influence of extracellular Na+ on the calcium influx stimulated by P4. Capacitated, Fura-2-loaded spermatozoa were incubated in mBWW+Na+ or in mBWW+Cho with 10 µM P4 with (solid bars) or without (open bars) pretreatment for 5 min with 10 µM flunarizine (n = 5). Results are expressed as the Ca2+ peak (mean ± SEM; top) and Ca2+ plateau phase (mean ± SEM; bottom) induced by P4 for five sperm samples. *P < 0.01; Mann and Whitney's test

We tested whether the calcium response of these samples to P4 was sensitive to Na+ and Ca2+ channel inhibitor in Na+-depleted medium. Prior incubation of five sperm samples with 10 µM flunarizine inhibited neither the calcium peak nor the calcium plateau phase induced by P4 in mBWW+Cho (n = 5; Fig. 6). However, flunarizine significantly inhibited the calcium peak (P < 0.01; Mann and Whitney's test) and the calcium plateau phase (P < 0.01; Mann and Whitney's test) in Na+-containing medium (n = 5; Fig. 6). So the effect of flunarizine on the P4-induced calcium influx was significantly different in Na+-containing and Na+-depleted media.

Ten micromolar flunarizine by itself increased basal intracellular calcium concentration, with the same amplitude whatever the incubation medium, mBWW+Na+ or mBWW+Cho (87.0 ± 25.0 nM vs. 104.0 ± 33.7 nM, respectively).

Consequences of Extracellular Na+ Depletion on the AR Induced by P4

The rate of spontaneous AR was similar when the spermatozoa were incubated in mBWW with or without Na+ (10.6 ± 2.8% and 10.3 ± 2.1%, respectively, n = 6). Exposure of spermatozoa to 10 µM P4 induced more AR when sperm samples were incubated in mBWW+Na+ (18.6 ± 2.6%, n = 6) than when incubated in mBWW+Cho (13.0 ± 2.3%, n = 6). The ability of P4 to induce the AR was significantly higher in a Na+-containing than in a Na+-depleted medium (P < 0.01 with paired Student's t-test).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
P4 Simulates a Sodium Influx in Human Spermatozoa

Our results show that P4 stimulates a sodium influx in capacitated human spermatozoa. Although already suggested by Foresta et al. [7] from the observation that the depolarizing effect of P4 was partially abolished in a sodium-depleted medium, the [Na+]i increase promoted by P4 in human spermatozoa had never been directly investigated and measured. In this study, we found that P4 initiates a mean influx of 10 mM Na+ above the basal level of intracellular sodium. To date, no physiological AR inducer has ever been shown to cause an increase in mammalian sperm [Na+]i. Only speract has been reported to stimulate a Na+ influx via the activation of a Na+/H+ exchanger in marine invertebrate sperm [15]. The basal [Na+]i found in this study for human spermatozoa (18 mM) was similar to the basal [Na+]i previously reported for bovine sperm (14 mM) [16] and for other nongerminal cell types [11].

What Is the Mechanism for Na+ Influx in Human Spermatozoa?

The pathway implicated in Na+ entry in human spermatozoa upon P4 addition remains to be clarified. The sodium channels are poorly characterized in human spermatozoa. Selective Na+ channels and cation channels with a low specificity have both been evidenced in mammalian spermatozoa [17, 18]. A Na+ channel inhibited by tetrodotoxin (TTX) has also been identified in the lipid bilayer of human sperm plasma membrane [19]. In the present study, the inhibition of the P4-stimulated Na+ influx by flunarizine, a diphenylpiperazine derivative known to inhibit TTX-sensitive and voltage-dependent Na+ channel [14], suggests that this type of Na+ channel could be involved in this process. In this hypothesis, a primary P4 action on membrane potential must precede the Na+ entry to open voltage-dependent Na+ channels. This primary effect of P4 could be due to events, independent of ion fluxes, such as motion perturbations in sperm plasma membrane due to P4 partitioning into sperm plasma membrane [20, 21]. Alternatively, the Cl- efflux stimulated in few seconds by P4 through a gamma aminobutyric acidA-like receptor activation [6] could also lead to this first and rapid plasma membrane depolarization. The other part of P4-promoted depolarization would be due to Na+ influx itself [7]. Using the Nernst equation, the extent of the mean depolarization of the sperm population due to the [Na+]i change would correspond to about +10 mV. Further investigations must be carried out at the cellular level to better characterize and understand all the effects of P4 on sperm membrane potential involving ionic and nonionic events.

Another pathway that may account for the Na+ influx could be the activation of the Na+/H+ exchanger by P4. Recent observations are consistent with the presence of such an exchanger in human capacitated spermatozoa [22]. The fact that no intracellular alkalinization, which should be logically associated with Na+ entry, was observed during P4 action by Garcia and Meizel [8] and by us (unpublished results) rules out a direct involvement of Na+/H+-exchanger in Na+ influx promoted by P4.

What Is the Mechanism for Calcium Influx in Human Spermatozoa?

We found that flunarizine and pimozide inhibited the P4-promoted Ca2+ peak and plateau phase.

This result confirms the inhibition of the P4-stimulated intracellular Ca2+ peak by pimozide, recently reported in human spermatozoa [9], with a similar potency of inhibition (IC50 = 8 µM). Comparing the time course of the two ion fluxes promoted by P4, we found that Na+ influx does not precede the intracellular Ca2+ peak since it increases gradually over 1 min, a time during which the calcium peak fully develops. Consequently, it is not likely that the inhibitory effect of flunarizine on the P4-induced Ca2+ peak results from a primary blocking action on Na+ influx; more probably it results from its other blocking effect on voltage-dependent Ca2+ channels as observed in neurons [14]. At the micromolar dose in our experiments, flunarizine exerts a blocking effect more pronounced on the T-type than on the L-type Ca2+ current of the nerve cell [23]. However, a total specificity of this drug for T- or L-type VOCC cannot be ascertained, as already pointed out by Garcia and Meizel [9]. Moreover, both testis-specific L- and T-type channels have been described in rat testis and mouse spermatogenetic cells [24–26]. Finally, two studies testing the effects of the same T-type VOCC inhibitor, mibefradil, on the calcium peak in response to P4 have recently presented different data [9, 10]. Thus the involvement of T-type rather than L-type channels in P4 action in human spermatozoa cannot be concluded with certainty. Our present findings are only additional proof for the implication of VOCC in the calcium peak stimulated by P4 in human spermatozoa.

Previously, using the selective tyrosine kinase inhibitor genistein, it was reported that the P4-stimulated plateau phase of calcium was dependent on a tyrosine kinase activity [27]. At first sight, this observation is not contradictory with the putative involvement of VOCC. Indeed, it is known in somatic cells that VOCC are regulated not only by membrane potential alterations but also possibly by tyrosine kinase activity [28]. However, in mouse spermatogenetic cells, T-type VOCC are activated by a tyrosine phosphatase and not a tyrosine kinase activity [26]. Therefore VOCC do not seem be involved in the P4-stimulated Ca2+ plateau phase. Thus the inhibitory effect of flunarizine on P4-induced Ca2+ plateau phase could be due to the primary blocking action on the Ca2+ peak, according to the hypothesis in which this latter is a prerequisite event for full development for the Ca2+ plateau phase.

Relationship Between Na+ and Ca2+ Fluxes in Human Spermatozoa

The increase in [Na+]i induced by P4 is not a necessary event for the calcium response of capacitated spermatozoa to P4, since this latter is observed in a Na+-depleted medium. This finding confirms two previous studies [7, 8]. However some comments can be made. In contrast to the observations of Foresta et al. [7], no enhancement of the calcium peak in Na+-depleted medium as compared to the calcium peak observed in Na+-containing medium was found in the present study [7]. Our results seem to exclude the involvement of a nonspecific channel conducting Ca2+ in place of Na+ in the occurrence of the calcium peak. In the same way, the different patterns of the time course for Na+ and Ca2+ entries stimulated by P4 into spermatozoa (peak for Ca2+ and sustained plateau for Na+) do not support the implication of the same channel for these two ion influxes.

Unexpectedly, contrary to the observation made in Na+-containing medium, the P4-induced calcium response in spermatozoa incubated in Na+-depleted medium was not inhibited by flunarizine and pimozide. This absence of inhibitory effect of flunarizine or pimozide cannot be explained by their proper effect on basal [Ca2+]i, since this latter was similarly expressed in Na+-containing and Na+-depleted media. It is possible that the sperm membrane is hyperpolarized when extracellular Na+ is replaced by choline in incubation medium, leading to an absence of an inhibitory effect of flunarizine. Such a voltage–dependent blocking activity of flunarizine and pimozide has already been reported in somatic cells [29]. Garcia and Meizel [9] have found that pimozide increased the blocking activity when spermatozoa were depolarized by 100 mM KCl. This is in accordance with the general observation that when the channel-blocking activity of drugs is voltage dependent, a depolarization enhances the block whereas it is less pronounced at hyperpolarized membrane potentials.

Importance of Na+ Influx in the Physiological Role of P4 in Human Sperm

We have shown that, in human sperm, P4 was able to induce more AR in a Na+-containing medium than a Na+-depleted medium. These results confirm previous data from Garcia and Meizel [8] and ascertain a physiological relevance of P4-triggered Na+ influx in the occurrence of the AR in human sperm. A Na+ influx via the Na+/H+ exchanger has already been described during the AR in response to speract in sea urchin [15]. A putative involvement of the Na+/H+ exchanger in ZP-induced AR has also been suggested in mammals [30]. However, as the ability of P4 to induce the AR in human spermatozoa is weak, it is possible that the Na+-dependent mechanism is rather involved in the sensitizing effect of P4 on the subsequent action of the ZP.

In conclusion, we have demonstrated that P4 actually initiates Na+ influx. The increase in [Na+]i was progressive, in contrast to the rapid Ca2+ entry. This suggests that Na+ influx does not precede Ca2+ entry and therefore is not a prerequisite event for the Ca2+ peak promoted by P4. The difference between entry patterns for these two ions indicates that Na+ and Ca2+ ions likely enter spermatozoa by distinct channels. The Na+ channels operated by P4 would be voltage dependent, such as the calcium channels involved in the calcium influx. Lastly, the Na+ dependence of P4-induced AR evidences the physiological importance of the Na+ influx in this exocytotic event.


   ACKNOWLEDGMENTS
 
The authors thank J. Knight for correction of the English text.


   FOOTNOTES
 
First decision: 23 September 1999.

1 This work was supported by grant no. 1752 from the Direction de la Recherche et des Etudes Doctorales. Back

2 Correspondence: Catherine Patrat, Service d'Histologie-Embryologie-Biologie de la Reproduction, Université Paris V-Cochin, 24, rue du Faubourg Saint-Jacques, 75014 Paris, France. FAX: 33 01 58 41 15 75;catherine.patrat{at}cch.ap-hop-paris.fr Back

Accepted: December 13, 1999.

Received: August 3, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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