Properties of a Novel pH-dependent Ca²⁺ Permeation Pathway Present in Male Germ Cells with Possible Roles in Spermatogenesis and Mature Sperm Function

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.

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.

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).

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 (ΔF/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.

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.

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.

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 ΔF/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.

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).

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).

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.

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.

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.

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 × 10⁻⁸) 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.

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).

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.

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.

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.

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.

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.

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 obtained at different potentials and their time constants of inactivation (τ_h) were measured. The results are shown in Fig. 14,B. Data points corresponding to pH_o 8.1 were plotted with a 5-mV shift in the positive direction to compensate for the negative shift observed in the voltage dependence of the channels. As shown earlier (Santi et al., 1996), τ_h decreases markedly with depolarization, showing a strong voltage dependence in the voltage range between −45 and 0 mV. In the group of control cells, τ_h showed an e-fold reduction per 6.18 mV. This value was 6.86 mV for cells recorded in external medium at pH 8.1 and 10.77 mV for cells recorded with a pipette internal solution at pH 8.1. At −40 mV, the inactivation time constant of cells recorded in both external and internal alkaline solutions was significantly faster than under control conditions: 15.07 ± 1.19 ms (mean ± SEM, n = 8, pH_o 8.1) and 14.57 ± 0.47 ms (mean ± SEM, n = 13, pH_i 8.1) vs. 20.19 ± 3.12 ms (mean ± SEM, n = 8, control). Nevertheless, they became virtually identical at potentials positive to −30 mV regardless of the pH (see Fig. 14,B). This finding is further illustrated in Fig. 14,C, where current traces obtained at the peak of the three I-V curves shown in Fig. 14 A were normalized. The traces virtually superimpose onto one another. The main conclusion from this group of experiments is that the kinetics of activation and inactivation of Ca²⁺ currents are not affected to a large extent by alkalinization of either side of the channel. Thus, our data do not support the notion that, upon elevation of pH_i, the properties of T-type Ca²⁺ channels change so much as to allow sustained Ca²⁺ influx.

Intracellular pH can influence a wide variety of cellular processes (Putnam, 1995). In the adluminal compartment of seminiferous tubules, spermatogenic cells grow and differentiate in close contact and under the influence of Sertoli cells (reviewed in Jegou, 1993). Some aspects of these processes are sensitive to pH_i (Boron, 1986; Moolenaar, 1986), which is well regulated by spermatogenic cells (Osses et al., 1997). It is well established that in various systems increases in pH_i can elevate [Ca²⁺]_i. Thus, one mechanism by which pH_i changes may affect differentiation is by modulating [Ca²⁺]_i. In addition, elevations of pH_i modulate sperm flagellar motility in many species (Shapiro et al., 1990; Darszon et al., 1996) are required for capacitation in mammalian sperm (Meizel and Deamer, 1978; Working and Meizel, 1983; Vredenburgh and Parrish, 1995; Zeng et al., 1996) and are involved in acrosomal exocytosis in a diversity of organisms (reviewed in Darszon et al., 1996).

The information stated above suggests that a pH_i-modulated Ca²⁺ permeability pathway participates both in spermatogenesis and in sperm physiology. Since sperm are terminal cells, their ion transport systems are synthesized during spermatogenesis (Hetch, 1988). Therefore, late spermatocytes and spermatids, interesting in their own right, are good models to study the properties and regulation of ion transport systems that will be present in mature sperm (Arnoult et al., 1996b; Liévano et al., 1996; Santi et al., 1996). The present experiments were conducted to evaluate the influence of intracellular alkalinization on the Ca²⁺ levels in spermatogenic cells isolated from the testes of adult mice. Our results indicate that cytosolic alkalinization elicited by exposure to the cell-permeant weak base NH₄Cl increases [Ca²⁺]_i mainly by opening a plasmalemmal Ca²⁺ influx pathway. The magnitude of this response increases gradually as maturation advances, suggesting a possible role in the physiology of mature sperm. This Ca²⁺ permeation pathway allows the passage of Sr²⁺, Ba²⁺, and Mn²⁺ and is blocked by inorganic Ca²⁺ channel blockers Ni²⁺ and Cd²⁺, but not by nifedipine. It is likely that, if present in mature sperm, this pH_i-regulated Ca²⁺ pathway may contribute, perhaps in concert with voltage-gated Ca²⁺ channels, to capacitation and the sustained Ca²⁺ influx required to initiate the AR.

Cytosolic pH might influence resting [Ca²⁺]_i of spermatogenic cells by way of several mechanisms. The following discussion assumes that the Ca²⁺ permeation pathway constitutes a single, elementary mechanism. Clearly, the interpretation of our results would be different if several pH-dependent processes contribute to the response.

It is conceivable that upon intracellular alkalinization Ca²⁺ is released from intracellular reservoirs of spermatogenic cells. Ca²⁺-induced Ca²⁺ release and [³H]ryanodine binding is pH-sensitive, with an optimal pH in the alkaline range (>7.2; Meissner, 1994). Similarly, pH elevations enhance the rate of IP₃-induced Ca²⁺ release in skinned smooth muscle cells (Tsukioka et al., 1994). However, as shown in this study, the major source of Ca²⁺ mobilization induced by cytosolic alkalinization is plasmalemmal Ca²⁺ influx, with little or no apparent direct contribution from internal sources. In a related study (Treviño et al., 1998), we examined the density and spatial distribution of Ca²⁺ stores in primary spermatocytes and spermatids using confocal microscopy and fluorescent derivatives of thapsigargin and ryanodine. Both fluorescent analogs showed sparse but distinct labeling of endoplasmic reticulum cisternae, nuclear membrane, and Golgi complex. The scant labeling agrees with the apparently poor Ca²⁺ release that can be induced pharmacologically, suggesting that perhaps the abundance of Ca²⁺ stores is the limiting factor, rather than their filling status. These findings indicate that the amount of Ca²⁺ available for release from rapidly exchanging intracellular reservoirs in spermatogenic cells is too small to directly contribute to the Ca²⁺ signal induced by cytosolic alkalinization. Also, these results, especially those obtained with thapsigargin and cyclopiazonic acid, rule out a major role of Ca²⁺-ATPase of endoplasmic reticulum in the generation of alkalinization-induced Ca²⁺ transients.

Hydrogen ions are important physiological regulators of ion flux through voltage-gated Ca²⁺ channels. As established by numerous studies, proton concentration on either side of the channel modulates L-type Ca²⁺ currents (Kaibara and Kameyama, 1988; Krafte and Kass, 1988; Klockner and Isenberg, 1994). By comparison, very little information is available on the pH modulation of T-type channels, the class of voltage-gated Ca²⁺ channels present in spermatogenic cells. In heart cells, Tytgat et al. (1990) found that external acidification reduces currents carried by T-type Ca²⁺ channels, while alkalinization enlarges them. Thus, raising pH_o from 7.4 to 8.0 increased both peak current density and maximum conductance (g_max) twofold. Furthermore, both the peak of the I-V curve and the half-maximal activation parameter shifted 5 mV negatively. Conversely, T-type Ca²⁺ channels were not significantly modulated by internal changes in pH in the range 6.5–8.0.

Our results with spermatogenic cells are in almost perfect agreement with those obtained in heart cells regarding external pH regulation. Also, cytosolic alkalinization slightly increased peak Ca²⁺ current density without affecting the voltage dependence of the channels. It has been shown that T-type Ca²⁺ current modulation is due to (a) titration of fixed surface charges near the channels, and (b) changes both in single channel conductance and probability of the channel being open (Tytgat et al., 1990). Given the remarkable similarities in the pH dependence of the T-type currents in heart and spermatogenic cells, it is likely that the same mechanisms are involved, although this conclusion awaits experimental confirmation. Experiments in mutated L-type Ca²⁺ channels indicate that external protons block the channel by interacting with a site along the permeation pathway rather than at an external regulatory site outside the pore (Chen et al., 1996). Since the protonation site lies within the pore, and the crucial P-region glutamates in repeats I, II, and III are perfectly conserved in all known α₁ Ca²⁺ channel subunits, it is likely that the same molecular basis underlies the pH sensitivity of T-type Ca²⁺ channels.

Our results indicate that upon internal alkalinization both voltage dependence and kinetics of the Ca²⁺ channels remain virtually unaltered. The mild increase in peak Ca²⁺ current density upon cytosolic alkalinization could slightly augment the capability of this pathway to contribute to a sustained [Ca²⁺]_i elevation. However, external alkalinization is expected to have more repercussion, not only because it increases more Ca²⁺ current density, but also because it shifts the activation threshold to more negative potentials. In this regard, it should be stressed that, in cardiac cells, the external pH-induced changes in g_max showed an apparent pK_a in the range 7.1–7.5 for T-type Ca²⁺ channels, while L-type Ca²⁺ channels of the same cells had a more acidic apparent pK_a ∼5.2. This difference implies that near the physiological pH, T-type Ca²⁺ channels are much more sensitive to variations in external pH than L-type Ca²⁺ channels. This divergence may be crucial in sperm, given the substantial environmental pH changes it experiences through the epididymis and during its journey along the female genital tract.

These findings, together with the inability of nifedipine to significantly inhibit alkalinization-induced Ca²⁺ transients, make unlikely that voltage-gated Ca²⁺ channels constitute a major component of the permeation pathway made available by internal alkalinization. Regardless of this conclusion, T-type Ca²⁺ channels, if present in sperm, are likely to participate in the Ca²⁺ influx required to initiate the acrosome reaction (Arnoult et al., 1996b; Liévano et al., 1996; Santi et al., 1996).

It has been shown that Na⁺/Ca²⁺ exchange is extremely sensitive to cytoplasmic pH, particularly in the physiological range. The activity of the exchanger is partially inhibited at physiological pH, is completely inhibited below pH 6.0, and is maximal at pH 9.0 (Doering and Lederer, 1993; Doering et al., 1996). Although external and internal pH have opposite effects on the Na⁺/Ca²⁺ exchanger, its activity is about three times more sensitive to changes in pH_i than to changes in pH_o (Doering et al., 1996). Thus, under physiological conditions, where external pH changes may result in parallel pH_i changes, the inhibitory effect of H⁺ is expected to dominate. Plasmalemmal Ca²⁺ influx carried by Na⁺/Ca²⁺ exchange is possible under its “reverse” mode of operation (Na⁺-dependent Ca²⁺ influx). This occurs when membrane potential becomes more positive than the equilibrium potential of the exchanger, E_Na/Ca. The resting membrane potential of rat spermatids in suspension has been estimated using a membrane potential sensitive dye (−22 mV; Reyes et al., 1994). The equilibrium potential of the exchanger in spermatogenic cells is not known. Nevertheless, since the resting [Ca²⁺]_i remains low and invariant for extended periods of observation (this study), it is probably safe to assume that the cell's resting potential is more negative than the exchanger's reversal potential. It could be argued that Na⁺/Ca²⁺ exchange may participate in the alkalinization-induced Ca²⁺ influx based on the following similarities: (a) the Na⁺/Ca²⁺ activity increases upon cytosolic alkalinization (see above); (b) Sr²⁺ and Ba²⁺ can also be transported by the exchanger, thus allowing these divalents to enter the cell (Tibbits and Philipson, 1985; Condrescu et al., 1997); (c) Ni²⁺ inhibits the Na⁺/Ca²⁺ exchanger at low millimolar concentrations, but not nifedipine (Trosper and Philipson, 1983); and (d) monensin, which enhances alkalinization-induced responses, also stimulates the reverse mode of operation of the Na⁺/Ca²⁺ exchange by increasing [Na⁺]_i (Santi et al., 1995).

In spite of these similarities, alkalinization-induced Ca²⁺ entry differs from Na⁺/Ca²⁺ exchange in several crucial aspects. First, the former readily allows Mn²⁺ influx, while the exchanger cannot transport this divalent (Trosper and Philipson, 1983). Furthermore, superfusion with 10 μM 2-4 dichlorobenzamil, a relatively specific inhibitor of the Na⁺/Ca²⁺ exchanger, does not suppress alkalinization-induced Ca²⁺ transients (data not shown). Finally, ouabain (30 μM) does not enhance alkalinization-induced Ca²⁺ transients even though, like monensin, it increases [Na⁺]_i by inhibiting the Na⁺/K⁺-ATPase (data not shown). These findings are inconsistent with a significant participation of the Na⁺/ Ca²⁺ exchanger in the pH-dependent Ca²⁺ entry of spermatogenic cells.

In nonexcitable cells, intracellular calcium release is followed by a sustained “store-operated Ca²⁺ influx” (Putney, 1986, 1997; reviewed in Parekh and Penner, 1997). The associated inward currents, termed I_CRAC (calcium-release activated currents) or I_SOC (store operated channel) are highly selective for Ca²⁺ over monovalents (Hoth and Penner, 1993; Yao and Tsien, 1997). Another calcium entry pathway, clearly distinct from I_CRAC exists in some cell types (Putney, 1997), and could correspond to InsP₃ receptors present in the plasma membrane (Vaca and Kunze, 1995). The properties of these Ca²⁺ entry mechanisms resemble those of the alkalinization-induced Ca²⁺ influx pathway: I_CRAC and I_SOC display high selectivity for Ca²⁺ ions, although other divalents, such as Ba²⁺ and Sr²⁺, are also permeant (Hoth and Penner, 1993; Yao and Tsien, 1997). Ni²⁺ and Cd²⁺ block both currents (Premack et al., 1994; Zweifach and Lewis, 1993; Yao and Tsien, 1997). Similarly, the InsP₃-induced permeation across the plasma membrane of Jurkat T cells has relative cation conductances Ca²⁺ > Ba²⁺ > Sr²⁺, and La³⁺ and Cd²⁺ have been used as nonspecific blockers of InsP₃ receptors. Both I_CRAC and InsP₃ receptors, although Ca²⁺ selective, allow the passage of Mn²⁺ (Fasolato et al., 1993; Hoth and Penner, 1993; Premack et al., 1994; Zweifach and Lewis, 1993). Interestingly, pH is an important modulator of the activity of both mechanisms (Tsukioka et al., 1994; Iwasawa et al., 1997).

As shown earlier, Ca²⁺ release from internal stores does not play a major role in directly generating the alkalinization-induced Ca²⁺ transients in spermatogenic cells, probably because their Ca²⁺ stores are not very abundant. Nonetheless, these findings do not rule out the possibility that pH-induced depletion of Ca²⁺ stores may lead to opening of SOCs. Since the activity of this Ca²⁺ influx pathway is itself regulated by intracellular pH, it is conceivable that it could participate or even be responsible for the alkalinization-induced Ca²⁺ rise.

The expression of SOCs in spermatogenic cells has not been thoroughly examined. Nevertheless, the experiments shown in Fig. 9,E, and particularly those in Fig. 10, are consistent with the presence of such channels. The slow and sustained Ca²⁺ influx observed upon the readmission of external Ca²⁺ after thapsigargin application indicates that spermatogenic cells contain thapsigargin-sensitive sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPases. The absence of a significant Ca²⁺ rise during thapsigargin application could be due to the presence of a mechanism that extrudes Ca²⁺ as quickly as it is released. Secondly, these experiments strongly suggest that spermatogenic cells express SOCs. It remains to be explored if this Ca²⁺ permeation pathway is responsible for the alkalinization-induced Ca²⁺ entry. Alternatively, this route could represent a novel pH-regulated channel, predominantly expressed in late spermatogenic cells, not unlike Slo3, a novel potassium channel abundantly expressed in mammalian spermatocytes and regulated by both intracellular pH and membrane voltage (Schreiber et al., 1998).

As shown here, testicular sperm undergo large [Ca²⁺]_i increases when exposed to NH₄Cl. In this regard, it is worth considering how a pH_i-dependent Ca²⁺ permeability pathway could influence sperm physiology.

Sperm travel through very different environments in an excursion that lasts many days from the seminiferous tubules to the oviduct. Clearly, environmental changes, pH_o among them, can influence their physiological state and rate of survival. These changes select the fittest sperm and induce the maturation processes required to achieve fertilization at the appropriate site (reviewed in Harper, 1988; Setchell and Brooks, 1988). In this context, it is not difficult to imagine that the pH-dependent Ca²⁺ permeability pathway described here, along with T-type Ca²⁺ channels, which are also sensitive to external pH, may modulate [Ca²⁺]_i, thus influencing sperm function along the tortuous path towards the egg.

Before they can acquire the capacity to fertilize the egg, sperm need to become capacitated, a process that involves a series of ill defined functional and biophysical modifications (Florman and Babcock, 1991; Baldi et al., 1996). An obligatory step during in vitro capacitation is internal alkalinization from pH ∼6.5 to 6.7 (Parrish et al., 1989; Zeng et al., 1996; reviewed in Baldi et al., 1996). Interestingly, this is the same range of pH change required for spermatogenic cells to respond to alkalinization with Ca²⁺ rises. It can be speculated that the “pH threshold” phenomenon, along with the use-dependent facilitation here described, is relevant for the mechanism of capacitation by allowing cells to respond more vigorously to subsequent episodes of intracellular alkalinization.

Several models exist to explain the sperm AR, a Ca²⁺-dependent exocytotic event required for fertilization, but the precise order of events and the molecular identity of the participants remains elusive (Arnoult et al., 1996a; Darszon et al., 1996). In a striking similarity to sea urchin sperm (Guerrero and Darszon, 1989a, 1989b), at least two different Ca²⁺ channels are believed to participate in the mammalian sperm AR (Florman, 1994, Tiwari-Woodruff et al., 1995). It has been proposed (Arnoult et al., 1996a) that a cation channel that allows Ca²⁺ influx depolarizes the spermatozoon, thus opening T-type Ca²⁺ channels (Arnoult et al., 1996b; Liévano et al., 1996; Santi et al., 1996). The ensuing Ca²⁺ rise, together with the pH_i increase, would trigger the AR. One problem with this model is that Ca²⁺ channels only activate transiently, thus precluding sustained [Ca²⁺]_i elevations. As shown here, this shortcoming of the voltage-gated pathway is not lessened by internal alkalinization. On the other hand, the pH_i-regulated Ca²⁺ pathway described here appears more suitable for supporting sustained Ca²⁺ elevations.

Sperm are devoted to the generation of a new individual. An exquisitely choreographed signaling cascade is required to reach this goal, which may use specific ion-transport variations not evident in other cell types. This novel pH-dependent Ca²⁺ permeation pathway, assuming its presence in mature sperm, could be one of the ion-transport variations responsible for the dihydropyridine-insensitive Ca²⁺ influx that precedes and triggers the acrosome reaction.

The authors thank Drs. Agustín Guerrero and Javier Alvarez-Leefmans for many useful discussions, and Drs. Larry Salkoff and Luis Vaca for reviewing an earlier version of this manuscript. Dr. Reinaldo Dipolo made many helpful suggestions in the early stages of this project. We also thank Dr. José Luis Molinari for providing healthy mice from his colony and Nicolás Jiménez for help in conducting some experiments.

This work was supported by grants from DGAPA-UNAM (IN206395, IN204497) and CONACyT (2366PN, 25261-N; México) to A. Hernández-Cruz, and by the Howard Hughes Medical Institute and the International Centre for Genetic Engineering and Biotechnology to A. Darszon. This work was conducted by C.M. Santi in partial fulfillment of her Ph.D. Thesis. For that purpose, she was awarded a Ph.D. fellowship from DGAPA-UNAM.

AR

acrosome reaction

I-V

current to voltage

SOC

store operated channel