Molecular Human Reproduction

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Molecular Human Reproduction, Vol. 7, No. 2, 119-128, February 2001
© 2001 European Society of Human Reproduction and Embryology

Testis and spermatogenesis

Intracellular calcium store depletion and acrosome reaction in human spermatozoa: role of calcium and plasma membrane potential

M. Rossato¹, F.Di Virgilio², R. Rizzuto², C. Galeazzi¹ and C. Foresta^1,3

¹ University of Padova, Department of Medical and Surgical Sciences, Clinica Medica 3, Via Ospedale 105, 35128 Padova and ² University of Ferrara, Department of Experimental and Diagnostic Medicine, Section of General Pathology, Via Borsari 46, 44100 Ferrara, Italy

Abstract

We evaluated the presence and role of internal calcium stores in human uncapacitated spermatozoa by determining the effects of two inhibitors of Ca²⁺ ATPase of the sarco-endoplasmic reticulum (SERCA–ATPase), thapsigargin and cyclopiazonic acid (CPA) on intracellular calcium concentrations, [Ca²⁺]_i, plasma membrane potential and acrosome reaction. Using a fluorescent conjugate of thapsigargin, we localized internal Ca²⁺ stores on the acrosome, post-acrosomal region and sperm midpiece. SERCA–ATPase inhibitors induced a rise in [Ca²⁺]_i both in Ca²⁺ and Ca²⁺-free media but under these latter conditions it was reduced with a progressive decline to baseline values; the re-addition of Ca²⁺-stimulated a rise in [Ca²⁺]_i. This demonstrated that internal Ca²⁺ store depletion can evoke the opening of Ca²⁺-channels on sperm plasma membrane, thus showing the existence of `capacitative' Ca²⁺ entry into these specialized cells. The addition of thapsigargin to human spematozoa induced a dose-dependent increase in acrosome reaction percentages, but only when Ca²⁺ was present in the external medium. Plasma membrane potential monitoring showed that these inhibitors induced a depolarization dependent on Ca²⁺ influx from external medium and that this was preceded by a transient hyperpolarization caused by activation of Ca²⁺-dependent K⁺ channels. When K⁺-dependent plasma membrane hyperpolarization was inhibited, the thapsigargin- and CPA-stimulated rise in [Ca²⁺]_i plasma membrane depolarization and acrosome reaction were abolished. In conclusion, the present study demonstrates that human spermatozoa possess internal Ca²⁺ stores and that the capacitative Ca²⁺ entry pathway present in these cells regulates important biological processes that are fundamental for the acrosome reaction.

calcium/channels/internal stores/membrane potential/spermatozoa

Introduction

In all eukaryotic cells Ca²⁺ signal transduction regulates a number of different cellular functions. In this respect, there is extensive evidence that the sperm acrosome reaction, a specialized exocytotic event occurring as spermatozoa approach the oocyte, is a Ca²⁺-dependent process (Wassarmann, 1987; Thomas and Meizel, 1989; Kopf and Gerton, 1991). Progesterone, a well-known activator of the acrosome reaction in human and other mammalian spermatozoa, induces a rapid rise of intracellular calcium concentrations, [Ca²⁺]_i, activating an influx of Ca²⁺ from the external medium (Osman et al., 1989; Blackmore et al., 1990; Foresta et al., 1993). ZP3, a glycoprotein of the oocyte zona pellucida, stimulates Ca²⁺ influx and acrosome reaction in human and other mammalian spermatozoa (Tesarik et al., 1993; Arnoult et al., 1999). Sperm exposure to hypotonic media induces an important and rapid influx of Ca²⁺ from the external medium leading to acrosome reaction and fertilization (Rossato et al., 1996). All these studies point to extracellular space as the only source of Ca²⁺ for the increase in [Ca²⁺]_i induced by putative `physiological' stimuli and, in this respect, it is generally accepted that external Ca²⁺ is fundamental for the acrosome reaction to occur (Foresta and Rossato, 1997). To date, little is known about the role, if any, of internal Ca²⁺ stores in the regulation of sperm functions. Only recently some authors demonstrated the presence of inositol trisphosphate (IP₃) receptors, a hallmark for the presence of internal Ca²⁺ stores, in spermatozoa from different mammalian species (Walensky and Snyder, 1995; Kuroda et al., 1999).

Thapsigargin is a tumour-promoting drug that has been shown to increase [Ca²⁺]_i in a number of different cell types by releasing Ca²⁺ from the internal stores, through the inhibition of the Ca²⁺–ATPase pump localized on the membrane of the internal Ca²⁺ stores, without the production of IP₃ (Thastrup et al., 1990). Ca²⁺ leakage from internal stores, induced by thapsigargin and due to inhibition of Ca²⁺–ATPase, is accompanied by an important phenomenon called `capacitative' Ca²⁺ entry consisting of the gating of Ca²⁺ permeable channels on the cell membrane with a massive Ca²⁺ influx and an important rise in [Ca²⁺]_i (Putney 1990). Different laboratories have demonstrated that thapsigargin induces a rise in [Ca²⁺]_i in human spermatozoa through an influx of Ca²⁺ from the external medium leading to acrosome reaction (Blackmore 1993; Meizel and Turner, 1993; Perry et al., 1997). Furthermore, previous studies have demonstrated that thapsigargin potentiates the stimulatory effects of progesterone in human spermatozoa (Mendoza and Tesarik, 1993).

The influx of Ca²⁺ from the external medium induced by thapsigargin cannot be explained by inhibition of plasma membrane Ca²⁺–ATPases, since they are not sensitive to this agent (Thastrup et al., 1990). Thus, the mechanisms activated by thapsigargin to determine Ca²⁺ influx in human spermatozoa remain obscure.

In the present study, we have evaluated the effects of two different SERCA–ATPase inhibitors, thapsigargin and cyclopiazonic acid (CPA), on [Ca²⁺]_i and plasma membrane potential in human spermatozoa. The biological effects of these two inhibitors on the sperm acrosome reaction were also evaluated.

Materials and methods

Reagents
Fura-2/AM, DiBAC4(3), SBFI/AM, BAPTA/AM and BODIPY-FL-thapsigargin were purchased from Molecular Probes (Eugene, OR, USA). Nifedipine, gramicidin D, tetra-ethyl-ammonium (TEA), choline chloride, methylglucamine and sucrose were obtained from Sigma (St Louis, MO, USA). Thapsigargin, charybdotoxin and ionomycin were obtained from Calbiochem (La Jolla, CA, USA) and Verapamil from Knoll AG (Liestal, Switzerland). All other chemicals were of analytical grade.

Sperm collection
Sperm samples were obtained from 10 healthy fertile sperm donors (age range 20–33 years) after 3 days of sexual abstinence. Semen samples were allowed to liquefy at room temperature for 30 min; then they were analysed for semen volume, pH, sperm concentration, morphology, motility and viability. Samples with motility of >60% and viability of >80% were used. All experiments were carried out using uncapacitated spermatozoa isolated by the swim-up technique (Foresta et al., 1992). Spermatozoa isolated by this method were collected and centrifuged for 10 min at 800 g. The final pellets were resuspended in standard saline containing: 125 mmol/l NaCl, 4.8 mmol/l KCl, 1.2 mmol/l MgSO₄, 1.2 mmol/l KH₂PO₄, 5.6 mmol/l glucose, 25 mmol/l NaHCO₃, 1.7 mmol/l CaCl₂, 20 mmol/l HEPES (pH 7.4, 37°C), 10⁴ IU/ml penicillin, 10 mg/ml streptomycin. The sperm concentration was adjusted to 15x10⁶/ml. The sperm motility of each sample was assessed by light microscopy at the beginning and end of each experiment. Sperm motility and viability were unaffected by thapsigargin up to 10 µmol/l and by CPA up to 100 µmol/l. At higher concentrations, these two drugs produced a significant loss of sperm motility leaving sperm viability unaffected. Sperm viability was determined by red–eosin exclusion test and was >90% at the end of all experiments. In some experiments extracellular sodium (Na⁺) was isotonically replaced with N-methyl-D-glucamine (225 mmol/l).

[Ca²⁺]_i measurement
[Ca²⁺]_i was measured using the fluorescent probe fura-2/AM (Foresta et al., 1992). Aliquots (1 ml) of spermatozoa (isolated as above) were incubated for 30 min at 37°C in the presence of fura-2/AM (2 µmol/l). After loading, spermatozoa were washed by centrifugation at 800 g for 10 min, resuspended in standard saline at a concentration of 1.5x10⁶/ml and maintained at room temperature until used. [Ca²⁺]_i was measured in a LS50B Perkin-Elmer fluorometer equipped with a thermostatically controlled and magnetically-stirred cuvette holder and using 1.0 ml loaded sperm aliquots. The excitation wavelength alternated between 350 and 380 nm and emission fluorescence was continuously monitored at 505 nm. In experiments evaluating the involvement of voltage-operated Ca²⁺ channels (VOCCs) in thapsigargin-induced Ca²⁺ influx, fura-2 loaded sperm samples were incubated for 15 min in the presence of nifedipine (1.0 µmol/l), verapamil (10 µmol/l), TEA (10 mmol/l) or charybdotoxin (100 nmol/l) before thapsigargin addition. In some experiments, intracellular Ca²⁺ was chelated by incubating sperm aliquots with the intracellular Ca²⁺ chelator BAPTA/AM (2 µmol/l for 30 min at 37°C). After loading, spermatozoa were washed by centrifugation and resuspended in standard saline as described for fura/2 loading.

Evaluation of sperm plasma membrane potential changes
Sperm plasma membrane changes were monitored using the potential sensitive fluorescent dye DiBAC₄(3) as previously described (Foresta et al., 1992). Briefly, 1.5x10⁶ spermatozoa isolated as described above were placed in a thermostatically-controlled cuvette at 37°C containing the DiBAC₄(3) solution (200 nmol/l) in standard saline. After stabilization of the fluorescent signal additions of thapsigargin and CPA at different doses were made. Excitation and emission wavelengths were 540 and 580 nm respectively. In some experiments evaluating the role of K⁺ channel-blockers on thapsigargin-induced plasma membrane variations, sperm suspensions were pre-incubated with each specific channel blocker for 15 min before addition of thapsigargin.

Determination of Mn²⁺ influx
Mn²⁺ uptake was measured by monitoring the rate of fluorescence quenching at the excitation wavelength of 360 nm (isosbestic point). When measured at the isosbestic wavelength, the rate of fura-2 fluorescence decrease is insensitive to [Ca²⁺]_i changes and proportional to the rate of Mn²⁺ influx (Sargeant et al., 1992).

Measurement of [Na⁺]_i in sperm suspensions
Intracellular free Na⁺ was evaluated using the fluorescent sodium-binding dye, benzofuran isophtalate acetomethylester (SBFI/AM), as previously described (Foresta et al., 1996). Spermatozoa, suspended in standard saline, were incubated with 5 µmol/l SBFI/AM in the presence of the non-ionic detergent pluronic acid (20% in dimethylsulphoxide, 1:1 to SBFI/AM) for 60 min at 37°C with continuous stirring. Cells were then washed by centrifugation (twice at 250 g for 10 min at room temperature) in standard saline. After centrifugation the supernatant was discarded, cells were resuspended in standard saline at a concentration of 1.5x10⁶/ml and kept at room temperature until used. All experiments were performed within 90 min of the dye loading. [Na⁺]_i was measured in a LS50B Perkin–Elmer fluorometer equipped with a thermostatically-controlled, magnetically-stirred cuvette holder and using 1.0 ml loaded sperm aliquots. SBFI fluorescence was monitored at the wavelength pair 345 and 490 nm for excitation and emission respectively.

SERCA–ATPase localization
SERCA–ATPase were localized using a membrane-permeant fluorescent derivative of thapsigargin, BODIPY-FL-thapsigargin. Aliquots of human spermatozoa isolated as described above were incubated with BODIPY-FL-thapsigargin (1.0 µmol/l) for different times. After incubation cells were washed free of the dye, suspended in saline and observed under fluorescent microscope. Parallel experiments were performed incubating sperm aliquots with BODIPY-FL alone, as control.

Acrosome reaction evaluation
Sperm aliquots for evaluation of acrosome reaction were retrieved before and after incubation with thapsigargin and CPA for 30 min in the different experimental conditions as described above. After fixation with formaldehyde the percentage of acrosome-reacted spermatozoa was assayed using an indirect fluorescence technique with fluorescein isothiocyanate (FITC)-conjugated lectins from Pisum sativum which selectively bind to the intact acrosome (Cross and Meizel, 1989). According to their recommendations, spermatozoa were scored as acrosome-reacted only if a bright fluorescence showed on the equatorial segment. A total of 200 spermatozoa were scored in each sample to evaluate the percentage of acrosome-reacted spermatozoa.

Statistical analysis
Experimental data were analysed using the Stat View II (Abacus Concepts, Berkeley, CA, USA) statistical package. Statistical analyses were carried out using analysis of variance (ANOVA) and Student's t-test. P < 0.05 was considered to be statistically significant.

Results

Localization of SERCA–ATPase in human spermatozoa
The existence and then the localization of internal Ca²⁺ stores in human spermatozoa has always been a matter of debate. The possible localization of these Ca²⁺ pumps in human spermatozoa was investigated by means of a fluorescent derivative of thapsigargin, BODIPY-thapsigargin. As shown in Figure 1A, this fluorescent thapsigargin-conjugate is clearly localized on the acrosome and midpiece, as demonstrated by a strong fluorescence well evident on these sperm regions in all spermatozoa observed in our preparations. As shown in Figure 1B, thapsigargin induced rapid modifications of the fluorescence pattern with a strong reduction on the acrosomal region so that only a faint fluorescence was visible in the post-acrosomal region. No significant variations were observed in the pattern of fluorescence on the midpiece.

View larger version (65K): [in this window] [in a new window]   Figure 1 . Localization of internal Ca2+ stores in human spermatozoa. Spermatozoa were incubated in the presence of fluorescent thapsigargin conjugate BODIPY-thapsigargin to localize SERCA–ATPase of internal Ca2+ stores. (A) Spermatozoa under resting conditions. Fluorescence is localized only on the sperm acrosome (ac) and midpiece (mp). The tail appears to be completely negative. (B) Spermatozoa incubated in the presence of thapsigargin for 15 min. Fluorescence is localized mainly at the equatorial segment in the post-acrosomal region (es, arrowheads) and in the midpiece. It was not possible to observe significant fluorescence on sperm acrosome. (C) Control spermatozoa incubated with BODIPY alone (original magnification x1250; bar = 4 µm).

 
Effects of thapsigargin on sperm [Ca²⁺]_i
Addition of thapsigargin to sperm suspensions induced a rapid and dose-dependent rise in [Ca²⁺]_i with maximal effect at 100 nmol/l (Figure 2A). Addition of EGTA after [Ca²⁺]_i rise induced a rapid return to basal [Ca²⁺]_i, thus supporting the hypothesis that thapsigargin induced an influx of Ca²⁺ from the external medium. The addition of thapsigargin to spermatozoa suspended in Ca²⁺-free medium (no Ca²⁺ added + 0.1 mmol/l EGTA) induced a dose-dependent rise in [Ca²⁺]_i that was slower and reduced in comparison with that observed in Ca²⁺-containing medium (Figure 2B) and was transient, since [Ca²⁺]_i returned to basal levels after ~8–9 min. Re-addition of Ca²⁺ to external medium at this time provoked a prompt and important rise of [Ca²⁺]_i. The dose–response curves for [Ca²⁺]_i obtained by stimulating spermatozoa with thapsigargin in the presence and absence of external Ca²⁺ are similar (Figure 2A and B, inset) with maximal effects at a dose of 100 nmol/l. These data suggest that thapsigargin activates a signal that induces Ca²⁺ rise due to the emptying of internal Ca²⁺ stores and the subsequent opening of Ca²⁺ channels on the sperm plasma membrane allowing Ca²⁺ influx and further [Ca²⁺]_i rise.

View larger version (20K): [in this window] [in a new window]   Figure 2 . Effects of thapsigargin on [Ca2+]i in the presence and absence of Ca2+ in the extracellular medium. (A) Fura-2-loaded spermatozoa were suspended in standard saline and then treated with thapsigargin. Where indicated, thapsigargin (Tg, 100 nmol/l) or EGTA (2.0 mmol/l) were added. Representative results from five similar experiments are shown. The inset shows the dose-dependent effects of thapsigargin on [Ca2+]i. Peak [Ca2+]i increases above basal levels were plotted against thapsigargin concentrations. Results are means ± SD of five separate experiments. (B) Fura-2 loaded spermatozoa were incubated in Ca2+-free medium (no Ca2+ added and EGTA 0.1 mmol/l) prior to the addition of thapsigargin (Tg, 100 nmol/l). Where indicated Ca2+ (2.0 mmol/l) was added. Representative results from five similar experiments are shown. The inset shows the dose-dependent effects of thapsigargin on [Ca2+]i. Peak [Ca2+]i increases above basal levels were plotted against thapsigargin concentrations. Results are means ± SD of five separate experiments.

 
To further evaluate the role of Ca²⁺ stores emptying in the activation of Ca²⁺ influx from external medium, we performed experiments with CPA, another well-known inhibitor of SERCA–ATPase but one that is chemically unrelated to thapsigargin. In the presence of Ca²⁺ in the extracellular medium, CPA induced a dose-dependent increase in [Ca²⁺]_i which resembled that produced by thapsigargin although at higher concentrations, with maximal effect at 100 µmol/l (data not shown). As observed for thapsigargin, in the absence of extracellular Ca²⁺, CPA produced a lower and transient rise in [Ca²⁺]_i confirming that this drug also releases Ca²⁺ from intracellular stores (data not shown).

To confirm that thapsigargin and CPA open a plasma membrane channel permeable to Ca²⁺, we utilized the ability of the divalent cation Mn²⁺ to permeate through Ca²⁺ channels quenching cytoplasmic fura-2 fluorescence. Figure 3 shows that these two SERCA–ATPase inhibitors induce a rapid Mn²⁺ influx demonstrating that these agents activate a cascade of events leading to the opening of Ca²⁺-permeable channels in human sperm plasma membrane.

View larger version (8K): [in this window] [in a new window]   Figure 3 . Effects of thapsigargin and cyclopiazonic acid on Mn2+ influx in human spermatozoa. Fura-2 loaded spermatozoa were suspended in standard saline containing 200 µmol/l MnCl2. Fluorescence was monitored at the Ca2+-insensitive excitation wavelength (360 nmol/l). Where indicated, thapsigargin (Tg, 100 nmol/l, trace a) and cyclopiazonic acid (CPA, 10 µmol/l, trace b) were added. Traces are representative of three similar experiments.

 
Effects of thapsigargin on sperm plasma membrane potential
The Ca²⁺ entry induced by Ca²⁺-stores depletion by thapsigargin was investigated by evaluating ion currents across the sperm plasma membrane as determined by the monitoring of sperm plasma membrane potential using the potential sensitive fluorescent dye bis-oxonol. As reported in Figure 4A (trace a), thapsigargin addition to sperm suspensions induced a rapid plasma membrane hyperpolarization followed by a progressive plasma membrane depolarization. We ascertained whether Na⁺ was the ionic species involved in thapsigargin-induced plasma membrane depolarization. As shown in Figure 4A (trace b), when spermatozoa were suspended in Na⁺-free medium (in which extracellular Na⁺ was iso-osmotically substituted with N-methyl-D-glucamine), thapsigargin was still able to induce a depolarization of the plasma membrane that was preceded by the rapid and transient hyperpolarization as observed in Na⁺-containing medium. These results seem to exclude that Na⁺ is the ionic charge responsible for thapsigargin-induced plasma membrane potential variations. To strengthen this hypothesis, we determined the [Na⁺]_i in spermatozoa loaded with the Na⁺-sensitive fluorescent dye SBFI. As shown in Figure 4A (inset), thapsigargin was not able to induce any variation of sperm [Na⁺]_i, while extracellular ATP induced a prompt rise of [Na⁺]_i as previously demonstrated (Foresta et al., 1992). Then we focused on Ca²⁺ as the charge-carrying ion involved in these depolarizing effects. Figure 4B (trace a) shows that EGTA addition after plasma membrane depolarization induced by thapsigargin returned the plasma membrane potential to basal levels. Furthermore, thapsigargin addition to spermatozoa suspended in Ca²⁺-free medium did not induce any depolarizing effect while the first rapid phase of hyperpolarization was not affected (Figure 4B, trace b). Interestingly, when Ca²⁺ was added back to the medium under these experimental conditions, a rapid depolarization of the plasma membrane was observed. Together, these data point to Ca²⁺ as the ion responsible for the plasma membrane depolarization induced by thapsigargin. As previously demonstrated with CPA on sperm [Ca²⁺]_i, this agent induced sperm plasma membrane effects similar to those obtained with thapsigargin (data not shown).

View larger version (12K): [in this window] [in a new window]   Figure 4 . (A) Effects of thapsigargin on sperm plasma membrane potential. Sperm suspensions were incubated in the presence of bis-oxonol (200 nmol/l) for 10 min before thapsigargin addition. In trace a, spermatozoa were suspended in Ca2+-medium. In trace b, spermatozoa were suspended in Na+-free, N-methyl-D-glucamine containing medium. The inset shows that thapsigargin does not influence [Na+]i in human spermatozoa. SBFI loaded spermatozoa were suspended in standard saline and then stimulated with thapsigargin. Where indicated thapsigargin (Tg, 100 nmol/l) and ATP (2.5 mmol/l) were added. (B) Effects of Ca2+ on thapsigargin-induced plasma membrane potential variations. In trace a, spermatozoa were suspended in Ca2+-containing medium while, in trace b, spermatozoa were suspended in Ca2+-free medium. Where indicated thapsigargin (Tg, 100 nmol/l), EGTA (2.0 mmol/l) or Ca2+ (2.0 mmol/l) were added. Traces are representative of five similar experiments.

 
While the nature of the thapsigargin-induced plasma membrane depolarizing effects has been elucidated, the origin of the thapsigargin-induced plasma membrane hyperpolarization observed after thapsigargin addition remains to be clarified. When thapsigargin was added after previous internal Ca²⁺ store depletion (with prolonged incubation in Ca²⁺-free medium or following a previous addition of a maximal efficacious thapsigargin dose) or after intracellular Ca²⁺ chelation with BAPTA we did not observe any hyperpolarization of the plasma membrane (Figure 5) while valinomycin induced a prompt plasma membrane hyperpolarization. These observations suggest that plasma membrane hyperpolarization may be due to internal Ca²⁺ store depletion.

View larger version (11K): [in this window] [in a new window]   Figure 5 . Role of internal Ca2+-store depletion on plasma membrane hyperpolarization induced by thapsigargin. Spermatoza were treated with different experimental protocols and then suspended in the presence of bis-oxonol (200 nmol/l). Trace a: spermatozoa were suspended in Ca2+ free medium and then stimulated with thapsigargin. Trace b: spermatozoa were suspended in Ca2+-free medium (no Ca2+-added and 1.0 mmol/l EGTA) for 60 min before incubation with bis-oxonol (200 nmol/l). Trace c: spermatozoa were suspended in Ca2+-free medium and then stimulated with thapsigargin after the addition of a maximal efficacious dose of thapsigargin. Trace d: spermatozoa loaded with a Ca2+ chelator, BAPTA, were suspended in Ca2+-free medium in the presence of bis-oxonol (200 nmol/l). Where indicated thapsigargin (Tg, 100 nmol/l) and valinomycin (Val, 1.0 µmol/l) were added.

 
It is possible that the hyperpolarization observed after thapsigargin addition is induced by Ca²⁺ store depletion which in turn can activate the Ca²⁺-dependent K⁺ channels that are present in all eukaryote cells (Darszon et al., 1999). To explore this hypothesis, we used different experimental approaches: in the presence of 60 mmol/l K⁺ in the external medium, to reduce the driving force for K⁺ efflux, the hyperpolarizing effects of thapsigargin were strongly reduced (Figure 6, trace b). Sperm incubation with TEA (10 mmol/l) and with charybdotoxin (1 µmol/l), two well-known blockers of K⁺ channels (Findlay et al., 1985; Gimenez-Gallego et al., 1988), abolished the hyperpolarization induced by thapsigargin (Figure 6, traces c and d respectively). Interestingly, in these experimental conditions, i.e. when thapsigargin-dependent plasma membrane hyperpolarization was prevented (by incubation with high K⁺-containing medium, TEA or charybdotoxin), the plasma membrane depolarization dependent on Ca²⁺ influx was abolished while progesterone was fully competent in depolarizing the sperm plasma membrane, as previously reported (Foresta et al., 1993).

View larger version (10K): [in this window] [in a new window]   Figure 6. Effects of K+-channel inhibition on thapsigargin-induced plasma membrane potential variations. Trace a: spermatozoa were suspended in standard saline. Trace b: spermatozoa were suspended in high K+-containing medium (60 mmol/l). Trace c: spermatozoa were pre-incubated in the presence of tetra-ethyl-ammonium (TEA; 10 mmol/l) for 15 min before thapsigargin addition. Trace d: spermatozoa were pre-incubated in the presence of charybdotoxin (100 nmol/l) for 15 mins before thapsigargin addition. Where indicated, thapsigargin (Tg, 100 nmol/l) or progesterone (Pg, 3.18 µmol/l) were added. Traces are representative of three similar experiments.

 
The reduction of the depolarizing effect of progesterone observed in Figure 6 (trace b), is probably due to the fact that in these experimental conditions (high external K⁺ concentration) sperm plasma membrane potential has been previously depolarized by K⁺ addition so that progesterone addition cannot much further depolarize the sperm plasma membrane.

In high K⁺-containing medium thapsigargin induced a rise in [Ca²⁺]_i with kinetic characteristics resembling those of Ca²⁺-free medium (data not shown), thus confirming that inhibition of plasma membrane hyperpolarization blocks the thapsigargin-induced Ca²⁺ influx in human spermatozoa.

Role of voltage-operated Ca²⁺ channels (VOCCs) in thapsigargin-induced [Ca²⁺]_i rise
Plasma membrane depolarization induced by thapsigargin, although determined by Ca²⁺ influx, may activate VOCCs, thus inducing further Ca²⁺ influx through these channels in addition to that through store-operated channels. Pre-incubation with VOCCs antagonists verapamil (10 µmol/l) and nifedipine (1.0 µmol/l) induced a reduction in the rise of [Ca²⁺]_i observed after thapsigargin addition of ~20%, with respect to that observed in control experiments (Figure 7A). In Figure 7B it is shown that the depolarization of sperm plasma membrane by gramicidin addition (0.1 µg/ml) did not modify the [Ca²⁺]_i (trace a) while progesterone induced a rapid rise of [Ca²⁺]_i, as previously described (Foresta et al., 1993). When plasma membrane depolarization induced by gramicidin was preceded by hyperpolarization, by the addition of valinomycin, the depolarization by gramicidin induced a rise in [Ca²⁺]_i (Figure 7B, trace b) that was inhibited by sperm pre-incubation with verapamil and nifedipine (data not shown). Thus, sperm plasma membrane hyperpolarization may play a role in the activability of VOCCs in human spermatozoa.

View larger version (12K): [in this window] [in a new window]   Figure 7 . Effects of voltage-operated Ca2+ channel antagonists on sperm [Ca2+]i elevations induced by thapsigargin. (A) Trace a: spermatozoa suspended in standard saline and then treated with thapsigargin (Tg, 100 nmol/l); trace b and trace c: spermatozoa pre-incubated in the presence of verapamil (10 µmol/l for 15 mins) and nifedipine (1.0 µmol/l for 15 min) respectively before thapsigargin addition. (B) Trace a: spermatozoa suspended in standard saline; trace b, sperm were pre-treated with valinomycin (Val, 1.0 µmol/l) before gramicidin (Gr, 0.1 µg/ml) addition. Traces are representative of three similar experiments.

 
Effects of thapsigargin and CPA on sperm acrosome reaction
Figure 8 shows the percentages of acrosome-reacted spermatozoa obtained after incubation with SERCA–ATPase inhibitors. Thapsigargin and CPA induced a rapid and dose-dependent increase in sperm acrosome reaction that was evident only when Ca²⁺ was present in the external medium (Figure 8A,B). Then we evaluated the role of Ca²⁺-activated K⁺ channels in thapsigargin and CPA induced acrosome reaction. As shown in Figure 8C, sperm pre-incubation in the presence of TEA (10 mmol/l) or charybdotoxin (1.0 µmol/l) inhibited the acrosome reaction induced by thapsigargin and CPA.

View larger version (20K): [in this window] [in a new window]   Figure 8 . Effects of thapsigargin on sperm acrosome reaction. (A and B) Thapsigargin and cyclopiazonic acid (CPA) stimulate the acrosome reaction in a dose-dependent manner but only when Ca2+ was present in the external medium. (C) K+ channel blockers inhibit the thapsigargin-stimulated sperm acrosome reaction. White bars = saline; black bars = thapsigargin. Bars corresponding to control (C) represent percentages of acrosome-reacted spermatozoa before addition of saline and thapsigargin.

 
Discussion

In the present study we have demonstrated that thapsigargin and CPA, two structurally unrelated SERCA–ATPase inhibitors (Thastrup et al., 1990), mobilize Ca²⁺ from internal stores in human spermatozoa thus suggesting the presence of Ca²⁺-sequestering organelles in these specialized cells. For a long time, the existence of Ca²⁺ stores in mammalian spermatozoa has been matter of debate. It has been previously suggested that Ca²⁺ accumulation can occur within mitocondria or in the sperm cytoplasmic droplet (Blackmore, 1993; Meizel and Turner, 1993) but only recently the acrosomal vescicle from different mammalian spermatozoa has been shown to possess IP₃ receptors (Walensky and Snyder, 1995; Kuroda et al., 1999) as found in Ca²⁺ stores of different somatic cells. Furthermore, calreticulin, a Ca²⁺-binding protein localized within Ca²⁺ stores, has been demonstrated in the acrosome of the rat (Nakamura et al., 1992) thus suggesting that mammalian spermatozoa possess internal Ca²⁺ stores (Walensky and Snyder, 1995; Kuroda et al., 1999). In the present study, we have attempted to localize internal Ca²⁺ stores in human spermatozoa using a fluorescent conjugate of thapsigargin, BODIPY-FL-thapsigargin. This fluorescent probe (after binding to SERCA–ATPase of internal Ca²⁺ stores that are different from those of the plasma membrane), emits a fluorescence that allows localization of SERCA–ATPase and thus putative internal Ca²⁺ stores. This experimental approach allowed us to identify some sperm regions where thapsigargin-binding sites (and possibly internal Ca²⁺ stores) are located, these being the acrosomal vescicle, the posterior portion of the sperm head, the equatorial segment, and the midpiece. After the acrosome reaction, the fluorescence previously observed in the acrosome was progressively lost, to further support the hypothesis that this vescicle may be an internal Ca²⁺ store in human spermatozoa. On the contrary, the fluorescence localized in the midpiece was unmodified after acrosome reaction induction with residual fluorescence also at the post-acrosomal region. These results are in perfect agreement with those recently obtained in human spermatozoa (Kuroda et al., 1999), which demonstrate the presence of IP₃ type 1 receptor in the acrosome and of IP₃ type 3 receptor in the posterior region of the head, midpiece and tail. These authors also demonstrated that the expression of IP₃ type 1 receptor in the acrosome was diminished after the acrosome reaction, while the expression of IP₃ type 3 receptor in the other regions was unmodified (Kuroda et al., 1999).

It is well known that mammalian sperm capacitation and the acrosome reaction depend on extracellular Ca²⁺ (Wassarmann, 1987; Thomas and Meizel, 1989; Kopf and Gerton, 1991; Florman et al., 1992; Foresta and Rossato, 1997). The role of internal Ca²⁺ stores in the regulation of sperm functions has not been evaluated extensively and, to date, the presence of Ca²⁺ sequestering organelles in human spermatozoa is still a matter of debate. Experiments with anti-IP₃ receptor antibodies have demonstrated that intracellular Ca²⁺ sequestering organelles are also present in mature spermatozoa from different mammalian species and in the human (Walensky and Snyder, 1995; Kuroda et al., 1999). The present study, showing the localization of SERCA–ATPase specifically present on internal Ca²⁺ stores, confirms that human spermatozoa possess internal Ca²⁺ sequestering organelles, possibly localized on the acrosome and midpiece.

The results of the present study clearly show that the rise in [Ca²⁺]_i induced by thapsigargin and CPA, two chemically unrelated inhibitors of SERCA–ATPases (Thastrup et al., 1990), was characterized by two components: a first phase observable both in the presence and absence of external Ca²⁺ due to internal Ca²⁺ store depletion and a second plateau phase that was present only in Ca²⁺ medium and that was due to Ca²⁺ influx from the extracellular space. Since thapsigargin and cyclopiazonic acid do not stimulate IP₃ production (Thastrup et al., 1990), the results of the present study resemble the characteristics of the so called `capacitative' Ca²⁺ entry model (Putney, 1990) thus demonstrating that this pathway of Ca²⁺ entry is active also in a mature spermatozoa. The possible involvement of capacitative Ca²⁺ entry in human sperm function regulation was previously also proposed for the stimulatory effects of progesterone (Tesarik et al., 1996; Kirkman-Brown et al., 2000). Previous studies failed to demonstrate a rise in [Ca²⁺]_i after thapsigargin addition in Ca²⁺-free medium (Blackmore, 1993; Meizel and Turner, 1993); the reasons for this discrepancy are not clear but could be due to the detector system used or to the different experimental approaches used in sperm incubation procedures. Furthermore, in a previous study, Meizel and Turner did not observe the acrosome reaction after thapsigargin addition in human uncapacitated spermatozoa. Different sperm selection and incubation procedures might explain these different results. On the other hand, different studies have demonstrated that thapsigargin can also induce the acrosome reaction in uncapacitated spermatozoa (Blackmore, 1993; Williams and Ford, 1997).

We further analysed the effects of SERCA–ATPase inhibitors on sperm ion homeostasis monitoring their effects on plasma membrane potential. As mentioned previously, thapsigargin and CPA produced important effects on plasma membrane potential: an initial fast and transient plasma membrane hyperpolarization followed by a long-lasting plasma membrane depolarization. This depolarization phase was clearly dependent on Ca²⁺ influx from the external medium since it was completely reversed by EGTA addition and it was absent in Ca²⁺-free medium. Ca²⁺ currents activated by internal Ca²⁺-store depletion have been described in a number of different non-excitable cells and have been called Ca²⁺-release activated Ca²⁺ currents (I_CRAC) (Parekh and Penner, 1997). A Ca²⁺-dependent plasma membrane depolarization preceded by a fast and transient hyperpolarization has been previously described in sea urchin spermatozoa after stimulation with the peptide speract (Gonzalez-Martinez and Darszon, 1987). External Na⁺ has no significant role on ionic currents activated by intracellular Ca²⁺-store emptying in human spermatozoa, confirming observations by other authors in different cell types (Hoth and Penner, 1993; Lepple-Wienhues and Cahalan, 1996).

In a cell as small as the human spermatozoon, it is difficult to perform patch-clamp techniques but, despite the purely suggestive nature of plasma membrane potential studies with fluorescent probes, it is possible to suggest that the ionic currents described in this study after internal Ca²⁺ stores depletion may resemble the I_CRAC described in other cell types using patch-clamp (Parekh and Penner, 1997).

While the origin of the plasma membrane depolarization induced by internal Ca²⁺ store depletion is clear, the nature of the first rapid phase of hyperpolarization induced by Ca²⁺ store depletion is obscure. It could be observed both in the presence and absence of Ca²⁺ in the external medium and thus it does not depend on the influx of Ca²⁺ from the external space. The close correlation between the thapsigargin-induced transient rise of [Ca²⁺]_i due to internal Ca²⁺ store emptying and plasma membrane hyperpolarization suggest a tight link between these events. One hypothesis is that plasma membrane hyperpolarization may be due to an increased K⁺ permeability induced by the opening of Ca²⁺-activated K⁺ channels on the sperm plasma membrane. Sperm incubation in the presence of high [K⁺] in the external medium, TEA and charybdotoxin, two well-known inhibitors of K⁺ channels (Findlay et al., 1985; Gimenez-Gallego et al., 1988), inhibited plasma membrane hyperpolarization induced by Ca²⁺ store depletion, thus demonstrating that sperm plasma membrane hyperpolarization depends on the activation of K⁺ efflux, possibly through Ca²⁺-activated K⁺ channels. These channels, whose existence has been reported in all eukaryotic cells, have been recently described in rat spermatozoa (Chan et al., 1998) and other spermatogenetic cells (Darszon et al., 1999). In the same experimental conditions, when K⁺-dependent plasma membrane hyperpolarization was prevented, both Ca²⁺ influx from the external medium and the acrosome reaction were abolished, suggesting a possible key role for plasma membrane hyperpolarization in the activation of the mechanisms triggering capacitative Ca²⁺ entry and acrosomal exocytosis in human spermatozoa.

The same results were observed when plasma membrane depolarization due to Ca²⁺ influx from the external medium after thapsigargin-induced store depletion was abolished by sperm incubation with the intracellular Ca²⁺ chelator BAPTA. In these experimental conditions, [Ca²⁺]_i variations are prevented as well as plasma membrane potential variations and the acrosome reaction. These results seem to suggest that plasma membrane hyperpolarization induced by Ca²⁺ exit from internal stores is the focal event in the activation of Ca²⁺ influx from the external medium and thus for the sperm acrosome reaction occurrence induced by thapsigargin and CPA.

Gonzalez-Martinez and Darszon demonstrated that a fast transient hyperpolarization caused by the opening of K⁺ channels occurred during the sperm acrosome reaction in the sea urchin after egg jelly addition and that the inhibition of the hyperpolarization phase blocked the plasma membrane depolarization and the induction of the acrosome reaction by egg jelly (Gonzalez-Martinez and Darszon, 1987). The results of the present study in human spermatozoa seem to fit very well with those from other studies on invertebrate spermatozoa and with those reported in bovine and mouse spermatozoa demonstrating that plasma membrane hyperpolarization regulates ZP3-dependent acrosome reaction (Wassarmann, 1987; Zeng et al., 1995). Therefore (from the cited studies and the results of the present paper), it seems that plasma membrane hyperpolarization plays a pivotal role in regulating the biological events leading to sperm acrosome reaction in both humans and other species, and thus it may have a primary role in the fertilization process (Llanos, 1994).

Recent studies have also demonstrated that injection of mRNA from rat spermatogenic cells induces the expression of Ca²⁺-activated K⁺ channel in Xenopus oocytes (Chan et al., 1998). Finally, a subunit of a K⁺ channel has been identified in rat mature spermatozoa (Salvatore et al., 1999) and recently a novel K⁺ channel named SLO3, abundantly expressed in mouse and human spermatogenic cells (Schreiber et al., 1998), has been cloned and shown to possess a protein sequence similar to that of SLO1, a Ca²⁺-gated K⁺ channel (Schreiber et al., 1998). On the other hand, it has been long known that spermatogenic cells express K⁺-selective and TEA sensitive channels (Hagiwara and Kawa, 1984). Together, these data suggest that Ca²⁺-activated K⁺ channels are present in mammalian and human spermatozoa possibly playing an important role in regulating early events fundamental for the occurrence of acrosome reaction and fertilization. Furthermore, as well as the priming of ion channels, plasma membrane hyperpolarization may provide a route to potentiate Ca²⁺ influx from the external medium by increasing the driving force for Ca²⁺ entry within sperm cytoplasm. It is possible that activation of K⁺ channels responsible for plasma membrane hyperpolarization may be important for the recruitment of VOCCs from an inactivated state to an activatable one from which they are available for the opening during physiological events occurring at fertilization, as previously suggested (Zeng et al., 1995; Arnoult et al., 1999). In fact, following internal Ca²⁺ store depletion, an influx of Ca²⁺ from the external medium is activated, partially reduced in the presence of VOCC antagonists suggesting some involvement of VOCCs in the [Ca²⁺]_i rise observed after thapsigargin-induced internal Ca²⁺ store depletion.

Interestingly, the first phase of hyperpolarization activated by internal Ca²⁺ stores depletion plays an important role in the activation of Ca²⁺ influx since, when absent (by incubating spermatozoa with intracellular Ca²⁺ chelator, with high K⁺-medium or with K⁺-channel inhibitors), the Ca²⁺ influx was not observed and the acrosome reaction was inhibited. These findings suggest that plasma membrane K⁺ channels and plasma membrane hyperpolarization may be important determinants in the cascade of events leading to Ca²⁺ influx and perhaps to the activation of `capacitative' Ca²⁺ entry in human spermatozoa. Further studies are needed to confirm this hypothesis. Finally, the inability of sperm plasma membrane to undergo hyperpolarization in the presence of high K⁺ concentrations in the external medium, fits well with the high K⁺ concentrations present in the seminal plasma (Darszon et al., 1999), where spermatozoa have to be quiescent to prevent activation before the approach of the oocyte in the female genital tract. In this respect, the progressive reduction of [K⁺] in the spermatozoa bathing milieu following their transit from seminal tract to female genital tract secretions (Darszon et al., 1999), could cause a plasma membrane hyperpolarization that could be the first step of the cascade of events for sperm activation or activability.

In conclusion, the results of the present study demonstrate that: (i) human spermatozoa possess internal Ca²⁺ stores localized mainly at the acrosomal vescicle and midpiece; (ii) Ca²⁺ emptying from these stores by SERCA–ATPase inhibitors leads to [Ca²⁺]i rise and activation of the so called `capacitative' Ca²⁺ entry demonstrating that this Ca²⁺ entry pathway is operative also in human spermatozoa; (iii) internal Ca²⁺ store depletion activates Ca²⁺-dependent K⁺ channels inducing a plasma membrane hyperpolarization that may play an important role in the ionic events following store depletion; (iv) K⁺-dependent hyperpolarization is essential for the induction of Ca²⁺ influx activated by Ca²⁺ store depletion and for the acrosome reaction; and (v) a [Ca²⁺]_i rise induced by internal store depletion leads to exocytosis of the acrosome only when Ca²⁺ is present in the external medium.

Notes

³ To whom correspondence should be addressed. E-mail: forestac{at}protec.it

References

Arnoult, C., Kazam, I.G., Visconti, P.E. et al. (1999) Control of the low voltage-activated calcium channel of mouse sperm by egg ZP3 and by membrane hyperpolarization during capacitation. Proc. Natl Acad. Sci. USA, 96, 6757–6762.[Abstract/Free Full Text]

Blackmore, P.F. (1993) Thapsigargin elevates and potentiates the ability of progesterone to increase intracellular free calcium in human sperm: possible role of perinuclear calcium. Cell. Calcium, 14, 53–60.[ISI][Medline]

Blackmore, P.F., Beebe, S.J., Danforth. D.R. et al. (1990) Progesterone and 17 alpha-hydroxyprogesterone. Novel stimulators of calcium influx in human sperm. J. Biol. Chem., 256, 1376–1380.

Chan, H.C., Wu, W.L., Sun, Y.P. et al. (1998) Expression of sperm Ca²⁺-activated K⁺ channels in Xenopus oocytes and their modulation by extracellular ATP. FEBS Lett., 438, 177–182.[ISI][Medline]

Cross, N.L. and Meizel, S. (1989) Methods for evaluating the acrosomal status of mammalian sperm. Biol. Reprod., 41, 635–641.[Abstract]

Darszon, A., Labarca, P., Nishigaki et al. (1999) Ion channels in sperm physiology. Physiol. Rev., 79, 481–510.[Abstract/Free Full Text]

Findlay, I., Dunne, M.J., Ullrich, S. et al. (1985) Quinine inhibits Ca²⁺-independent K⁺ channels whereas tetraethylammonium inhibits Ca²⁺-activated K⁺ channels in insulin-secreting cells. FEBS Lett., 185, 4–8.[ISI][Medline]

Florman, H.M., Corron, M.E., Kim, T.D. et al. (1992) Activation of voltage-dependent calcium channels of mammalian sperm is required for zona pellucida-induced acrosomal exocytosis. Dev. Biol., 152, 304–314.[ISI][Medline]

Foresta, C. and Rossato, M. (1997) Calcium influx pathways in human spermatozoa. Mol. Hum. Reprod., 3, 1–4.[Free Full Text]

Foresta, C., Rossato, M., Chiozzi, P. et al. (1996) Mechanism of human sperm activation by extracellular ATP. Am. J. Physiol., 270, C1709–C1714.[Abstract/Free Full Text]

Foresta, C., Rossato, M. and Di Virgilio, F. (1992) Extracellular ATP is a trigger for the acrosome reaction in human spermatozoa. J. Biol. Chem., 267, 19443–19447.[Abstract/Free Full Text]

Foresta, C., Rossato, M. and Di Virgilio, F (1993) Ion fluxes through the progesterone-activated channel of the sperm plasma membrane. Biochem. J., 294, 279–283.

Gimenez-Gallego, G., Navia, M.A., Reuben, J.P. et al. (1988) Purification, sequence, and model structure of charybdotoxin, a potent selective inhibitor of calcium-activated potassium channels. Proc. Natl Acad. Sci. USA, 85, 3329–3333.[Abstract/Free Full Text]

Gonzalez-Martinez, M. and Darszon, A. (1987) A fast transient hyperpolarization occurs during the sea urchin sperm acrosome reaction induced by egg jelly. FEBS Lett., 218, 247–250.[ISI][Medline]

Hagiwara, S. and Kawa, K. (1984) Calcium and potassium currents in spermatogenic cells dissociated from rat seminiferous tubules. J. Physiol. (London), 356, 135–149.[Abstract/Free Full Text]

Hoth, M. and Penner, R. (1993) Calcium release-activated calcium current in rat mast cells. J. Physiol. (London), 465, 359–386.[Abstract/Free Full Text]

Kirkman-Brown, J.C., Bray, C., Stewart P.M. et al. (2000) Biphasis elevation of [Ca²⁺]_i in individual human spermatozoa exposed to progesterone. Dev. Biol., 222, 326–335.[ISI][Medline]

Kopf, G.S. and Gerton, G.L. (1991) Elements of Mammalian Fertilization, CRC Press, Boca Raton, USA.

Kuroda, Y., Kaneko, S., Yoshimura, Y. et al. (1999) Are there inositol 1,4,5-triphosphate (IP₃) receptors in human sperm? Life Sci., 65, 135–143.[ISI][Medline]

Lepple-Wienhues, A. and Cahalan, M.D. (1996) Conductance and permeation of monovalent cations through depletion-activated Ca²⁺ channels (I_CRAC) in Jurkat T cells. Biophys. J., 71, 787–794.[Abstract/Free Full Text]

Llanos, M.N. (1994) Evidence in support of a role for Ca²⁺-activated K⁺ channels in the hamster sperm acrosome reaction. J. Exp. Zool., 269, 484–488.[ISI][Medline]

Meizel, S. and Turner, K.O. (1993) Initiation of the human sperm acrosome reaction by thapsigargin. J. Exp. Zool., 267, 350–355.[ISI][Medline]

Mendoza, C. and Tesarik, J. (1993) A plasma-membrane progesterone receptor in human sperm is switched on by increasing intracellular free calcium. FEBS Lett., 330, 57–60.[ISI][Medline]

Nakamura, M., Michikawa, Y., Baba, T. et al. (1992) Calreticulin is present in the acrosome of spermatids of rat testis. Biochem. Biophys Res. Commun., 186, 668–673.[ISI][Medline]

Osman, R.A., Andria, M.L., Jones, A.D. et al. (1989) Steroid induced exocytosis: the human sperm acrosome reaction. Biochem. Biophys Res. Commun., 160, 828–833.[ISI][Medline]

Parekh, A.B. and Penner, R. (1997) Store depletion and calcium influx. Physiol. Rev., 77, 901–930.[Abstract/Free Full Text]

Perry, R.L., Barrat, C.L.R., Warren, M.A. et al. (1997) Elevating intracellular calcium levels in human sperm using an internal calcium ATPase inhibitor, 2,5-di(tert-butyl) hydroquinone (TBQ), initiates capacitation and the acrosome reaction but only in the presence of extracellular calcium. J. Exp. Zool., 279, 291–300.[ISI][Medline]

Putney, J.W. Jr. (1990) Capacitative calcium entry revisited. Cell Calcium, 11, 611–624.[ISI][Medline]

Rossato, M., Di Virgilio, F. and Foresta, C. (1996) Calcium influx pathways in human spermatozoa. Mol. Hum. Reprod., 2, 903–909.[Abstract/Free Full Text]

Salvatore, L., D'Adamo, M.C., Polinshchuk, R. et al. (1999) Localization and age-dependent expression of the inward rectifier K⁺ channel subunit Kir 5.1 in a mammalian reproductive system. FEBS Lett., 449, 146–152.[ISI][Medline]

Sargeant, P., Clarkson, W.D., Sage, S.O. et al. (1992) Calcium influx evoked by Ca²⁺ store depletion in human platelets is more susceptible to cytochrome P-450 inhibitors than receptor-mediated calcium entry. Cell Calcium, 13, 553–564.[ISI][Medline]

Schreiber, M., Wei, A., Yuan, A. et al. (1998) Slo3, a novel pH-sensitive K⁺ channel from mammalian spermatocytes. J. Biol. Chem., 273, 3509–3516.[Abstract/Free Full Text]

Tesarik, J., Carreras, A. and Mendoza, C. (1993) Differential sensitivity of progesterone- and zona pellucida-induced acrosome reactions to pertussis toxin. Mol. Reprod. Dev., 34, 183–189.[ISI][Medline]

Tesarik, J., Carreras, A. and Mendoza, C. (1996) Single cell analysis of tyrosine kinase dependent and independent Ca²⁺ fluxes in progesterone induced acrosome reaction. Mol. Hum. Reprod., 2, 225–232.[Abstract/Free Full Text]

Thastrup, O., Cullen, P.J., Drobak, B.K. et al. (1990) Thapsigargin, a tumor promoter, discharges intracellular Ca²⁺ stores by specific inhibition of the endoplasmic reticulum Ca²⁺-ATPase. Proc. Natl Acad. Sci. USA, 87, 2466–2470.[Abstract/Free Full Text]

Thomas, P. and Meizel, S. (1988) An influx of extracellular calcium is required for initiation of the human sperm acrosome reaction induced by human follicular fluid. Gamete Res., 20, 397–411.[ISI][Medline]

Walensky, L.D. and Snyder, S.H. (1995) Inositol 1,4,5-trisphosphate receptors selectively localized to the acrosomes of mammalian sperm. J. Cell. Biol., 130, 857–869.[Abstract/Free Full Text]

Wassarmann, P.M. (1987) The biology and chemistry of fertilization. Science, 235, 553–560.[Abstract/Free Full Text]

Williams, K.M. and Ford, W.C.L. (1997) Effect of thapsigargin on intracellular calcium, motility and acrosome reaction of human sperm. [Abstr. P5] Int. J. Androl. 20 (Suppl. 1).

Zeng, Y., Clark, E.N. and Florman, H.M. (1995) Sperm membrane potential: hyperpolarization during capacitation regulates zona pellucida-dependent acrosomal secretion. Dev. Biol., 171, 554–563.[ISI][Medline]

Submitted on October 5, 2000; accepted on November 16, 2000.

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