Effect of intracellular pH on depolarization-evoked calcium influx in human sperm

Juan J. Fraire-Zamora and Marco T. González-Martínez

Departamento de Farmacología, Facultad de Medicina, Universidad Nacional Autónoma de México, Ciudad Universitaria, CP 04510, Mexico City, Mexico

Submitted 16 March 2004 ; accepted in final form 9 August 2004


    ABSTRACT
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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Human sperm are endowed with putative voltage-dependent calcium channels (VDCC) that produce measurable increases in intracellular calcium concentration ([Ca2+]i) in response to membrane depolarization with potassium. These channels are blocked by nickel, inactivate in 1–2 min in calcium-deprived medium, and are remarkably stimulated by NH4Cl, suggesting a role for intracellular pH (pHi). In a previous work, we showed that calcium permeability through these channels increases approximately onefold during in vitro "capacitation," a calcium-dependent process that sperm require to fertilize eggs. In this work, we have determined the pHi dependence of sperm VDCC. Simultaneous depolarization and pHi alkalinization with NH4Cl induced an [Ca2+]i increase that depended on the amount of NH4Cl added. VDCC stimulation as a function of pHi showed a sigmoid curve in the 6.6–7.2 pHi range, with a half-maximum stimulation at pH ~7.00. At higher pHi (≥7.3), a further stimulation occurred. Calcium release from internal stores did not contribute to the stimulating effect of pHi because the [Ca2+]i increase induced by progesterone, which opens a calcium permeability pathway that does not involve gating of VDCC, was unaffected by ammonium. The ratio of pHi-stimulated-to-nonstimulated calcium influx was nearly constant at different test depolarization values. Likewise, depolarization-induced calcium influx in pHi-stimulated and nonstimulated cells was equally blocked by nickel. In our capacitating conditions pHi increased 0.11 pH units, suggesting that the calcium influx stimulation observed during sperm capacitation might be partially caused by pHi alkalinization. Additionally, a calcium permeability pathway triggered exclusively by pHi alkalinization was detected.

mammalian sperm; capacitation; intracellular calcium


DURING ITS SHORT LIFE, the mature sperm must accomplish a series of cellular processes that require activation of calcium entry mechanisms. For instance, in mammals, ejaculated sperm go through a complex process called "capacitation," which makes them capable of undergoing acrosomal exocytosis (AE) on contact with the egg zona pellucida glycoprotein ZP3 (3). AE is essential for fertilization because only sperm that have undergone AE are able to fuse with the egg. Capacitation occurs in the female genital tract and involves, among other processes, the activation of a biochemical route that increases cAMP, which in turn depends on calcium (17), bicarbonate (8), or cholesterol removal from the plasma membrane (25). As a consequence, a tyrosine kinase activates (3). Concomitantly, the resting intracellular pH (pHi) increases 0.14 units (9), and the resting intracellular calcium concentration ([Ca2+]i) increases ~100 nM (2). In this regard, maintenance of adequate values of pHi is required for bovine (19) and mouse (35) sperm motility. Furthermore, the hyperactivation of motility, which sperm require to reach the egg, is regulated by [Ca2+]i (7, 16, 27, 37).

In sperm of all species, the identification of voltage-dependent calcium entry mechanisms has been hampered by the size of the head, which makes the use of patch-clamp methods very difficult (10). Nevertheless, our group has shown (23) that step depolarization, induced by potassium in the presence of the potassium ionophore valinomycin, induces an increase in [Ca2+]i in fura-2-loaded human sperm populations that is totally prevented in medium without calcium. This strongly indicates that human sperm are endowed with functional voltage-dependent calcium channels (VDCC). These putative channels are insensitive to nifedipine and verapamil but sensitive to nickel (23) and mibefradil (4). Furthermore, they inactivate in ~1.5 min in calcium-deprived medium and are stimulated by ammonium, suggesting a role for pHi (23). It is clear that these data are not sufficient to properly classify the putative channels. Nevertheless, their physiological role and regulation, based on the functional test reported here, deserve to be explored in light of the potential role of VDCC in AE induction by ZP3 and in sperm motility.

During capacitation the calcium influx through these channels increases about onefold (12). In addition, in noncapacitated and capacitated sperm, progesterone, which is present in the follicular fluid and induces calcium influx through nongenomic receptors (5), enhances the [Ca2+]i increase induced by depolarization (12). These observations have led to the hypothesis that "in vivo," a progressive modification of VDCC caused by capacitation and progesterone action may increase as much as four times the levels of calcium permeation found in noncapacitated sperm. Thus, when capacitated sperm reach the egg zona pellucida, the ZP3-induced AE would occur more efficiently because the mechanism of calcium mobilization induced by this glycoprotein may involve gating of VDCC (1).

Here we present evidence indicating that the putative VDCC are modulated by pHi. We found that pHi alkalinization with NH4Cl stimulated the calcium influx induced by depolarization to an extent that may explain, in considerable part, the stimulation of VDCC observed during sperm capacitation.


    METHODS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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All reagents were obtained from Sigma and Molecular Probes. Capacitating human sperm medium (HSM) had the following composition (29) in mM: 117.5 NaCl, 8.6 KCl, 2.5 CaCl2, 0.3 NaH2PO4, 0.49 MgCl2, 0.3 Na-pyruvate, 19 Na-lactate, 2 glucose, and 25 NaHCO3, with 3 mg/ml bovine serum albumin (BSA, fraction V; Sigma). The medium used for sperm purification and dye loading procedure (H-HSM) had the same composition except that BSA was removed and NaHCO3 was replaced by 25 mM HEPES, adjusted to pH 7.6. Fluorescence was recorded from samples in H-HSM medium supplemented with 5 mM NaHCO3 (H-HSM1). H-HSM1 medium was supplemented with bicarbonate because the presence of this anion (1–15 mM) stimulates calcium transport in mouse sperm (Ref. 37; see DISCUSSION). Thus NaHCO3 was included in the measurements medium, despite the fact that our group previously showed (12, 14, 23) that the calcium influx induced by depolarization can be triggered in medium without bicarbonate.

Sperm purification, dye loading, and capacitation. Human semen was obtained from a panel of sixteen 18- to 34-year-old healthy donors who gave informed consent under a protocol approved by the institutional review board. Sperm purification was performed with Percoll gradients as described previously (33). Purified sperm (1–2 x 108 cells) were loaded with 2 µM fura-2-AM (Sigma) in 2 ml of H-HSM medium at 36°C for 40 min. Once washed, cells were either resuspended in 25 ml of H-HSM medium and used immediately for fluorescence recordings (noncapacitated sperm) or resuspended in 25 ml of HSM (capacitating medium) for 4–6 h at 36°C under 3% CO2-97% air and 100% humidity. These conditions kept the pH of the HSM medium between 7.6 and 7.7.

The pHi measurements were performed with BCECF in fura-2-loaded sperm. Loading human sperm with both fura-2 and BCECF did not affect BCECF fluorescence, but it did interfere with fura-2 fluorescence (not shown). Even though the pHi measurements in fura-2-loaded and fura-2-free sperm were practically identical (not shown), fura-2-loaded cells were preferred for pHi determinations to properly compare pHi with calcium transport results.

Noncapacitated or capacitated fura-2-loaded sperm were loaded with 3 µM BCECF-AM for 30 min in 5 ml of H-HSM (36°C) or HSM (36°C; under 3% CO2–97% air, 100% humidity) medium, respectively. The cells were washed, and pHi was determined as described in Measurement of [Ca2+]i, pHi, and membrane potential.

Measurement of [Ca2+]i, pHi, and membrane potential. Fluorescence recordings were performed with a Photon Technology International (PTI) spectrofluorometer equipped with an excitation monochromator and two photomultiplier tubes (PMTs) positioned at 90° with respect to the xenon source. The use of both PMTs and optical interference filters permits measurement of two fluorescence signals simultaneously.

[Ca2+]i measurements. [Ca2+]i was detected and calibrated in fura-2-loaded sperm as described by Linares-Hernández et al. (23). Sperm (1–2 x 107 cells) were centrifuged at 300 g for 5 min, and the pellet (~100 µl) was immediately added to the fluorescence cuvette, containing 2.5 ml of H-HSM1, at 36°C and under constant magnetic stirring. The cells were alternately excited at 340 and 380 nm, and the fluorescence was detected at 488 nm with an optical filter (band pass 10.0 ± 2 nm; Andover); the ratios were acquired at 0.86 Hz. To calibrate, the maximum fluorescence ratio (Rmax) and, subsequently, the minimum fluorescence ratio (Rmin) were determined by adding 8 µM ionomycin and a mixture of 6 mM EGTA + 0.12% Triton X-100, respectively. Triton X-100 was necessary to obtain a rapid value of Rmin, because intracellular calcium removal with EGTA is extremely slow in the presence of ionomycin (~20 min). Rmin reached similar values in the presence and in the absence of the detergent (not shown).

It has been reported that the Kd of fura-2 for calcium slightly decreases as pH increases (26). Because the [Ca2+]i increase induced by depolarization was assessed while pHi was increasing, the shift in the Kd for calcium could produce increases in fura-2 fluorescence independent of calcium. However, in HSM-H1 medium without calcium + 0.5 mM EGTA, a pHi alkalinization induced by 60 mM NH4Cl (from ~6.7 to ~7.34, the highest alkalinization studied here) did not induce change in intracellular calcium (not shown), indicating that under our conditions the small effect of pH on fura-2 Kd for calcium was undetectable.

Membrane potential measurements. In some experiments, simultaneous recordings of membrane potential (Vm) and [Ca2+]i were performed in fura-2-loaded sperm with the Vm-sensitive dye 3,3'-dipropylthiacarbocyanine iodide [diSC3(5); 500 nM] as described previously (23). In these experiments, fura-2-loaded sperm were added to a fluorescence cuvette containing 2.5 ml of H-HSM1 + 0.5 µM diSC3(5) at 36°C under constant magnetic stirring. On addition of cells, diSC3(5) fluorescence decreased because of the electrophoretic uptake by sperm, reaching nearly constant values in ~3 min. In human sperm incubated in the presence of the potassium ionophore valinomycin, the plasma Vm sets near the Nernst potential for potassium distribution [Ek = –61.54 mV log([K]i/[K]e), where [K]i and [K]e are intracellular and extracellular potassium concentrations, respectively] (23). Thus Vm can be calculated as the Ek, taking into consideration that [K]i = 120 mM (23). There is a linear relationship between the fractional change of diSC3(5) fluorescence, f – fo/fo (where fo is fluorescence in the presence of valinomycin addition and f are actual fluorescence values), and Ek (23). Consequently, f can be converted to Vm according to the following equation (11): Vm = f/mfo – 1/mb/m, where m and b are parameters of the linear calibration curve, that is, the slope and the fractional change of fluorescence at 0 mV, respectively.

It should be mentioned that we have not detected diSC3(5) fluorescence signal from mitochondria when valinomycin or the proton ionophore CCCP is added to human sperm (13, 23). This may be related to the fact that sperm motility is unaffected by mitochondrial inhibitors (17) or by anaerobiosis (24), which suggests low mitochondrial activity. Thus the uncoupling effect of valinomycin on mitochondria does not influence the results presented here.

Simultaneous recordings of Vm and intracellular calcium. Simultaneous recordings of fura-2 and diSC3(5) fluorescence were performed with two PMTs of the PTI system. One PMT, with the 488-nm filter, was used to detect fura-2 fluorescence as described above, and the other PMT, with a 670-nm filter (band pass 10 ± 2 nm; Andover), was used to detect diSC3(5) fluorescence simultaneously. Just in front of the xenon source an additional halide lamp (tri-lite; WPI) and a 600-nm filter (band pass 10 nm; Hansatech) were set to excite diSC3(5). The data were collected and analyzed with the PTI computer interface at 0.86 Hz.

pHi measurements. BCECF fluorescence was detected at 550 nm with an Andover filter (band pass 10.0 ± 2 nm) exciting at 500 and 439 nm, at 36°C and under magnetic stirring. The 500-to-439 ratios (acquired at 0.6 Hz) were calibrated at the end of each trace by adding 0.12% Triton X-100 and then by modifying the pH of the medium with HCl. The addition of Triton X-100 increased the fluorescence ratio to a value corresponding to the pH of H-HSM1 (with almost no effect at 439 nm; not shown). Three consecutive amounts of HCl were then added to the cuvette, which resulted in step decreases in fluorescence ratios. At each step (including the one obtained after detergent addition), the pH was determined with a conventional pH electrode and corresponded to 7.6, ~7.10, ~6.50, and ~5.45 (see Fig. 1A). The calibration curve shows the sigmoid relationship between fluorescence ratio and pH (Fig. 1B). The BCECF ratio values were converted to pHi with the software (FeliX, version 1.41) of the PTI spectrofluorometer.



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Fig. 1. Effect of NH4Cl on human sperm intracellular pH (pHi). Fura-2-loaded, noncapacitated sperm were loaded with 3 µM BCECF-AM. Once washed, the sperm pellet (~1 x 107) was poured into the fluorescence cell containing 2.5 ml of human sperm medium (HSM) used for purification and dye loading (H-HSM) + NaHCO3 (H-HSM1) at 36°C and under magnetic stirring. Fluorescence was detected at 550 nm, exciting at 500 and 349 nm. A: calibration procedure. The signal was calibrated by adding 0.12% Triton X-100 (TX-100), which permeabilized the cells and brought the ratio to a pH corresponding to the pH of the H-HSM1 medium. Different amounts of HCl were then added as indicated, which produced concomitant step decreases in BCECF ratios. At each step, pH was determined with a pH electrode. pH values are indicated at right. B: BCECF ratio as a function of pH (obtained from data of A; a calibration point at pH 8.35 is included). The line corresponds to the sigmoid equation ratio = 11.03 pH units – 10.38 pH units/{1 + exp[(pH – pKa)/0.59]}, with acidic dissociation constant (pKa) = 7.08. C: time course of pHi changes induced by the sequential addition of valinomycin and the 0–60 mM NH4Cl-30 mM KCl mixture. Three minutes after sperm were poured into the fluorescence cell, 0.4 µM valinomycin (V) was added; 1 min later, different amounts of NH4Cl (0–60 mM) and 30 mM KCl were added simultaneously. After 30 s, the increased pHi tended to decrease slowly. D: pHi (means ± SE, n = 7) as a function of the ammonium concentration added, according to the protocol established in C. The pHi shown in this graph was the average obtained during the first 30 s after NH4Cl + 30 mM KCl addition.

 
The pHi buffer capacity ({beta}), measured as a sperm response to the ammonium-induced pHi alkalinization, was estimated. An approximation of {beta} was determined with the equation (20) {beta} = /{Delta}pHi, where A is [NH4Cl] added, pH is the pH of the H-HSM1 medium (pH 7.6), pHi is the pHi reached on NH4Cl addition, and {Delta}pHi is the difference between pHi obtained on NH4Cl addition and resting pHi (pHi 6.7).

Measurement of voltage-dependent calcium influx. To detect the voltage-dependent calcium influx, sperm was depolarized with different amounts of KCl in the presence of 0.4 µM valinomycin (added 1 min before potassium). Valinomycin was required to bring Vm to approximately –71 mV, which removes VDCC from the inactivated state and makes the membrane mainly dependent on potassium permeability. The voltage-dependent calcium influx was measured as the difference between the [Ca2+]i reached at the peak and the resting [Ca2+]i. This difference is an appropriate estimation of a calcium influx because calcium removal activities are rather slow in sperm (12, 35).

Statistical analysis. Numeric results are expressed as means ± SE; n indicates the number of individuals tested. ANOVA and paired Student's t-test were used. P values ≤0.05 were considered statistically significant.


    RESULTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Effect of pHi on calcium influx induced by depolarization. To establish the pHi dependence of the voltage-dependent calcium influx, we first determined the effect of NH4Cl on pHi in human sperm loaded with both fura-2 and BCECF. pHi was measured in the presence of valinomycin, the potassium ionophore required to modify Vm and so trigger voltage-dependent calcium influx with potassium (23). The resting pHi was 6.70 ± 0.01 (n = 7) and was not affected by valinomycin addition (Fig. 1C). One minute after valinomycin addition, different amounts of NH4Cl and 30 mM KCl (which was the amount of potassium most frequently used to depolarize sperm) were added simultaneously. In the absence of NH4Cl, 30 mM KCl induced a slight, slow alkalinization (Fig. 1C, bottom trace), as previously reported (23). In contrast, NH4Cl caused a concentration-dependent, fast pHi alkalinization (Fig. 1C) that was linear up to 10 mM, with a slope of ~0.02 pH units/mM (Fig. 1D). At concentrations >10 mM, the slope diminished abruptly (~0.003 pH units/mM). The {beta}-value obtained with 2.5–20 mM ammonium ranged between 93 and 109 mM/pHi, and at 40 and 60 mM NH4Cl {beta}-values increased to ~146 mM/pHi. These values are rather high compared with other sperm species (30), suggesting that human sperm have a strong pHi buffer system at high pH, particularly at pHi >7.2. In this regard, the pHi alkalinization induced by 60 mM NH4Cl in the absence of bicarbonate was similar to that obtained in H-HSM1 medium (5 mM bicarbonate; not shown), indicating that supplementation of this salt in the medium did not further contribute to the high {beta}-value. Alternatively, the high {beta}-values obtained here could be overestimated if dampening of the NH3-induced alkalinization occurred because of ammonium permeation.

In the presence of valinomycin, addition of 30 mM K+ brings Vm from around –71 mV to around –30 mV and concomitantly induces a transient increase in [Ca2+]i (23). When 10 mM NH4Cl was added 10 s before 30 mM KCl, the voltage-dependent calcium influx was markedly stimulated (Fig. 2). The simultaneous addition of ammonium and KCl produced a similar result (Fig. 2), indicating that the effect of ammonium was practically immediate. This result was consistent with the ability of ammonium to rapidly increase human sperm pHi. The rest of the experiments reported here were performed with the simultaneous addition procedure.



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Fig. 2. Effect of NH4Cl on the calcium influx induced by depolarization in human sperm. Noncapacitated fura-2-loaded sperm were poured into the fluorescence cell containing 2.5 ml of H-HSM1 at 36°C and under constant magnetic stirring. Fluorescence was detected at 488 nM, exciting at 340 and 380 nM. After 3-min incubation, 0.4 µM valinomycin was added (not shown on traces). In the trace on the left, 30 mM KCl was added 1 min after valinomycin. The trace in the middle is like the trace on the left, except that 10 mM NH4Cl was added 10 s before KCl. In the trace on the right, 30 mM KCl and 10 mM NH4Cl were added simultaneously 1 min after valinomycin. Traces are representative of 5 experiments. [Ca2+]i, intracellular calcium concentration.

 
The stimulating effect of NH4Cl depended on the concentration: the higher the amount of NH4Cl added, the higher the [Ca2+]i increased (Fig. 3, top). At 10 and 20 mM, the stimulation seemed to reach a nearly constant value; however, when NH4Cl was raised to 40 and 60 mM a further stimulation occurred, at levels that were frequently close to fura-2 saturation. Interestingly, addition of ammonium alone (without KCl), at concentrations >10 mM (>pHi 7.05), also induced small, slow, concentration-dependent increases in [Ca2+]i (Fig. 3, bottom; see Fig. 5) that, unlike VDCC, were nontransient. This finding suggested that human sperm is endowed with calcium channels gated by pHi alkalinization that would not inactivate. In H-HSM1 medium without calcium + 0.5 mM EGTA, 60 mM NH4Cl did not produce any change in [Ca2+]i, suggesting that in normal medium, ammonium induces calcium influx from the external medium (trace not shown). These putative channels may be related to a calcium permeability pathway that is opened by pHi alkalinization, found in the plasma membrane of mouse spermatocytes and mouse testicular sperm (31).



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Fig. 3. Effect of increasing NH4Cl concentration ([NH4Cl]) on the calcium influx induced by depolarization in human sperm. Noncapacitated fura-2-loaded sperm were poured into the fluorescence cell containing 2.5 ml of H-HSM1 at 36°C and under magnetic stirring. Fluorescence was detected at 488 nM, exciting at 340 and 380 nM. After 3-min incubation, 0.4 µM valinomycin was added (not shown on trace); 1 min later, the indicated amount of NH4Cl (0–60 mM) + 30 mM KCl was simultaneously added (top). Bottom: effect of NH4Cl alone on human sperm [Ca2+]i. One minute before NH4Cl addition, 0.4 µM valinomycin was added (not shown on trace). At the end of each trace the 340- to 380-nm ratio was calibrated as described in METHODS.

 


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Fig. 5. [Ca2+] increase induced by depolarization (–71 to –30 mV) as a function of the simultaneous pHi alkalinization induced by ammonium ({bullet}). {square}, Data obtained at pHe 7.0 (pHi 6.59) and pH 8.0 (pHi 6.9). The continuous line takes into consideration the medium values obtained up to 20 mM ammonium and fits the sigmoid equation (Boltzmann equation) [Ca2+]i = (256 µM – 1,200 µM)/{1 + exp[(pHi – pHmed)/0.07]} + 1,200 µM, where the pH value at which half-maximum intracellular calcium increase stimulation occurred (pHmed) = 7.0. {circ}, Experiments made with NH4Cl alone. All data are means ± SE (n = 6). *P = 0.09, **P = 0.013 (paired t-test) compared with 30 mM K + 20 mM NH4Cl. {Delta}[Ca2+]i, [Ca2+]i obtained at the peak of [Ca2+]i (induced by depolarization) minus resting [Ca2+]i.

 
To determine the whole range of pHi values that affected the depolarization-induced [Ca2+]i increase, the effect of low pHi was explored. We attempted to lower pHi with a weak acid; however, the addition of propionic acid, even at high concentrations (50 mM), barely lower pHi (not shown). Alternatively, pHi was changed by modifying the pH of the medium (15). In this regard, in a previous report (23) our group explored the effect of nifedipine on VDCC in medium buffered at pH 6.5 and, incidentally, did not observe any evident effect on the depolarization-induced calcium influx compared with normal pH. In the present work we found that when the pH of the H-HSM1 medium was adjusted to 7.0, the sperm pHi decreased to 6.59 ± 0.02 (n = 6), that is, 0.11 pH units more acidic than found at normal pH (pHi = 6.70). Conversely, when the pH of the H-HSM1 medium was brought to 8.0, the pHi increased to 6.90 ± 0.01 (n = 6). As expected, the [Ca2+]i increase induced by depolarization at pHi 6.59 was slightly smaller than that observed at normal pHi, and at external pH 8.0 the [Ca2+]i increase was stimulated (Fig. 4). This indicated that the lower limit for the effect of pHi is situated at values close to resting pHi. Interestingly, the small [Ca2+]i increase induced by valinomycin (23) was stimulated at external pH 8.0 (Fig. 4).



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Fig. 4. Effect of the pH of the medium (pHe) on the calcium influx induced by depolarization in human sperm. A: noncapacitated, fura-2-loaded sperm were added to the fluorescence cuvette containing 2.5 ml of H-HSM1, adjusted to pH 7.0, 7.6, or 8.0, at 36°C and under magnetic stirring. After 3 min, 0.4 µM valinomycin (V) and 30 mM KCl (K) were sequentially added as indicated. B: [Ca2+]i increase induced by depolarization at the indicated pHe values (means ± SE; n = 6). pHi values obtained in fura-2- and BCECF-loaded sperm are indicated at bottom (means ± SE; n = 6). *P < 0.05 (paired t-test) compared with pHi 6.70. {Delta}[Ca2+]i, [Ca2+]i obtained at the peak of [Ca2+]i (induced by depolarization) minus resting [Ca2+]i.

 
The voltage-dependent [Ca2+]i increase induced by depolarization from –71 to –30 mV as a function of pHi, modified either with NH4Cl or by changing the pH of the medium, is shown in Fig. 5; the open squares are the results obtained at external pH 7.0 (pHi = 6.59) and 8.0 (pHi = 6.9). The curve has apparently two components, which may reflect different pHi sensitivities of the channels (see below). The left component fitted a sigmoid curve, with effect starting at pHi 6.6 and tending to reach saturation at pHi 7.2. In this range of pHi, the calcium permeation through the channels was stimulated as much as approximately sixfold, with apparent half-maximum effect near pHi 7.0. Interestingly, the calcium influx induced by depolarization at pH 8, which increased the pHi to 6.9, sited on the sigmoid curve, suggesting that the stimulation at pH 8 could be exclusively related to the change in pHi, with no influence of extracellular pH. In this regard, in mouse sperm the induction of VDCC opening requires extracellular pH alkalinization (38). Further studies are required to establish whether external pH alkalinization influences gating of human sperm VDCC.

The overstimulation observed at pHi ~7.28 (with 40 mM NH4Cl; Fig. 5) seemed to result from the sum of the pHi-stimulated voltage-dependent calcium influx that would occur at saturation and that triggered by pHi alone. However, at pHi ~7.35 (60 mM NH4Cl) the stimulation was higher than the sum of both calcium permeability pathways. This result raised the possibility that 60 mM NH4Cl could stimulate the [Ca2+]i increase by inducing a stronger depolarization contributed by the cationic form [98% ammonium, corresponding to acidic dissociation constant (pKa) = 9.3]. In a previous paper (23) our group reported that 10 mM NH4Cl did not depolarize human sperm incubated in the presence of valinomycin; however, the higher concentrations of ammonium used here could depolarize and so contribute to [Ca2+]i. To assess this hypothesis, we investigated the effect of 60 mM NH4Cl on membrane potential and [Ca2+]i (detected simultaneously) in fura-2-loaded sperm. Figure 6 shows that even when 60 mM ammonium produced a small, slow depolarization, it was slower than the initial [Ca2+]i increase rate. This result suggested that the [Ca2+]i induced by 60 mM NH4Cl alone was related to a direct effect of pHi alkalinization, with some late contribution of VDCC. Additionally, 60 mM ammonium did not affect the depolarization induced by 30 mM KCl, indicating that the overstimulation produced by 60 NH4Cl on the depolarization-induced [Ca2+]i increase was not related to contribution of ammonium to membrane potential. In this regard, the small effect of 60 mM NH4Cl on membrane potential recordings strongly suggested that the sperm plasma membrane remained highly impermeable, and, hence, toxic effects of NH4Cl should not produce unspecific calcium permeation.



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Fig. 6. Effect of 60 mM NH4Cl on membrane potential in human sperm. Fura-2-loaded sperm were added to the fluorescence cuvette containing 500 nM 3,3'-dipropylthiacarbocyanine iodide [diSC3(5)]. Fura-2 (340- to 380-nm ratios) and diSC3(5) (excitation, 600 nm; emission, 670 nm) fluorescence were simultaneously detected at 36°C and under magnetic stirring as described in METHODS. Four minutes after sperm addition to the fluorescence cell, valinomycin and 30 mM KCl (A), 60 mM NH4Cl (B; bold traces), or 30 mM KCl + 60 mM NH4Cl (C) were sequentially added as indicated. Top: membrane potential. Bottom: [Ca2+]i.

 
Together these results supported the notion that the biphasic effect of pHi on depolarization-evoked calcium influx may involve two underlying processes with different pKa values. The experiments described in the remainder of this report were performed with 10 mM NH4Cl because this concentration was within the lower part of the biphasic curve and it also produced a change of pHi that was relatively close to the pHi alkalinization that occurs during capacitation (see below).

Does pHi contribute to calcium release from internal stores? It has been found that calcium influx through ryanodine receptors may be stimulated by pH alkalinization (23). Thus the possibility that calcium-induced calcium release through these channels contributed to the high pHi-stimulated depolarization-evoked [Ca2+]i increase was considered. We induced [Ca2+]i increase by activating a permeability pathway different from VDCC, such as that triggered by progesterone (6, 14), and investigated the effect of pHi alkalinization. Figure 7 shows that 10 mM NH4Cl barely affected the [Ca2+]i increase induced by progesterone, indicating that the stimulating effect of pHi (at least that induced with NH4Cl ≤10 mM) on the [Ca2+]i increase induced by depolarization was not related to calcium-induced calcium release from internal stores. Another potential source of calcium could involve calcium release from mitochondria, via activation of the mitochondrial Na+/Ca+ exchanger. However, in our experimental conditions, valinomycin, the potassium ionophore used to modify Vm with potassium, also uncouples mitochondria and, consequently, impairs the ability of this organelle to accumulate calcium (29). This strongly suggested that calcium release from mitochondria did not contribute to the calcium signals reported here. Interestingly, in mouse sperm, mitochondria do not accumulate calcium in response to intracellular calcium load (36).



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Fig. 7. Effect of pHi alkalinization on the progesterone-induced calcium influx in human sperm. A: fura-2-loaded sperm were added to the fluorescence cell, and [Ca2+]i was recorded as described in METHODS at 36°C and under magnetic stirring. After 4 min, 4 µM progesterone (P) or 4 µM progesterone + 10 mM ammonium (bold trace) were added. The effects of 30 mM KCl (K) and 10 mM NH4Cl + 30 mM KCl are shown for comparison. The calcium influx induced by depolarization was measured in the presence of 0.4 µM valinomycin as described in Fig. 2. B: [Ca2+]i increase (means ± SE; n = 6) obtained from traces in A. *P < 0.05 compared with K (paired t-test). {Delta}[Ca2+]i, [Ca2+]i obtained at the peak of [Ca2+]i (induced by depolarization or by progesterone) minus resting [Ca2+]i.

 
Effect of pHi on calcium influx induced by different test Vm: effect of calcium channel blocker nickel. The effect of pHi on voltage-dependent calcium influx was explored at different test Vm. As expected, depolarization-induced calcium influx depended on the extent of depolarization, both in pHi-stimulated and in nonstimulated sperm (Fig. 8, A and B). The stimulating effect of pHi alkalinization, in terms of fractional change of stimulation over nonstimulated cells, was statistically indistinct for each depolarization (P = 0.58, ANOVA; Fig. 8C). The blocking effect of nickel was also determined for the high pHi-stimulated calcium influx (Fig. 9). The IC50 values for the pHi-stimulated and nonstimulated VDCC were 133 ± 30 and 197 ± 25 µM (n = 6), respectively; the differences between these IC50 values were not statistically significant (P = 0.152, paired t-test). Furthermore, in both nonstimulated and pHi-stimulated VDCC the maximum inhibitory effect of nickel (1 mM) was close to 70% (Fig. 8B). Interestingly, the resistant fraction was stimulated by pHi alkalinization. In the presence of 1 mM nickel, the calcium influx induced by 30 mM KCl was 128 ± 16 nM (n = 6, SE), whereas that the calcium influx induced by high pHi depolarization (with 10 mM NH4Cl) was significantly increased to 233 ± 43 nM (P = 0.046, paired t-test).



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Fig. 8. A: effect of pHi alkalinization on the calcium influx induced at different step depolarizations in fura-2-loaded sperm. Traces a, b, c, and d correspond to high pHi depolarization with the indicated amounts of KCl, and a', b', c', and d' are controls without NH4Cl. B: [Ca2+]i increase (means ± SE; n = 6 except for –30 mV, where n = 10) as a function of test membrane potential calculated as the Nernst potential for potassium distribution (Ek) as described in METHODS. {Delta}[Ca2+]i, [Ca2+]i obtained at the peak of [Ca2+]i (induced by depolarization) minus resting [Ca2+]i. pHi alkalinization significantly increased the calcium influx induced by each depolarization (P ≤ 0.05, paired t-test). C: stimulation of voltage-dependent calcium influx by 10 mM NH4Cl, expressed as a fractional change of pHi-stimulated over nonstimulated cells, as a function of test membrane potential. The fractional change (means ± SE) was calculated from data shown in B.

 


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Fig. 9. Blocking effect of nickel on the high pHi-stimulated calcium influx induced by depolarization in human sperm. A: fura-2-loaded sperm were added to the fluorescence cell containing 2.5 ml of H-HSM1 (at 36°C and under magnetic stirring), and fluorescence was detected as described in METHODS. After 3-min incubation, the indicated amounts of NiCl2 and, 1 min later, valinomycin (0.4 µM) were consecutively added (not shown in traces). After 1 min, calcium influx was triggered either with 30 mM KCl (bottom) or with 30 mM KCl + 10 mM NH4Cl (top). Traces without nickel are on left. B: fractional change of inhibition as a function of [NiCl2], both in pHi-stimulated ({bullet}) and nonstimulated ({circ}) cells (means ± SE; n = 6).

 
Role of pHi in stimulation of depolarization-evoked calcium influx observed during capacitation. Our group previously reported (12) that the calcium influx induced by depolarization is stimulated during sperm capacitation. On the other hand, Cross and Razy-Faulkner (9) found that human sperm incubated for 24 h in capacitating conditions undergo a 0.14-pH unit alkalinization. Thus the stimulation of the [Ca2+]i increase induced by depolarization observed in capacitated cells could be related to pHi. Consistent with this, the [Ca2+]i increase induced by depolarization in capacitated sperm was strikingly similar to that observed in noncapacitated sperm when depolarization and alkalinization to pHi 7.05 (with 10 mM NH4Cl) were simultaneously triggered (Fig. 10). Under our capacitating conditions (see METHODS), the pHi of fura-2-loaded sperm increased from 6.70 ± 0.01 to 6.81 ± 0.03 (n = 8). These values are slightly different from others previously reported by our group (14), perhaps because in these experiments the measurements were made in the absence of bicarbonate. The alkalinization reported here is sufficient to produce near-30% stimulation in the calcium influx induced by depolarization (see Fig. 2). Thus the increase in pHi occurring during capacitation may contribute considerably to the stimulation of VDCC in human sperm via a direct action of pHi on these channels.



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Fig. 10. Similarity between the pHi-stimulated calcium influx induced by depolarization in noncapacitated sperm and the calcium influx induced by depolarization in capacitated sperm. A: fura-2-loaded sperm were capacitated in HSM medium (at 36°C, 3% CO2-97% air, and 100% humidity) for 4 h. A sample containing ~1 x 107 cells was centrifuged, and the pellet was added to the fluorescence cell containing 2.5 ml of H-HSM1 at 36°C and under constant magnetic stirring. After 3 min, 0.4 µM valinomycin was added to the sample (not shown in traces); 1 min later, calcium influx was triggered with 30 mM KCl (Cap). For comparison, the calcium influx induced by pHi-alkaline depolarization (with 30 mM KCl + 10 mM NH4Cl) and by depolarization alone (30 mM KCl) determined in sperm before incubation under capacitating conditions (No Cap) is shown. B: [Ca2+]i increase obtained from the traces in A (means ± SE; n = 5). *P < 0.05 compared with noncapacitated sperm (paired t-test).

 

    DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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In this paper we analyze the effect of pHi on putative VDCC present in human sperm. Our results indicate that the calcium influx induced by depolarization is markedly stimulated by pHi, in a biphasic mode, in the range of 6.6 to 7.4. The biphasic effect of pHi suggests that one VDCC with two sites of pH regulation or two different VDCC with different pKa values might be involved in the stimulation by pHi. Within the low pHi range (the range studied), the effect of pHi does not involve calcium release from internal stores and both pHi-stimulated and nonstimulated VDCC are blocked by nickel with similar Ki and maximum inhibition of ~70%. Altogether, this evidence strongly suggests that proton removal directly from VDCC on the cytoplasm side, or from a putative regulator of them, enhances the calcium permeability when the channels are gated by depolarization.

In mature mouse sperm there are putative VDCC, detected via intracellular calcium measurements, the gating of which (induced with potassium) strictly depends on simultaneous external medium alkalinization, from pH 7.4 to pH 8.6 (38). In these cells, the calcium influx induced by alkaline depolarization is markedly enhanced by repeated stimuli and also by NaHCO3 (37). These manipulations increase the cAMP levels because of the activation of a peculiar form of adenylate cyclase, which is in turn stimulated by calcium ions (18) or by bicarbonate (8). Consequently, it has been proposed that cAMP may enhance the calcium influx induced by alkaline depolarization (37). Accordingly, because of the induction of biochemical changes, the stimulating effect of bicarbonate on the calcium entry in mouse sperm requires seconds to occur (half-time ~60 s; Ref. 37). In contrast, the stimulating effect of pHi on human sperm VDCC reported here is practically immediate, suggesting that biochemical changes induced by pHi alkalinization are not involved in this phenomenon. Furthermore, it was recently reported that mouse sperm lacking catsper1, a putative VDCC present in mouse sperm flagella (27), are not able to hyperactivate their motility (28), and, interestingly, they do not increase [Ca2+]i in response to alkaline depolarization (7). This led to the hypothesis that catsper1 might be the calcium channel that allows the alkaline depolarization-induced calcium influx. Whether or not the putative pHi-dependent VDCC described here is a form of catsper1 remains to be established.

It should be noted that the effect of pHi on VDCC has been documented in different excitable cells. Stimulating effects of pHi alkalinization on calcium permeation have been observed in T- and N-type VDCC currents in neurons (21, 34), the T-type VDCC in mouse spermatocytes (31), and the L-type VDCC present in smooth muscle (32, 39). pHi does not affect the gating of the channel (21, 32); instead, the pHi sensitivity of L-type Ca2+ channels is conferred by the {beta}-subunit of the channel complex (32). Accordingly, in this study we have observed that the pHi-stimulated calcium influx induced by depolarization does not depend on the activation of the channels.

The physiological role of pHi on VDCC in excitable cells is not clear. For instance, it has been speculated that pathological conditions leading to pHi acidification, such as ischemia, would decrease neuronal excitability because of the lower opening of VDCC. Consequently, maintenance of [Ca2+]i at resting levels would be favored, keeping the cell alive under such a stressing situation (21). In human sperm, the role of pHi on VDCC could be directly related to normal sperm function. Indeed, during sperm capacitation the calcium influx through VDCC is stimulated (Ref. 12 and this study) and the pHi increases ~0.11–0.14 pH units (Ref. 9 and this study). The pHi sensitivity of the depolarization-induced calcium influx described here shows that the pHi alkalinization occurring in human sperm would be enough to produce ~30% of the stimulation observed during capacitation. Thus the pHi alkalinization observed during capacitation is not sufficient to explain the level of calcium influx stimulation observed in capacitated sperm. However, the similarity between the calcium influx induced by depolarization in capacitated cells and that induced by depolarization + 0.35-pH unit pHi alkalinization (with 10 mM NH4Cl) in noncapacitated cells strongly suggests that pHi still has a major role. We have the impression that a biochemical modification of the channel, which may be produced by cAMP or a tyrosine kinase, the activities of which increase during capacitation, might enhance the effect of pHi on the channels.

Finally, it should be noted that calcium permeation can be further stimulated by progesterone (12), which is normally produced by the follicular cells that surround the egg. Under our conditions, progesterone does not induce pHi changes in sperm, indicating a different stimulatory mechanism on VDCC. Even though the physiological role of human sperm VDCC has not been established, the remarkable enhancement of calcium permeation on these channels produced during its journey to the egg may have a major role for the ZP3-induced AE, where gating of VDCC has been postulated to occur. Furthermore, the possible contribution of VDCC to resting [Ca2+]i remains to be explored. In light of the results presented here, if calcium influx through VDCC contributes to resting [Ca2+]i, the small but biologically significant increase in [Ca2+]i observed during capacitation could be related to the VDCC stimulation reported here. Other putative non-voltage-dependent calcium channels have been considered as mechanisms to set the resting [Ca2+]i (11).


    GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
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This work was supported by Grant IN202702 from Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT-DGAPA) of Universidad Nacional Autónoma de México. J. J. Fraire-Zamora obtained a scholarship from the same foundation.


    ACKNOWLEDGMENTS
 
We acknowledge the excellent technical assistance of Nicolas Alejandro Martínez Parra.


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. T. González-Martínez, Departamento de Farmacología, Facultad de Medicina, Universidad Nacional Autónoma de México. Ciudad Universitaria, CP 04510, Apartado Postal 70-297 Mexico City, Mexico (E-mail: tuliog{at}servidor.unam.mx)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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