INTRODUCTION

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Spermatogenesis is a complex and highly coordinated process by which spermatogonia proliferate and differentiate to produce mature sperm. This unique process depends on the capacity of spermatogonia to undergo proliferation and to enter into a differentiation program that includes a meiotic cycle. There is ample evidence indicating that elevations of intracellular Ca²⁺ concentration ([Ca²⁺]_i) are key signals for cell division, differentiation, and maturation of somatic cells. Thus, [Ca²⁺]_i may be important for spermatogenesis, although very little is known about its regulation in spermatogenic cells (reviewed in Jegou, 1993). An important step towards understanding germ cell [Ca²⁺]_i regulation was the demonstration that T-type Ca²⁺ channels constitute their only voltage-gated Ca²⁺ permeation pathway (Arnoult et al., 1996b; Liévano et al., 1996; Santi et al., 1996). These channels, either alone or in combination with other Ca²⁺ signaling mechanisms, could play important roles in spermatogenesis (Santi et al., 1996).

Significantly more data exist in the role of [Ca²⁺]_i variations in mature sperm, although a universally accepted model has yet to be published. Changes in intracellular pH (pH_i) and [Ca²⁺]_i in mammalian sperm have been implicated in the control of capacitation and the acrosome reaction (AR),¹ a Ca²⁺-dependent exocytotic event required for fertilization. Evidence suggests that a Ca²⁺ channel modulated by pH_i also participates in the sea urchin sperm AR (García-Soto et al., 1987; Guerrero and Darszon, 1989a). The nature of the membrane events of this activation pathway is not clear yet, nor is it well understood how internal alkalinization can induce the elevation of intracellular Ca²⁺ required for the physiological response (Florman et al., 1989; Florman, 1994; Arnoult et al., 1996a).

A close interrelationship between pH_i and [Ca²⁺]_i has been demonstrated in a variety of somatic cell types. In particular, cytosolic alkalinization modestly increases [Ca²⁺]_i in cultured smooth muscle cells (Siskind et al., 1989), endothelial cells (Danthuluri et al., 1990), HT₂₉ colon carcinoma cells (Benning et al., 1996; Nitschke et al., 1996), rat lachrymal acinar cells (Yodozawa et al., 1997), rat lymphocytes (Grinstein and Goetz, 1985), and several neuronal or neuroendocrine cell types (Dickens et al., 1990; Shorte et al., 1991).

The [Ca²⁺]_i changes induced when ZP3 (a glycoprotein of the egg's zona pellucida) binds to its receptor on the sperm membrane have been recorded in fura-2-loaded bovine and mouse sperm (Florman, 1994; Arnoult et al., 1996a). However, the minute volume of these cells makes reliable detection of fura-2 fluorescence difficult and electrophysiology very arduous (Darszon et al., 1996). Using mouse spermatogenic cells can circumvent some of these obstacles. Their large volume improves signal to noise ratio of fluorescence measurements. Also, recordings of ionic currents are much simpler than in sperm (Santi et al., 1996; Arnoult et al., 1996b), and it is possible to apply strategies of molecular biology to learn about their ion channel composition (Liévano et al., 1996). Late spermatogenic cells can be considered a suitable approximation to mature sperm since they possess many of the membrane proteins present in these transcriptionally inactive, terminal cells (Hetch, 1988).

This study was designed to evaluate the influence of changes in pH_i on [Ca²⁺]_i homeostasis of spermatogenic cells from adult male mice. Our main goal was to determine if cytosolic alkalinization, elicited by the controlled exposure to a permeant weak base, induces changes in [Ca²⁺]_i and, if so, to understand the mechanism by which these changes occur. Furthermore, by taking advantage of the fact that cells from different stages of spermatogenesis and spermiogenesis can be readily identified, their individual responses to intracellular alkalinization were examined and compared.

Our results indicate that internal alkalinization consistently induces Ca²⁺ transients. Using maneuvers that inhibit plasmalemmal Ca²⁺ influx or Ca²⁺ release from internal stores, we concluded that the majority of Ca²⁺ rise results from plasmalemmal Ca²⁺ influx, although a small component that could be attributed to intracellular Ca²⁺ release was occasionally observed. The alkalinization- induced Ca²⁺ permeation pathway allows the passage of other divalents such as Sr²⁺, Ba²⁺, and Mn²⁺. In addition, the inorganic Ca²⁺ channel blockers Ni²⁺ and Cd²⁺ block this Ca²⁺ influx pathway, but not the organic blocker nifedipine. The pharmacological properties of this Ca²⁺ influx pathway virtually rule out a major contribution of voltage-gated Ca²⁺ channels or the Na⁺/Ca²⁺ exchanger.

The magnitude of the Ca²⁺ elevations increases as maturation advances, suggesting a significant role of this mechanism in sperm physiology. We postulate that this novel Ca²⁺ permeation pathway regulated by intracellular pH may contribute, in combination with voltage-gated Ca²⁺ channels, to support Ca²⁺ signals required for spermatogenesis and spermiogenesis. Also, if present in mature sperm, it could be involved in the generation of Ca²⁺ signals needed to initiate important functions such as capacitation and acrosome reaction.

	MATERIALS AND METHODS

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Dissociation Procedure

Spermatogenic cells were obtained as in Santi et al. (1996). Briefly, decapsulated testes of adult male mice (anesthetized with ether and killed by cervical dislocation) were washed with ice-cold saline and incubated for 15 min at 28°C in Ca²⁺-free saline containing 1 mg/ml collagenase type I (Sigma Chemical Co., St. Louis, MO) plus 0.4 mg/ml DNAse type I (Sigma Chemical Co.), pH 7.3. Washed seminiferous tubules were then incubated for 10 min in Ca²⁺-free saline containing 0.4 mg/ml trypsin (Worthington Biochemical Corp, Freehold, NJ). Thereafter, tubules were washed twice with Ca²⁺-free solution supplemented with 1% bovine serum albumin and mechanically dissociated. The resulting cell suspension was stored at 4°C. An aliquot of this suspension was seeded into a Plexiglas recording chamber (RC-25; Warner Instruments, Hamden, CT) placed on the stage of an inverted microscope equipped with phase contrast optics (Nikon Diaphot TMD; Nikon Corp., Tokyo, Japan). The cells remained undisturbed until they attached to the bottom of the recording chamber formed by a No. 1 glass coverslip previously coated with either poly-L-lysine (Sigma Chemical Co.) or Cell-Tack (Collaborative Biomedical Products, Bedford, MA). In early experiments, the pH of the Ca²⁺-free saline used for dissociation was adjusted to 7.3. However, we noticed that responses to internal alkalinization were more vigorous if the cells had been previously exposed to a slightly more alkaline solution. Thus, all cells used in this study were routinely dissociated and maintained at pH 7.7. Throughout the experiments, cells were superfused with saline whose pH was adjusted to 7.3. All experiments were carried out at room temperature.

Cell Identification

Cells obtained by dissociation of seminiferous tubules from adult testes belong to the most advanced stages of spermatogenesis. Spermatocytes, spermatids, and immature sperm are easily recognized under phase contrast by their size and characteristics of nuclei and nucleolus (Bellvé et al., 1977). Pachytene spermatocytes, identified by their condensed nuclear chromatin, are the largest germ cells found in adult testes. Their small size and pale nucleus with a single central nucleolus can recognize round spermatids. Fig. 1 illustrates micrographs, viewed under phase-contrast optics, of freshly dissociated pachytene spermatocytes (A), round and condensing spermatids (B), condensing spermatid with flagellum (C), and testicular sperm (D).

View larger version (102K): [in this window] [in a new window]   Fig. 1.   Phase contrast micrographs of acutely dissociated spermatogenic cells from adult mouse testis. (A) Pachytene spermatocytes, (B) round and condensing spermatids, (C) immature sperm with flagellum, (D) testicular sperm.

Ca²⁺ and pH_i Measurements

Cells were loaded by incubation with the membrane-permeant (AM) form of the Ca²⁺ indicator dye fura-2. In other experiments, the pH-sensitive dye BCECF [(2',7')-bis(carboxymethyl)- (5,6)-carboxyfluorescein]-AM was used instead (Molecular Probes, Inc., Eugene, OR). Incubation was initiated by adding to the recording chamber 300 µl of cell suspension and 300 µl of a solution of either 2 µM fura-2 AM or 4 µM BCECF-AM. Cells were allowed to load for 30 (fura-2) or 10 (BCECF) min at room temperature, and then rinsed continuously for another 15 min with recording medium before the beginning of the experiments. Fluorescence of both fura-2 and BCECF appeared diffusely distributed rather than punctate, suggesting that dye compartmentalization was minimal. During the experiments, cells were continuously superfused (~1 ml/min) with recording medium containing (mM): 130 NaCl, 3 KCl, 2 CaCl₂, 2 MgCl₂, 1 NaHCO₃, 0.5 NaH₂PO₄, 5 HEPES, and 5 glucose, pH 7.3.

Intracellular Ca²⁺ levels were determined as previously described (Hernández-Cruz et al., 1997). Briefly, fura-2-loaded cells were imaged with an inverted microscope (Nikon Diaphot TMD), and pairs of images were digitized and recorded using alternating illumination provided by two nitrogen dye lasers tuned at 340 and 380 nm. The pulsed lasers (3-ns pulse duration) were triggered alternately at frequencies ranging from 2 to 15 Hz under computer control. Images at 340 and 380 nm illumination were taken from an area of the coverslip free of cells and stored separately for on-line background subtraction. The key elements of the Ca²⁺ imaging system were a high numerical aperture objective (UV-F 100×, 1.3 NA; Nikon Corp.), an intensified charge coupled device camera (c2400-87; Hamamatsu, Bridgewater, NJ), and the imaging system running under the FL-2 software (Biolase Imaging System, Newton, MA). Ca²⁺ determinations in this study were obtained from individual, entire cells. In the case of sperm, fluorescence measurements were obtained from the head region. Ca²⁺ concentrations were calculated (Grynkiewicz et al., 1985) using the formula [Ca²⁺] = K_d (F_f /F_b) (R  R_min)/(R_max  R), where the K_d of fura-2 for Ca²⁺ is 250 nM, F_f/F_b is the ratio of fluorescence values for Ca²⁺-free/Ca²⁺-bound indicator at 380 nm excitation, R is the ratio of fluorescence at 340/380 nm for the unknown [Ca²⁺], and R_min and R_max are the ratio of fura-2 fluorescence at 340/380 nm of Ca²⁺-free and Ca²⁺-bound fura-2. The values of F_f/F_b, R_min, and R_max for Ca²⁺ were determined by measuring the fluorescence of a glass capillary 100 µm in external diameter containing calibration solutions with 50 µM fura-2 (pentapotassium salt; Molecular Probes, Inc.) and known Ca²⁺ concentrations in the range 10 nM to 40 µM. Although in situ calibrations were attempted, we found it very difficult to manipulate [Ca²⁺]_i over the required range. Our Ca²⁺ measurements, based only in the in vitro calibrations, could be underestimated to some extent because of effects of viscosity and dye binding to cytoplasmic constituents (Konishi et al., 1988). By comparing fluorescence levels attained 10 min after breaking-in with patch pipettes filled with known concentrations of fura-2 pentapotassium, we estimated that fura-2 reached intracellular concentrations between 20 and 50 µM in the AM-loaded cells.

Intracellular pH measurements were conducted either by single or dual wavelength excitation. In the first case, one of the nitrogen lasers was dye-tuned at 505 nm to illuminate BCECF-loaded spermatogenic cells. Emission light was collected at 520 nm by placing a 510-nm/520-nm dichroic/barrier cube into the microscope light pathway. In these experiments, intended for assessment of pH_i changes with high temporal resolution (<1 s), signals were plotted as differential changes in BCECF fluorescence regarding resting fluorescence (F/F_o). For quantitative pH_i determinations, a different fluorescence imaging system was used. Dual wavelength excitation at 440 and 500 nm was achieved by directing the output of an SLM 8000 spectrofluorometer (SLM Aminco, Rochester, NY) via a fiber optic cable into a B-2A Nikon cube (with the excitation filter removed), placed into the light pathway of an inverted microscope (emission wavelength 520 ± 10 nm). A field containing dye-loaded cells was imaged with a UV objective (UV-F 100×, 1.3 NA) and an intensified charge coupled device camera (c2400-87). Wavelength generation by the spectrofluorometer, as well as image acquisition and fluorescence determination from selected areas of interest, were controlled with the program package Image-1/FL (Universal Imaging Corp., West Chester, PA) running on a PC/AT 66 MHz computer. This system allows continuous ratio measurements of background-corrected BCECF fluorescence at 500/440-nm excitation at intervals of ~2 s. At the end of the experiment, a group of cells was imaged and their 500/440-nm fluorescence monitored during the application of 10 µM of the K⁺/H⁺ ionophore nigericin in a K⁺-rich medium (130 mM KCl, 10 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 10 mM K-HEPES). This protocol sets pH_i equal to pH_o and was used for in situ calibration of BCECF signals. Fluorescence ratios at 500/440-nm excitation were obtained while the cells were bathed with nigericin-containing external solutions with their pH adjusted with KOH in the range 6.2-9. These ratios were then plotted and the resulting graph was fitted to the Henderson-Hasselbach equation: pH = pK' + log (R  R_min/R_max R). The resulting values for R_max and R_min (minimum and maximum 500/400-nm ratios), and pK' were introduced in the same equation to compute pH values from the fluorescence ratios R obtained during the experiments.

Intracellular Alkalinization Procedure

The application of the weak base NH₃ was used to passively alkali-load spermatogenic cells. The solution used contained 25 mM NH₄Cl (osmolarity was maintained with appropriate changes in the amount of NaCl). The pH of the perfusate remained at 7.3. In solution, NH₃ is in chemical equilibrium with its conjugate weak acid, NH₄⁺, according to the formula: NH₃ + H⁺  NH₄⁺. When the cells are exposed to the NH₃-NH₄⁺ solution, the NH₃ freely crosses the cell membrane and associates with a proton to form NH₄⁺. The resulting decrease in free proton concentration causes an increase in pH_i, which continues until [NH₃]_i equals [NH₃]_o. The magnitude and rate of rise of the pH_i increase with this method depends on the buffering power of the cell, its initial pH_i, [NH₃]_o in the alkalinization solution, and the membrane permeability to NH₄⁺, which tends to acidify the cell. For comparison, experiments were conducted using 25 mM of the permeable weak base trimethylamine instead of NH₄Cl. Similar results were obtained using both methods.

Both the NH₃-NH₄ solution and other test solutions were pressure-applied (10 psi) to the cells by way of puffer pipettes positioned within 100 µm with the aid of manipulators. The solenoid valves of separate Picospritzer II devices (General Valve, Fairfield, NJ) controlled solution application. Control experiments showed that with this procedure, the extracellular medium surrounding the cell was replaced within ~100 ms. Drugs used for different purposes during these experiments were 10 mM caffeine, 10 µM thapsigargin, 50 µM cyclopiazonic acid, 1 µM ionomycin, 30 µM ouabain (Sigma Chemical Co.), 5 µM ryanodine, 20 µM monensin (Calbiochem Corp., San Diego, CA), 10 µM 2-4 dichlorobenzamil (Molecular Probes, Inc.), 5-20 µM nifedipine (Alomone, Jerusalem, Israel), 1 mM NiCl₂, 0.5 mM CdCl₂, 2 mM BaCl₂, 1 mM MnCl₂, and 2 mM SrCl₂. For experiments requiring Ca²⁺-free conditions in the external solution, CaCl₂ was omitted and the calcium chelator EGTA (0.5 mM) was added.

Electrophysiology

Whole-cell Ca²⁺ currents were recorded using the patch-clamp technique. The recording medium contained (mM): 130 NaCl, 3 KCl, 2 MgCl₂, 1 NaHCO₃, 0.5 NaH₂PO₄, 5 Na-HEPES, 5 glucose, 10 CaCl₂, pH 7.3. The composition of the pipette internal solution was (mM): 110 CsMe, 10 CsF, 15 CsCl, 2 Cs-BAPTA, 4 ATP-Mg, 10 mM phosphocreatine, 5 Cs-HEPES. The pH was adjusted in the range 6.5-8.1 with CsOH. The small liquid junction potential between these solutions (<5 mV; pipette potential negative against bath) was not corrected in this study. Internal Cs⁺ was sufficient to block the majority of the small outward K⁺ current expressed by these cells. Open-tip pipettes had resistances ranging between 2 and 5 Mohms when filled with pipette solution. Records were filtered (four-pole Bessel filter, bandpass 2 kHz), digitized, and stored. Pulse generation, data acquisition, and analysis were done with a PC computer governed by the pClamp program suite (Axon Instruments, Foster City, CA). Pipette capacitive currents were compensated before rupturing the patch. Once in the whole cell configuration, a holding potential of 80 mV was established, series resistance was electronically compensated, and currents elicited by brief 20-mV depolarizing pulses were averaged. These records were used to determine cell capacitance by digital integration of capacitive transients. Only cells exhibiting adequate voltage control were included in the analysis. Protocols for current-voltage (I-V) relationship and steady state inactivation were consistently used. A p/4 pulse protocol was routinely used to minimize leak and capacitive currents from the current records.

I_Ca activation curves were elaborated by converting peak current values from the I-V relationships to conductances using the equation g_Ca = I_Cap/(V_m  E_Ca), where I_Cap is the peak Ca²⁺ current, V_m the command pulse potential, and E_Ca the apparent reversal potential obtained by linear extrapolation of the current values in the ascending portion of the I-V curve. Conductance values were then normalized and fitted to a Boltzmann relation g/g_max = {1+ exp[(V  Va_1/2)/k_a]}¹, where g is the peak conductance, g_max the maximal peak Ca²⁺ conductance, Va_1/2 the midpoint of the activation curve, and k_a the activation steepness factor. The steady state inactivation curve was obtained by eliciting Ca²⁺ currents with a constant 24-ms test depolarization to 20 mV applied at the end of prepulses to different depolarization levels. The amplitude of these 200-ms prepulses was varied in 5-mV steps from 110 to 35 mV. Intervals of 10 s were allowed between consecutive trials to prevent accumulation of inactivation. Current to voltage curves were constructed by plotting peak currents vs. prepulse potentials, and steady state inactivation curves were obtained by normalizing the current values and fitting the data with a Boltzmann equation I/I_max = {1 + exp[(V  Vi_1/2)/k_i]}¹, where I is the peak current, I_max is the peak current when the prepulse was 110 mV, V is the prepulse potential, Vi_1/2 is the half-inactivation value, and k_i is the inactivation steepness parameter.

	RESULTS

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Controlled Rises of Intracellular pH During the Application of NH₄Cl

To test the speed of local perfusion achieved with the puffer pipette and the efficacy of NH₄Cl for inducing internal alkalinization of spermatogenic cells, single wavelength (505-nm excitation) fluorescence of BCECF-loaded cells was measured during the application of external solution containing 25 mM NH₄Cl. Fig. 2 A illustrates the effects of superfusing with NH₄Cl-containing solution a group of four BCECF-loaded spermatogenic cells. Cell alkalinization is indicated by an increase in the ratio F/F_o. pH_i increases rapidly, reaching within 7 s a plateau that remains during the application of NH₄Cl. Upon washout of NH₄Cl, pH_i returned monotonically to baseline. The decay phases of these records could be fitted to single exponential functions, with time constants of 11.4, 11.8, 9.0, and 9.9 s.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   (A) Time course of intracellular alkalinization induced by NH4Cl application in two pachytene spermatocytes and two round spermatids. Cells were loaded by incubation with 10 µM BCECF-AM for 20 min. Intracellular pH was monitored as changes of BCECF fluorescence emission (see MATERIALS AND METHODS). The horizontal bar indicates the duration of NH4Cl application. The internal pH of all cells increases rapidly upon exposure to NH4Cl, reaching a plateau. Upon removal of the NH4Cl, intracellular pH returns monotonically to resting levels without a rebound acidification. (B) Biphasic changes of intracellular calcium concentration induced by intracellular alkalinization in a fura-2-loaded pachytene spermatocyte. A NH4Cl-containing puffer pipette placed nearby was used for the controlled intracellular alkalinization. One or more separate pipettes were used for the application of additional test solutions. The horizontal bar represents the duration of NH4Cl application.

Dual wavelength determinations of BCECF signals showed that resting pH_i in spermatogenic cells varies over the range 6.0-7.2. These values are similar to those reported from ram and pig sperm (6.3-6.7; Babcock and Pfeiffer, 1987; Tajima et al., 1987). We did not find consistent pH_i differences among cells from different stages of differentiation (pachytene spermatocytes, 6.74 ± 0.06 [mean ± SEM], n = 18; round and condensed spermatids, 6.63 ± 0.04, n = 44). The application of 25 mM NH₄Cl increased pH_i by 1.35 ± 0.11 pH units, n = 13. This pH_i rise was dependent on the concentration of NH₄Cl (e.g., 5 mM = 0.85 ± 0.04; 15 mM = 1.06 ± 0.06). In contrast to many somatic cells, which exhibit an acidification shift after reaching a peak alkalinization, and a rebound acidification after the removal of NH₄Cl, spermatogenic cells rarely showed any acidification with the pulse duration used in these experiments (see Fig. 2 A). This suggests that their plasma membrane lacks an appreciable permeability to NH₄⁺ (Bevensee and Boron, 1995). Since the time course of intracellular alkalinization by NH₄Cl can be approximated to a sustained plateau with rapid onset and offset, a correlation between changes of [Ca²⁺]_i and pH_i changes is possible, making unnecessary the technically more demanding simultaneous measurement of [Ca²⁺]_i and pH_i (Martínez-Zaguilán et al., 1991, 1996; Wiegmann et al., 1993). Control experiments showed that the time course and amplitude of NH₄Cl-induced alkalinization recorded with BCECF were not greatly affected if the cells had been previously incubated in the presence of fura-2 AM (data not shown).

Alkalinization-induced Changes of Intracellular Ca²⁺ Concentration

In contrast to resting pH_i values, [Ca²⁺]_i at rest varied significantly among the population of spermatogenic cells: higher resting [Ca²⁺]_i was consistently determined in cells from more advanced stages of maturation. Thus, pachytene spermatocytes had a mean resting [Ca²⁺]_i of 56.5 ± 7.2 nM (n = 10), round spermatids of 122.2 ± 13.8 nM (n = 19), and condensing spermatids of 200.2 ± 46.3 nM (n = 5). These differences (P < 0.02) are likely to be related to differentiation and maturation of male germ cells. Resting [Ca²⁺]_i remained stable over periods of observation up to 30 min, suggesting the absence of spontaneous net Ca²⁺ fluxes under our recording conditions.

Alkalinization of spermatogenic cells was generally associated with a characteristic biphasic response composed of an initial [Ca²⁺]_i drop followed by a delayed rise. An example, recorded from a pachytene spermatocyte is shown in Fig. 2 B. Similar changes in [Ca²⁺]_i were produced by application of the membrane permeant base trimethylamine (25 mM; data not shown). As illustrated in Fig. 3, the initial [Ca²⁺]_i drop is more apparent in pachytene spermatocytes (A) than in round (B) or condensing (C) spermatids. The opposite is true for the delayed [Ca²⁺]_i rise. Fig. 3 also shows the progressive increase in the magnitude of the delayed Ca²⁺ rise frequently observed in spermatogenic cells upon repeated applications of NH₄Cl (see below). Although the variability of this facilitation makes the quantitative comparison of responses to different NH₄Cl concentrations difficult, it was clear that concentrations of 10, 15, and 25 mM NH₄Cl produced progressively larger intracellular Ca²⁺ elevations, while 5 mM was ineffective (data not shown). Interestingly, alkalinization with 25 mM NH₄Cl consistently produced similar elevations of [Ca²⁺]_i in the head region of testicular sperm. An example of these recordings is illustrated in Fig. 4. Some of the testicular sperm examined were immature (they exhibited a cytoplasmic bulge in the middle piece of the flagellum and limited motility; Fig. 4, inset), but others were virtually indistinguishable from mature, epididymal sperm. Alkalinization-induced large Ca²⁺ rises appear to be specific to spermatogenic cells from adult mice since they were absent in spermatogonia obtained from testes of 1-wk-old mice (only the initial Ca²⁺ drop component of the response was observed), as well as in other cells tested under similar conditions in our laboratory; i.e., rat sympathetic neurons, rat chromaffin, and pituitary cells (data not shown).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Ca2+ transients elicited in spermatogenic cells from different stages of maturation after successive applications of NH4Cl (pulse duration indicated by the horizontal bars). (A) Pachytene spermatocyte, (B) round spermatid, (C) condensing spermatid. Notice that the first application induces an initial [Ca2+]i drop in the pachytene spermatocyte, which is less noticeable in the round spermatid and virtually absent in the condensing spermatid. Also notice that the magnitude of the Ca2+ rise increases with repeated applications of NH4Cl.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   [Ca2+]i transients recorded from the head region of a testicular sperm similar to the one illustrated in the inset. The horizontal bar represents the duration of NH4Cl application. This response was obtained after the first application of NH4Cl.

It could be argued that [Ca²⁺]_i changes recorded with fura-2 may constitute an artifact resulting from pH_i effects on fura-2 dissociation constant for Ca²⁺ (Martínez-Zaguilán et al., 1991). However, this does not seem likely since most Ca²⁺ measurements of this study were performed at pH_i values where fura-2 properties are pH independent (Batlle et al., 1993; Nitschke et al., 1996). Moreover, experimental maneuvers that modified or even abolished the pH_i-induced Ca²⁺ transients (i.e., experiments done in the absence of external Ca²⁺; see Fig. 8 A) rule out a significant influence of pH on the binding properties of fura-2. Additional considerations emerge from comparisons of the kinetics of the responses. The half-rise time of Ca²⁺ transients among the population of spermatogenic cells ranged from 9.9 ± 1.7 s (pachytene spermatocytes, n = 4) to 15.3 ± 2.9 s (condensing spermatids, n = 4). In contrast, half-rise times of pH_i signals were faster, ranging from 2.4 ± 0.2 s (round spermatids, n = 5) and 2.9 ± 0.2 s (pachytene spermatocytes, n = 6) to 4.5 ± 0.4 s (condensing spermatids, n = 2). Also, the time constant of decay of [Ca²⁺]_i signals in pachytene spermatocytes was 12.9 ± 2.9 s (n = 4), while the time constant of decay of pH_i signal was 9.9 ± 0.9 s (n = 5). In Fig. 5, representative traces of [Ca²⁺]_i and pH_i from two pachytene spermatocytes are superimposed for comparison. Since effects of internal alkalinization on fura-2 dissociation constant should be immediate, the kinetic differences between [Ca²⁺]_i and pH_i responses suggest that the [Ca²⁺]_i rise constitutes a genuine physiological response.

View larger version (25K): [in this window] [in a new window]   Fig. 8.   Contribution of external Ca2+ to the alkalinization-induced [Ca2+]i transients in spermatogenic cells. Ca2+ rises were recorded first in normal recording solution, and then in a solution containing no added Ca2+ and 0.5 mM EGTA. Along with the standard puffer pipette, a second pipette containing NH4Cl and no Ca2+ was used to induce intracellular alkalinization while keeping Ca2+-free conditions. Responses were obtained from a pachytene spermatocyte (A) and a condensing spermatid (B). Notice that when NH4Cl is applied in the complete absence of external Ca2+, the delayed Ca2+ rise is either suppressed (A) or greatly reduced (B). Also, notice in A that the initial Ca2+ drop obliterated by the delayed Ca2+ rise becomes discernible upon removal of extracellular Ca2+.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Representative recordings of [Ca2+]i and pHi obtained from two different pachytene spermatocytes loaded with BCECF and fura-2, respectively. Traces were superimposed for comparison. Notice that the initial [Ca2+]i drop occurs almost simultaneously with the pHi rise elicited by NH4Cl application (horizontal bar), while the [Ca2+]i elevation appears with a significant delay.

Since alkalinization-induced Ca²⁺ rises are bigger and faster in condensing and round spermatids than in pachytene spermatocytes, we hypothesized that perhaps the rise was concealing the initial drop. Experiments shown in Figs. 3 and 6 appear to support this explanation. Fig. 6 A, obtained from a pachytene spermatocyte, shows the increasing magnitude of alkalinization-induced Ca²⁺ rises with repeated NH₄Cl applications. The four successive responses shown in Fig. 6 A were aligned and superimposed in Fig. 6 B for comparison. As these [Ca²⁺]_i records clearly show, the use-dependent facilitation of the Ca²⁺ rise determines the gradual obliteration of the initial Ca²⁺ drop. Also, notice that the magnitude of the initial Ca²⁺ drop does not facilitate as the Ca²⁺ rise does.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Facilitation of [Ca2+]i transients. (A) [Ca2+]i rises recorded from a pachytene spermatocyte show use-dependent facilitation upon repeated application of NH4Cl (horizontal bars). Notice that both the magnitude and the rate of rise of [Ca2+]i transients became larger. Conversely, [Ca2+]i transients decay more slowly after repeated intracellular alkalinizations. (B) Superimposed responses to the NH4Cl applications shown in A. Traces were aligned both vertically and horizontally for clarity. Notice that the magnitude of the initial [Ca2+]i drop remains invariant, becoming progressively obliterated by the delayed [Ca2+]i rise.

Alkalinization-induced Early Ca²⁺ Drop

The initial [Ca²⁺]_i drop, which is most clearly observed in pachytene spermatocytes, (a) develops without apparent delay regarding the pH_i increase, (b) does not exhibit facilitation, and (c) lasts for as long as the NH₄Cl application is maintained when the delayed rise is absent or has been eliminated (see Figs. 3 A, 7 B, and 8 A). These characteristics suggest that the initial Ca²⁺ drop has a different mechanism than the delayed Ca²⁺ rise. Several hypotheses can be offered to explain the initial Ca²⁺ drop. We favor the most parsimonious one; that is, upon entering the cell, NH₃ displaces H⁺ from Ca²⁺ binding sites on proteins, increasing the buffering power of the cell and hence diminishing resting [Ca²⁺]_i. Further experiments are being planned to determine the precise nature of the initial Ca²⁺ drop. The remainder of this paper will focus on the nature of the delayed [Ca²⁺]_i rise.

View larger version (29K): [in this window] [in a new window]   Fig. 7.   Development of [Ca2+]i responses after incubation with monensin. (A and B) Recordings obtained from two fura-2-loaded spermatogenic cells that initially failed to show a [Ca2+]i rise component upon stimulation with NH4Cl (pulses indicated with the filled horizontal bars). After 2-min incubation with 20 µM monensin, cells began to respond with small Ca2+ rises. These responses increased after a second monensin incubation for 4 min. Monensin-treated cells showed both Ca2+ rises and use-dependent facilitation of the responses. (C) Recording of fluorescence changes in a BCECF-loaded spermatogenic cell. The experiment was designed to determine if use-dependent facilitation results from a progressive increase in resting pHi. The first part of the record shows that pHi returns completely to resting levels between successive NH4Cl applications. The second part of the record shows that after monensin application, basal pHi rises without increasing the magnitude of the pHi rise elicited by NH4Cl application. Also notice that responses to NH4Cl after monensin incubation decay completely to prestimulation levels.

Use-dependent Facilitation of the [Ca²⁺]_i Rise

In early experiments, we noticed that preincubation of the cells in a slightly more alkaline medium promoted the appearance of [Ca²⁺]_i rises. Thus, cells incubated at external pH 7.7 had a pH_i = 6.6 ± 0.03 (n = 62) and quite often responded with [Ca²⁺]_i rises to the application of 25 mM NH₄Cl, while cells kept at pH 7.3 were significantly more acidic (6.4 ± 0.03, n = 100, P = 2.1 × 10⁸) and responded less frequently with increases in [Ca²⁺]_i (only the initial Ca²⁺ drop was observed). Therefore, we speculated that the appearance of alkalinization-induced [Ca²⁺]_i rises could be more likely to occur when pH_i reaches a critical value. Support for this "pH_i threshold hypothesis" comes from experiments such as the one illustrated in Fig. 7, A and B. Here, two initially unresponsive cells (notice the lack of [Ca²⁺]_i rises upon repeated pulsing with NH₄Cl), were incubated for 2 min in the presence of 20 µM monensin, a Na⁺ ionophore that exchanges external Na⁺ for internal H⁺, thus alkalinizing the cytoplasm. Shortly after incubation in the presence of monensin, the cells began to respond with small Ca²⁺ rises to the application of NH₄Cl. Moreover, after a second monensin application for 4 min, cells showed both Ca²⁺ rise and use-dependent facilitation of the responses. In separate experiments, we noticed that the pH_i of BCECF-loaded cells climbed from 6.57 ± 0.17 to 6.89 ± 0.20 (n = 3) after 4 min of monensin application. These data suggest that cells begin to show [Ca²⁺]_i rises in response to alkalinization when resting pH_i exceeds ~6.7. As explained later, this finding may be relevant for the sperm capacitation process.

As previously shown, spermatogenic cells display facilitation of the Ca²⁺ rise upon repeated applications of NH₄Cl. Sometimes this phenomenon is accompanied by a sustained increase in resting [Ca²⁺]_i, but not necessarily. In fact, normal facilitation is observed in cells that had been pulsed with NH₄Cl in Ca²⁺-free medium, thus preventing Ca²⁺ influx (data not shown). The question that immediately rises is, does use-dependent facilitation also result from a progressive, sustained increase in resting pH_i? The experiment shown in Fig. 7 C was designed to test this hypothesis. As shown in the first part of the record (initial two responses), the pH_i returns completely to resting levels between successive NH₄Cl pulses. Control experiments showed that both the sizes of pH_i deflections and the resting pH_i remained unchanged during repetitive NH₄Cl applications (data not shown). The second part illustrates the effects of monensin application. Clearly, monensin raises basal pH_i without increasing the magnitude of the pH_i change resulting from each NH₄Cl application. Also, the response to NH₄Cl in the presence of monensin decays completely to prestimulation levels. Similar results were obtained in five additional cells. These findings rule out an incremental rise of resting pH_i as an explanation to the use-dependent facilitation phenomenon. More likely, a pH-dependent regulatory mechanism capable of inducing long term modifications of the permeation pathway either directly or by way of second messengers (e.g., cyclic AMP; Trimmer and Vacquier, 1986; Gabers, 1989; Beltrán et al., 1996; Arnoult et al., 1997) underlies the use-dependent facilitation of the Ca²⁺ rise. These and other possible explanations to the facilitation phenomenon are yet to be explored.

Source of Ca²⁺ Underlying the [Ca²⁺]_i Rise

The [Ca²⁺]_i elevation secondary to internal alkalinization could result from plasmalemmal Ca²⁺ influx, intracellular Ca²⁺ release, or both. Experiments such as those exemplified in Fig. 8 explored the possible contribution of external Ca²⁺. Here, two puffer pipettes were positioned near the cell and the recording chamber was continuously bathed with Ca²⁺-free external solution when indicated. Both pipettes contained NH₄Cl, but only one of them contained 2 mM CaCl₂. As shown in Fig. 8 A, the application of NH₄Cl in the complete absence of external Ca²⁺ resulted only in the appearance of the [Ca²⁺]_i drop. When the pulse of NH₄Cl was delivered through the pipette containing 2 mM Ca²⁺, the [Ca²⁺]_i rise reappeared, masking the initial drop. In this case, as in most spermatocytes examined, Ca²⁺ influx is clearly required for the alkalinization-induced [Ca²⁺]_i rise. This result may have two possible explanations: either Ca²⁺ influx is the sole source of the Ca²⁺ transient or a combination of influx and release (that is, through the process termed Ca²⁺-induced Ca²⁺ release) is involved. Fig. 8 B illustrates the result of a similar experiment in a condensing spermatid. The main difference here is that in spite of the absence of external Ca²⁺, alkalinization can still produce a small [Ca²⁺]_i rise. The source of this [Ca²⁺]_i rise remains to be identified, although an intracellular Ca²⁺ source is the most likely explanation. It should be pointed out that responses such as those illustrated in Fig. 8 B were observed in the minority (~10%) of all spermatids examined.

To directly assess the availability of releasable Ca²⁺ from intracellular stores, we tested the effects of caffeine and ryanodine (10 mM and 5 µM, respectively). These plant alkaloids are agonist and antagonist, respectively, of ryanodine receptor/Ca²⁺ release channels. As shown in Fig. 9, A and B, neither caffeine nor ryanodine per se induced a significant Ca²⁺ rise in spermatogenic cells. We also tested the effects of thapsigargin and cyclopiazonic acid, specific inhibitors of the Ca²⁺-ATPase of the endoplasmic reticulum (10 and 50 µM, respectively). As shown in Fig. 9, C and D, when these drugs were applied, they produced either no effect or a modest Ca²⁺ release. Interestingly, these inhibitors only elicited noticeable Ca²⁺ rises in cells from less advanced stages of differentiation (e.g., pachytene spermatocytes). When internal alkalinization was induced after the application of these drugs (the effects of some of which are considered irreversible), Ca²⁺ signals of normal appearance were elicited (see Fig. 9, A-C). As shown in Fig. 9 E, even the application of 1 µM ionomycin (a divalent cation ionophore used to increase the permeability of biological membranes to Ca²⁺, which also depletes a variety of intracellular Ca²⁺ stores), without external Ca²⁺, produced small Ca²⁺ rises in spermatogenic cells. To test the efficacy of ionomycin, the same cells were exposed to the ionophore while bathed in Ca²⁺-free medium, and then perfused with a solution containing no ionomycin and 2 mM Ca²⁺. This was rapidly followed by a large Ca²⁺ rise (Fig. 9 E, middle). This rise may result from Ca²⁺ influx through ionomycin remaining in the plasma membrane, store-operated channels activated by depletion during the initial ionomycin application, or both (see below). When ionomycin was applied shortly after this substantial Ca²⁺ load, it was still ineffective to produce a large Ca²⁺ release, suggesting that Ca²⁺ uptake into intracellular stores is negligible within the time scale of this experiment. Taken together, these data suggest that the amount of Ca²⁺ available for release from rapidly exchanging intracellular reservoirs is too small to directly contribute to the Ca²⁺ transient induced by cytosolic alkalinization. Nonetheless, intracellular Ca²⁺ release could contribute indirectly, by activating store-operated channels (SOCs) whose presence has not been examined in spermatogenic cells. An alternative interpretation of the experiment shown in Fig. 9 E could be that, after the initial treatment with ionomycin, the stores become depleted, leading to the opening of SOCs. Subsequent addition of external Ca²⁺ would then result in a large Ca²⁺ influx mediated by SOCs rather than by ionomycin remaining in the plasmalemma. In fact, such a protocol (depletion of stores in Ca²⁺-free medium followed by readmission of external Ca²⁺) is often used to reveal SOCs (Parekh and Penner, 1997). To explore this possibility, we performed the classical protocol, but using thapsigargin instead of ionomycin. Thapsigargin was chosen because it specifically inhibits intracellular pumps and lacks ionophoric or detergent activity. The result of such an experiment is shown in Fig. 10. Here, a group of eight spermatogenic cells was exposed to thapsigargin (10 µM) for 3 min with the aid of a puffer pipette (Ca²⁺-free conditions effective for the whole time). A few minutes later, a medium containing 2 mM Ca²⁺ was applied to the cells with a second puffer pipette. This was followed by a slow and sustained Ca²⁺ influx that ceased upon removal of external Ca²⁺. These results suggested that (a) thapsigargin depleted the stores, albeit without an apparent Ca²⁺ rise, and (b) spermatogenic cells appear to express SOCs. It remains to be explored whether a pH-induced depletion of Ca²⁺ stores may lead to opening of SOCs and if this permeation pathway can account for the alkalinization-induced Ca²⁺ transient (see DISCUSSION).

View larger version (33K): [in this window] [in a new window]   Fig. 9.   Effects of agents affecting Ca2+ mobilization from intracellular stores. (A) Ca2+ recordings obtained from a pachytene spermatocyte and a round spermatid upon repeated applications of NH4Cl. When indicated, caffeine (10 mM) was applied from a second puffer pipette. Caffeine neither induced Ca2+ rises in spermatogenic cells nor affected their responses to subsequent NH4Cl applications. (B) In another cell pair, 5 µM ryanodine was also ineffective as a Ca2+ release agent. Although the responses to NH4Cl diminished slightly after ryanodine exposure, this result was not observed in other cells tested. (C) The effects of 10 µM thapsigargin were tested in a round spermatid and a pachytene spermatocyte. The figure is representative of more than 12 cells similarly examined. Notice that only the relatively less differentiated spermatocyte showed a modest Ca2+ rise. (D) Effects of cyclopiazonic acid (CPA) on resting Ca2+ levels and responses to alkalinization in three spermatogenic cells. CPA induced small Ca2+ rises in both pachytene spermatocytes, but not in the round spermatid. Responses to NH4Cl after incubation with thapsigargin or CPA appear similar to those of untreated cells. (E) Effects of ionomycin. When two spermatogenic cells were exposed to 1 µM ionomycin without external Ca2+, they exhibit small Ca2+ rises, suggesting that the Ca2+ content of the intracellular reservoirs is low. In contrast, the same stimulus induced a large increase of intracellular Ca2+ when external medium was switched to one containing normal external [Ca2+]. This elevation is likely due to plasmalemmal Ca2+ influx through ionomycin pores. Reapplication of ionomycin without external Ca2+ after such a large Ca2+ load is still unable to release a substantial amount of Ca2+.

View larger version (32K): [in this window] [in a new window]   Fig. 10.   Evidence for the presence of store-operated channels in spermatogenic cells. A group of eight spermatogenic cells, continuously bathed in Ca2+-free medium, were transiently exposed to Ca2+-free medium containing 10 µM thapsigargin when indicated (horizontal bar). Thapsigargin induced either no response or only a small Ca2+ rise, suggesting either that the Ca2+ content of the intracellular reservoirs is low or that Ca2+ is extruded from the cell as fast as it is released. When the cells were perfused with a puffer pipette filled with medium containing 2 mM Ca2+, an increase of intracellular Ca2+ was observed. The same maneuver had no effects on Ca2+ levels before the application of thapsigargin (not shown). Ca2+ elevation upon exposure to external Ca2+ is likely due to plasmalemmal Ca2+ influx mediated by store operated channels.

Permeation Properties of the pH-sensitive Ca²⁺ Influx Pathway in Spermatogenic Cells

To examine the selectivity of the Ca²⁺ permeation pathway, the recording chamber was continuously perfused with Ca²⁺-free external solution, and either Sr²⁺ or Ba²⁺ substituted Ca²⁺ in the NH₄Cl-containing puffer pipette. This protocol was used to warrant that the cells were only exposed to the test divalent cation during the episode of intracellular alkalinization. Fig. 11, A and B, shows results obtained from two round spermatids. Here, fluorescence ratios of 340/380 nm were plotted rather than intracellular concentrations of divalent cations because fura-2 dissociation constants for Sr²⁺ and Ba²⁺ differ considerably from that of Ca²⁺. Although these experiments only provide semi-quantitative information of divalent plasmalemmal fluxes, they clearly demonstrate that Ca²⁺, and to a large extent Sr²⁺ and Ba²⁺, can permeate through the influx pathway made available by intracellular alkalinization.

View larger version (23K): [in this window] [in a new window]   Fig. 11.   Selectivity of the permeation pathway induced by intracellular alkalinization. (A and B) Experiments were conducted in round spermatids to explore the effects of substituting Sr2+ or Ba2+ for Ca2+ in the NH4Cl-containing solution. Ca2+-free solution supplemented with 0.5 mM EGTA was continuously superfused to ensure that the cell was exposed only to the test divalent cation during intracellular alkalinization. Only fluorescence ratios 340/380 nm are shown since dissociation constants of fura-2 for Sr2+ and Ba2+ differ considerably from that of Ca2+. Both Sr2+ (A) and Ba2+ (B) can permeate through the Ca2+ influx pathway made available by intracellular alkalinization. (C) Mn2+ permeates the alkalinization-induced Ca2+ influx pathway. In this experiment, fluorescence of fura-2 at 340 nm was monitored. At this wavelength Ca2+ influx appears as a fluorescence increase, while Mn2+ entry appears as a decrease in fluorescence. Application of NH4Cl in the absence of Mn2+ (filled bar) induces the characteristic initial drop followed by delayed Ca2+ rise. In contrast, a fluorescence decrease is observed when alkalinization is produced with a puffer pipette also containing 1 mM Mn2+ (open bars). Notice that each application of NH4Cl in the presence of Mn2+ produces a step-like irreversible fluorescence loss, consistent with Mn2+ entering the cell and quenching a fraction of fura-2 fluorescence.

To further examine the permeation characteristics of the Ca²⁺ influx pathway, cells were bathed with Ca²⁺-free saline, and then exposed to the NH₄Cl-containing solution, first in the absence, and then in the presence of 1 mM Mn²⁺. Here, fura-2 fluorescence was monitored at 340-nm excitation. At this excitation wavelength, Ca²⁺ or Mn²⁺ influx should lead to opposite signals: Ca²⁺ influx producing a fluorescence increase, and Mn²⁺ entry producing a decrease because of quenching of fura-2 emission. As shown in Fig. 11 C, the application of NH₄Cl without Mn²⁺ (filled bar) induces the characteristic initial [Ca²⁺]_i drop followed by a delayed increase in [Ca²⁺]_i. In contrast, in the presence of 1 mM Mn²⁺ (open bars), alkalinization only produces a decrease in fluorescence. Moreover, a drop in fluorescence emission follows each application of NH₄Cl. This is consistent with Mn²⁺ entering the cell through the permeation pathway regulated by intracellular alkalinization, followed by the irreversible quenching of a fraction of fura-2 by Mn²⁺. It is noteworthy that fluorescence increases due to Ca²⁺ influx were not observed during internal alkalinization in the presence of Mn²⁺. One interpretation is that the pH-regulated permeation pathway is exceedingly more permeable to Mn²⁺ than to Ca²⁺. However, a comparison based on fluorescence changes is inappropriate because fura-2 has a 40-fold higher affinity for Mn²⁺ than for Ca²⁺ (Grynkiewicz et al., 1985), and a small Ca²⁺ influx could be vastly underestimated. Clearly, membrane current measurements would be the correct approach to decide this matter.

Role of Voltage-gated Ca²⁺ Channels in the Alkalinization-induced Ca²⁺ Influx (Effects of Inorganic and Organic Ca²⁺ Channel Blockers)

Recent reports have suggested that T-type Ca²⁺ channels expressed in late spermatogenic cells may contribute to the Ca²⁺ influx necessary to initiate the sperm acrosome reaction (Arnoult et al., 1996b; Liévano et al., 1996; Santi et al., 1996). This notion is supported by earlier observations that Ni²⁺, dihydropyridines such as PN200-110, and nifedipine (Florman et al., 1992), as well as amiloride and pimozide, which inhibit the acrosome reaction, also block spermatogenic cell Ca²⁺ currents (Arnoult et al., 1996b; Liévano et al., 1996; Santi et al., 1996).

To explore whether voltage-gated Ca²⁺ channels contribute to the Ca²⁺ permeation pathway induced by internal alkalinization, we tested the effect of inorganic divalent Ca²⁺ channel blockers Cd²⁺ and Ni²⁺. Cd²⁺ (0.5 mM) and Ni²⁺ (0.2 and 1 mM) invariably inhibited the delayed Ca²⁺ transient. Ni²⁺ was able to completely block Ca²⁺ rises when they were small, but when the responses became facilitated after repeated NH₄Cl applications, a progressively larger fraction of the Ca²⁺ rise remained unblocked (data not shown). Fig. 12 A shows the effects of pulsing with NH₄Cl, a pachytene spermatocyte, both with and without 1 mM Ni²⁺. This protocol was designed to take into account the facilitatory effect resulting from repeated alkalinization on the magnitude of the Ca²⁺ transients. Alkalinization-induced responses were smaller and rose more slowly in the presence of Ni²⁺. The effect of Ni²⁺ on the kinetics of Ca²⁺ transients in another pachytene spermatocyte is shown in Fig. 12 B. Here, responses to alternate applications of NH₄Cl with and without Ni²⁺ are superimposed for comparison. Upon the application of NH₄Cl plus Ni²⁺, the delayed Ca²⁺ rise was drastically inhibited, without affecting the kinetics or magnitude of the initial [Ca²⁺]_i drop. Ni²⁺ inhibited alkalinization-induced Ca²⁺ transients at concentrations as low as 200 µM.

View larger version (13K): [in this window] [in a new window]   Fig. 12.   Effects of Ni2+ and nifedipine on the alkalinization-induced Ca2+ entry pathway. (A) Ca2+ transients recorded from a pachytene spermatocyte upon alternate puffer applications of NH4Cl both in the absence (filled bars) and presence (open bars) of 1 mM NiCl2. The bathing external solution contained no Ca2+, while the solution in both pipettes contained 2 mM Ca2+. Alkalinization-induced Ca2+ transients were consistently smaller and had a slower rate of rise in the presence of Ni2+. (B) Comparison of responses obtained from a different spermatocyte upon alternate intracellular alkalinizations by NH4Cl applications, both in the absence (continuous line) and presence (dashed line) of 1 mM Ni2+. a-c are consecutive alternate responses. Notice that Ni2+ inhibits the delayed Ca2+ rise without affecting the initial Ca2+ drop. (C) Responses obtained from a pachytene spermatocyte to the repeated application of NH4Cl, first in the absence (open bars) and then presence (filled bars) of 20 µM nifedipine in the pipette solution. The last puffer application of NH4Cl and nifedipine was given with the laser illumination turned off to prevent the possible photolysis of the drug.

In separate experiments, we tested the effects of nifedipine (an organic blocker effective on the Ca²⁺ channels present in spermatogenic cells; Santi et al., 1996) on the Ca²⁺ transients elicited by internal alkalinization. Fig. 12 C illustrates responses of a pachytene spermatocyte to the repeated application of NH₄Cl, first in the absence, and then in the presence of 20 µM nifedipine. Nifedipine did not affect the magnitude of the Ca²⁺ transients or their use-dependent facilitation. Since dihydropyridines are very photolabile, the lack of effect on the alkalinization-induced Ca²⁺ permeation pathway could be due to its destruction by the laser illumination. To rule out this possibility, the last puffer application of NH₄Cl and nifedipine in Fig. 12 C was given with the laser illumination omitted. At the end of the NH₄Cl application, the laser illumination was turned on again. It is obvious from this recording that [Ca²⁺]_i had risen to an even higher level than in the previous NH₄Cl application, suggesting that photolysis of nifedipine is not responsible for the drug's lack of effect. Additional experiments, carried out with 20 µM nifedipine bathing the recording chamber for >10 min before the beginning of the recording, confirmed that nifedipine does not inhibit the Ca²⁺ permeation pathway. These pharmacological results were inconclusive as far as the role of T-type Ca²⁺ channels was concerned, since an effective blocker (Ni²⁺) inhibited the response to alkalinization, while the other (nifedipine) did not.

Effects of Intracellular Alkalinization on the Properties of T-type Ca²⁺ Currents

It has been pointed out that the fast inactivation of T-type Ca²⁺ channels upon membrane depolarization would prevent them from significantly contributing to the sustained elevation of [Ca²⁺]_i required for the acrosome reaction (Santi et al., 1996; Arnoult et al., 1996a, 1996b). Nevertheless, different mechanisms can be postulated by which the activity of T-type Ca²⁺ channels could allow more prolonged Ca²⁺ rises. T-type Ca²⁺ channels could contribute to a sustained Ca²⁺ rise if ZP3-induced intracellular signals, such as the elevation of pH_i (Florman et al., 1989; Florman, 1994) or a change in the phosphorylation state of the channels (Liévano et al., 1996; Arnoult et al., 1997), drastically affect one or more of their biophysical properties (e.g., activation threshold, rate of inactivation, "window current," number of active channels). For instance, a sustained Ca²⁺ influx carried by T-type Ca²⁺ channels could be expected if they become substantially less rapidly inactivating or if the window current (the area under the point of crossing of activation and steady state inactivation curves) increases.

To test directly the possibility that alkalinization affects Ca²⁺ currents, the currents were recorded using pipette internal solutions tailored at two different pH_i values (7.4 and 8.1), as well as external solution at pH 8.1. The results are summarized in Fig. 13 A, which illustrates peak Ca²⁺ current densities obtained for these experimental conditions (see Santi et al., 1996). When the intracellular pH was raised from 7.4 to 8.1 (keeping external pH constant), peak Ca²⁺ current density increased significantly (from 7.52 ± 0.41 to 9.06 ± 0.2 µA/cm²). A larger increase (to 9.52 ± 0.41 µA/cm²) was observed when external pH increased from 7.3 to 8.1 (keeping pH_i constant). Current density measured in cells recorded with an internal solution made to pH 6.5 (close to the cell's measured resting pH_i) was 7.45 ± 0.22 µA/cm², n = 3. This value is not different from that obtained at pH_i 7.4. Representative families of Ca²⁺ currents obtained at pH_i 7.4 and 8.1 and pH_o 8.1 are shown in Fig. 13 B. Mean I-V relationships obtained from the three experimental groups are shown in Fig. 14 A. Rising intracellular pH from 7.4 to 8.1 significantly increased peak Ca²⁺ current density (from 6.31 ± 0.83 to 8.49 ± 0.25 µA/cm²). A larger increase (to 9.62 ± 0.61 µA/cm²) was observed when external pH increased from 7.3 to 8.1. Besides the increase in peak current density, the peak of the I-V curve shifted ~5 mV in the hyperpolarizing direction when the external pH changed from 7.3 to 8.1 (see Fig. 14 A). No such shift was observed when internal pH changed from 7.4 to 8.1. The voltage dependence of steady state activation and inactivation of Ca²⁺ currents was determined for the three experimental groups as explained in MATERIALS AND METHODS (see also Santi et al., 1996). These results are summarized in Table I. Extracellular alkalinization from pH 7.3 to 8.1 produced a 5-mV negative shift in Va_1/2 (potential of half-maximal activation). Conversely, intracellular alkalinization in the same range did not affect voltage dependence of activation or inactivation. The corresponding steepness parameters k_a and k_i remained unaffected by either external or internal alkalinization.

View larger version (49K): [in this window] [in a new window]   Fig. 13.   (A) Peak Ca2+ current densities obtained at two different pHi (7.4 and 8.1), as well as at pHo 8.1. The current density increased from 7.52 ± 0.41 to 9.06 ± 0.2 µA/cm2 when the intracellular pH was raised from 7.4 to 8.1, and to 9.52 ± 0.41 µA/cm2 when external pH increased from 7.3 to 8.1. The level of significance of differences between groups is indicated. (B) Representative Ca2+ current families obtained at pHi 7.4 and 8.1 as well as at pHo 8.1. The membrane potential was stepped (100 ms) from 70 to 15 mV in 5-mV increments. The membrane potential was held at 80 mV.

View larger version (22K): [in this window] [in a new window]   Fig. 14.   Effects of pH on current to voltage relationship of Ca2+ currents in primary spermatocytes. (A) Peak I-V relationship recorded from spermatocytes in control conditions and after increasing either internal or external pH to 8.1. () pHi 7.4, () pHi 8.1, () pHo 8.1. When the external pH changed from 7.3 to 8.1, the peak current density increased significantly and the I-V curve shifted 5 mV in the hyperpolarizing direction. Conversely, intracellular alkalinization increased peak current density without affecting the voltage dependence of the channels. (B) Voltage dependence of the time constant of inactivation (h) of the decay of Ca2+ currents recorded under the three conditions shown in A. Dotted lines represent exponential fits to the data points. (C) Ca2+ current traces obtained at the peak of the I-V curves under control conditions (continuous trace) and after external (dotted trace) or internal (dashed trace) alkalinization to pH 8.1. Traces were normalized to their peak amplitude and superimposed for comparison. Notice that activation and inactivation kinetics of Ca2+ currents is not affected by alkalinization of either side of the channel.

T-type Ca²⁺ currents present in spermatogenic cells peak after a few milliseconds and decay rapidly with time courses well fitted by single exponential functions. The activation and inactivation kinetics of these currents are voltage dependent (Santi et al., 1996). To assess for possible internal and external pH effects on the voltage dependence of the rate of inactivation, Ca²⁺ records were obt