Departamento de Farmacología, Facultad de Medicina, Universidad Nacional Autónoma de México, Ciudad Universitaria, CP 04510, Mexico City, Mexico
Submitted 16 March 2004 ; accepted in final form 9 August 2004
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
mammalian sperm; capacitation; intracellular calcium
In sperm of all species, the identification of voltage-dependent calcium entry mechanisms has been hampered by the size of the head, which makes the use of patch-clamp methods very difficult (10). Nevertheless, our group has shown (23) that step depolarization, induced by potassium in the presence of the potassium ionophore valinomycin, induces an increase in [Ca2+]i in fura-2-loaded human sperm populations that is totally prevented in medium without calcium. This strongly indicates that human sperm are endowed with functional voltage-dependent calcium channels (VDCC). These putative channels are insensitive to nifedipine and verapamil but sensitive to nickel (23) and mibefradil (4). Furthermore, they inactivate in 1.5 min in calcium-deprived medium and are stimulated by ammonium, suggesting a role for pHi (23). It is clear that these data are not sufficient to properly classify the putative channels. Nevertheless, their physiological role and regulation, based on the functional test reported here, deserve to be explored in light of the potential role of VDCC in AE induction by ZP3 and in sperm motility.
During capacitation the calcium influx through these channels increases about onefold (12). In addition, in noncapacitated and capacitated sperm, progesterone, which is present in the follicular fluid and induces calcium influx through nongenomic receptors (5), enhances the [Ca2+]i increase induced by depolarization (12). These observations have led to the hypothesis that "in vivo," a progressive modification of VDCC caused by capacitation and progesterone action may increase as much as four times the levels of calcium permeation found in noncapacitated sperm. Thus, when capacitated sperm reach the egg zona pellucida, the ZP3-induced AE would occur more efficiently because the mechanism of calcium mobilization induced by this glycoprotein may involve gating of VDCC (1).
Here we present evidence indicating that the putative VDCC are modulated by pHi. We found that pHi alkalinization with NH4Cl stimulated the calcium influx induced by depolarization to an extent that may explain, in considerable part, the stimulation of VDCC observed during sperm capacitation.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sperm purification, dye loading, and capacitation. Human semen was obtained from a panel of sixteen 18- to 34-year-old healthy donors who gave informed consent under a protocol approved by the institutional review board. Sperm purification was performed with Percoll gradients as described previously (33). Purified sperm (12 x 108 cells) were loaded with 2 µM fura-2-AM (Sigma) in 2 ml of H-HSM medium at 36°C for 40 min. Once washed, cells were either resuspended in 25 ml of H-HSM medium and used immediately for fluorescence recordings (noncapacitated sperm) or resuspended in 25 ml of HSM (capacitating medium) for 46 h at 36°C under 3% CO2-97% air and 100% humidity. These conditions kept the pH of the HSM medium between 7.6 and 7.7.
The pHi measurements were performed with BCECF in fura-2-loaded sperm. Loading human sperm with both fura-2 and BCECF did not affect BCECF fluorescence, but it did interfere with fura-2 fluorescence (not shown). Even though the pHi measurements in fura-2-loaded and fura-2-free sperm were practically identical (not shown), fura-2-loaded cells were preferred for pHi determinations to properly compare pHi with calcium transport results.
Noncapacitated or capacitated fura-2-loaded sperm were loaded with 3 µM BCECF-AM for 30 min in 5 ml of H-HSM (36°C) or HSM (36°C; under 3% CO297% air, 100% humidity) medium, respectively. The cells were washed, and pHi was determined as described in Measurement of [Ca2+]i, pHi, and membrane potential.
Measurement of [Ca2+]i, pHi, and membrane potential. Fluorescence recordings were performed with a Photon Technology International (PTI) spectrofluorometer equipped with an excitation monochromator and two photomultiplier tubes (PMTs) positioned at 90° with respect to the xenon source. The use of both PMTs and optical interference filters permits measurement of two fluorescence signals simultaneously.
[Ca2+]i measurements.
[Ca2+]i was detected and calibrated in fura-2-loaded sperm as described by Linares-Hernández et al. (23). Sperm (12 x 107 cells) were centrifuged at 300 g for 5 min, and the pellet (100 µl) was immediately added to the fluorescence cuvette, containing 2.5 ml of H-HSM1, at 36°C and under constant magnetic stirring. The cells were alternately excited at 340 and 380 nm, and the fluorescence was detected at 488 nm with an optical filter (band pass 10.0 ± 2 nm; Andover); the ratios were acquired at 0.86 Hz. To calibrate, the maximum fluorescence ratio (Rmax) and, subsequently, the minimum fluorescence ratio (Rmin) were determined by adding 8 µM ionomycin and a mixture of 6 mM EGTA + 0.12% Triton X-100, respectively. Triton X-100 was necessary to obtain a rapid value of Rmin, because intracellular calcium removal with EGTA is extremely slow in the presence of ionomycin (
20 min). Rmin reached similar values in the presence and in the absence of the detergent (not shown).
It has been reported that the Kd of fura-2 for calcium slightly decreases as pH increases (26). Because the [Ca2+]i increase induced by depolarization was assessed while pHi was increasing, the shift in the Kd for calcium could produce increases in fura-2 fluorescence independent of calcium. However, in HSM-H1 medium without calcium + 0.5 mM EGTA, a pHi alkalinization induced by 60 mM NH4Cl (from 6.7 to
7.34, the highest alkalinization studied here) did not induce change in intracellular calcium (not shown), indicating that under our conditions the small effect of pH on fura-2 Kd for calcium was undetectable.
Membrane potential measurements.
In some experiments, simultaneous recordings of membrane potential (Vm) and [Ca2+]i were performed in fura-2-loaded sperm with the Vm-sensitive dye 3,3'-dipropylthiacarbocyanine iodide [diSC3(5); 500 nM] as described previously (23). In these experiments, fura-2-loaded sperm were added to a fluorescence cuvette containing 2.5 ml of H-HSM1 + 0.5 µM diSC3(5) at 36°C under constant magnetic stirring. On addition of cells, diSC3(5) fluorescence decreased because of the electrophoretic uptake by sperm, reaching nearly constant values in 3 min. In human sperm incubated in the presence of the potassium ionophore valinomycin, the plasma Vm sets near the Nernst potential for potassium distribution [Ek = 61.54 mV log([K]i/[K]e), where [K]i and [K]e are intracellular and extracellular potassium concentrations, respectively] (23). Thus Vm can be calculated as the Ek, taking into consideration that [K]i = 120 mM (23). There is a linear relationship between the fractional change of diSC3(5) fluorescence, f fo/fo (where fo is fluorescence in the presence of valinomycin addition and f are actual fluorescence values), and Ek (23). Consequently, f can be converted to Vm according to the following equation (11): Vm = f/mfo 1/m b/m, where m and b are parameters of the linear calibration curve, that is, the slope and the fractional change of fluorescence at 0 mV, respectively.
It should be mentioned that we have not detected diSC3(5) fluorescence signal from mitochondria when valinomycin or the proton ionophore CCCP is added to human sperm (13, 23). This may be related to the fact that sperm motility is unaffected by mitochondrial inhibitors (17) or by anaerobiosis (24), which suggests low mitochondrial activity. Thus the uncoupling effect of valinomycin on mitochondria does not influence the results presented here.
Simultaneous recordings of Vm and intracellular calcium. Simultaneous recordings of fura-2 and diSC3(5) fluorescence were performed with two PMTs of the PTI system. One PMT, with the 488-nm filter, was used to detect fura-2 fluorescence as described above, and the other PMT, with a 670-nm filter (band pass 10 ± 2 nm; Andover), was used to detect diSC3(5) fluorescence simultaneously. Just in front of the xenon source an additional halide lamp (tri-lite; WPI) and a 600-nm filter (band pass 10 nm; Hansatech) were set to excite diSC3(5). The data were collected and analyzed with the PTI computer interface at 0.86 Hz.
pHi measurements.
BCECF fluorescence was detected at 550 nm with an Andover filter (band pass 10.0 ± 2 nm) exciting at 500 and 439 nm, at 36°C and under magnetic stirring. The 500-to-439 ratios (acquired at 0.6 Hz) were calibrated at the end of each trace by adding 0.12% Triton X-100 and then by modifying the pH of the medium with HCl. The addition of Triton X-100 increased the fluorescence ratio to a value corresponding to the pH of H-HSM1 (with almost no effect at 439 nm; not shown). Three consecutive amounts of HCl were then added to the cuvette, which resulted in step decreases in fluorescence ratios. At each step (including the one obtained after detergent addition), the pH was determined with a conventional pH electrode and corresponded to 7.6, 7.10,
6.50, and
5.45 (see Fig. 1A). The calibration curve shows the sigmoid relationship between fluorescence ratio and pH (Fig. 1B). The BCECF ratio values were converted to pHi with the software (FeliX, version 1.41) of the PTI spectrofluorometer.
|
Measurement of voltage-dependent calcium influx. To detect the voltage-dependent calcium influx, sperm was depolarized with different amounts of KCl in the presence of 0.4 µM valinomycin (added 1 min before potassium). Valinomycin was required to bring Vm to approximately 71 mV, which removes VDCC from the inactivated state and makes the membrane mainly dependent on potassium permeability. The voltage-dependent calcium influx was measured as the difference between the [Ca2+]i reached at the peak and the resting [Ca2+]i. This difference is an appropriate estimation of a calcium influx because calcium removal activities are rather slow in sperm (12, 35).
Statistical analysis.
Numeric results are expressed as means ± SE; n indicates the number of individuals tested. ANOVA and paired Student's t-test were used. P values 0.05 were considered statistically significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the presence of valinomycin, addition of 30 mM K+ brings Vm from around 71 mV to around 30 mV and concomitantly induces a transient increase in [Ca2+]i (23). When 10 mM NH4Cl was added 10 s before 30 mM KCl, the voltage-dependent calcium influx was markedly stimulated (Fig. 2). The simultaneous addition of ammonium and KCl produced a similar result (Fig. 2), indicating that the effect of ammonium was practically immediate. This result was consistent with the ability of ammonium to rapidly increase human sperm pHi. The rest of the experiments reported here were performed with the simultaneous addition procedure.
|
|
|
|
The overstimulation observed at pHi 7.28 (with 40 mM NH4Cl; Fig. 5) seemed to result from the sum of the pHi-stimulated voltage-dependent calcium influx that would occur at saturation and that triggered by pHi alone. However, at pHi
7.35 (60 mM NH4Cl) the stimulation was higher than the sum of both calcium permeability pathways. This result raised the possibility that 60 mM NH4Cl could stimulate the [Ca2+]i increase by inducing a stronger depolarization contributed by the cationic form [98% ammonium, corresponding to acidic dissociation constant (pKa) = 9.3]. In a previous paper (23) our group reported that 10 mM NH4Cl did not depolarize human sperm incubated in the presence of valinomycin; however, the higher concentrations of ammonium used here could depolarize and so contribute to [Ca2+]i. To assess this hypothesis, we investigated the effect of 60 mM NH4Cl on membrane potential and [Ca2+]i (detected simultaneously) in fura-2-loaded sperm. Figure 6 shows that even when 60 mM ammonium produced a small, slow depolarization, it was slower than the initial [Ca2+]i increase rate. This result suggested that the [Ca2+]i induced by 60 mM NH4Cl alone was related to a direct effect of pHi alkalinization, with some late contribution of VDCC. Additionally, 60 mM ammonium did not affect the depolarization induced by 30 mM KCl, indicating that the overstimulation produced by 60 NH4Cl on the depolarization-induced [Ca2+]i increase was not related to contribution of ammonium to membrane potential. In this regard, the small effect of 60 mM NH4Cl on membrane potential recordings strongly suggested that the sperm plasma membrane remained highly impermeable, and, hence, toxic effects of NH4Cl should not produce unspecific calcium permeation.
|
Does pHi contribute to calcium release from internal stores?
It has been found that calcium influx through ryanodine receptors may be stimulated by pH alkalinization (23). Thus the possibility that calcium-induced calcium release through these channels contributed to the high pHi-stimulated depolarization-evoked [Ca2+]i increase was considered. We induced [Ca2+]i increase by activating a permeability pathway different from VDCC, such as that triggered by progesterone (6, 14), and investigated the effect of pHi alkalinization. Figure 7 shows that 10 mM NH4Cl barely affected the [Ca2+]i increase induced by progesterone, indicating that the stimulating effect of pHi (at least that induced with NH4Cl 10 mM) on the [Ca2+]i increase induced by depolarization was not related to calcium-induced calcium release from internal stores. Another potential source of calcium could involve calcium release from mitochondria, via activation of the mitochondrial Na+/Ca+ exchanger. However, in our experimental conditions, valinomycin, the potassium ionophore used to modify Vm with potassium, also uncouples mitochondria and, consequently, impairs the ability of this organelle to accumulate calcium (29). This strongly suggested that calcium release from mitochondria did not contribute to the calcium signals reported here. Interestingly, in mouse sperm, mitochondria do not accumulate calcium in response to intracellular calcium load (36).
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In mature mouse sperm there are putative VDCC, detected via intracellular calcium measurements, the gating of which (induced with potassium) strictly depends on simultaneous external medium alkalinization, from pH 7.4 to pH 8.6 (38). In these cells, the calcium influx induced by alkaline depolarization is markedly enhanced by repeated stimuli and also by NaHCO3 (37). These manipulations increase the cAMP levels because of the activation of a peculiar form of adenylate cyclase, which is in turn stimulated by calcium ions (18) or by bicarbonate (8). Consequently, it has been proposed that cAMP may enhance the calcium influx induced by alkaline depolarization (37). Accordingly, because of the induction of biochemical changes, the stimulating effect of bicarbonate on the calcium entry in mouse sperm requires seconds to occur (half-time 60 s; Ref. 37). In contrast, the stimulating effect of pHi on human sperm VDCC reported here is practically immediate, suggesting that biochemical changes induced by pHi alkalinization are not involved in this phenomenon. Furthermore, it was recently reported that mouse sperm lacking catsper1, a putative VDCC present in mouse sperm flagella (27), are not able to hyperactivate their motility (28), and, interestingly, they do not increase [Ca2+]i in response to alkaline depolarization (7). This led to the hypothesis that catsper1 might be the calcium channel that allows the alkaline depolarization-induced calcium influx. Whether or not the putative pHi-dependent VDCC described here is a form of catsper1 remains to be established.
It should be noted that the effect of pHi on VDCC has been documented in different excitable cells. Stimulating effects of pHi alkalinization on calcium permeation have been observed in T- and N-type VDCC currents in neurons (21, 34), the T-type VDCC in mouse spermatocytes (31), and the L-type VDCC present in smooth muscle (32, 39). pHi does not affect the gating of the channel (21, 32); instead, the pHi sensitivity of L-type Ca2+ channels is conferred by the -subunit of the channel complex (32). Accordingly, in this study we have observed that the pHi-stimulated calcium influx induced by depolarization does not depend on the activation of the channels.
The physiological role of pHi on VDCC in excitable cells is not clear. For instance, it has been speculated that pathological conditions leading to pHi acidification, such as ischemia, would decrease neuronal excitability because of the lower opening of VDCC. Consequently, maintenance of [Ca2+]i at resting levels would be favored, keeping the cell alive under such a stressing situation (21). In human sperm, the role of pHi on VDCC could be directly related to normal sperm function. Indeed, during sperm capacitation the calcium influx through VDCC is stimulated (Ref. 12 and this study) and the pHi increases 0.110.14 pH units (Ref. 9 and this study). The pHi sensitivity of the depolarization-induced calcium influx described here shows that the pHi alkalinization occurring in human sperm would be enough to produce
30% of the stimulation observed during capacitation. Thus the pHi alkalinization observed during capacitation is not sufficient to explain the level of calcium influx stimulation observed in capacitated sperm. However, the similarity between the calcium influx induced by depolarization in capacitated cells and that induced by depolarization + 0.35-pH unit pHi alkalinization (with 10 mM NH4Cl) in noncapacitated cells strongly suggests that pHi still has a major role. We have the impression that a biochemical modification of the channel, which may be produced by cAMP or a tyrosine kinase, the activities of which increase during capacitation, might enhance the effect of pHi on the channels.
Finally, it should be noted that calcium permeation can be further stimulated by progesterone (12), which is normally produced by the follicular cells that surround the egg. Under our conditions, progesterone does not induce pHi changes in sperm, indicating a different stimulatory mechanism on VDCC. Even though the physiological role of human sperm VDCC has not been established, the remarkable enhancement of calcium permeation on these channels produced during its journey to the egg may have a major role for the ZP3-induced AE, where gating of VDCC has been postulated to occur. Furthermore, the possible contribution of VDCC to resting [Ca2+]i remains to be explored. In light of the results presented here, if calcium influx through VDCC contributes to resting [Ca2+]i, the small but biologically significant increase in [Ca2+]i observed during capacitation could be related to the VDCC stimulation reported here. Other putative non-voltage-dependent calcium channels have been considered as mechanisms to set the resting [Ca2+]i (11).
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Baldi E, Casano R, Falsetti C, Krausz C, Maggi M, and Forti G. Intracellular calcium accumulation and responsiveness to progesterone in capacitating human spermatozoa. J Androl 12: 323330, 1991.[Abstract]
3. Baldi E, Luconi M, Bonaccorsi L, and Forti G. Signal transduction pathways in human spermatozoa. J Reprod Immunol 53: 121131, 2002.[CrossRef][ISI][Medline]
4. Blackmore PF and Eisoldt S. The neoglycoprotein mannose-bovine serum albumin, but not progesterone, activates T-type calcium channels in human spermatozoa. Mol Hum Reprod 5: 498506, 1999.
5. Blackmore PF, Neulen J, Lattanzio F, and Beebe SJ. Cell surface-binding sites for progesterone mediate calcium uptake in human sperm. J Biol Chem 266: 1865518659, 1991.
6. Bonaccorsi L, Forti G, and Baldi E. Low-voltage-activated calcium channels are not involved in capacitation and biological response to progesterone in human sperm. Int J Androl 24: 341351, 2001.[CrossRef][ISI][Medline]
7. Carlson AE, Westenbroek RE, Quill T, Ren D, Clapham DE, Hille B, Garbers DL, and Babcock DF. CatSper1 required for evoked Ca2+ entry and control of flagellar function in sperm. Proc Natl Acad Sci USA 100: 1486414868, 2003.
8. Chen Y, Cann MJ, Litvin TN, Iourgenko V, Sinclair ML, Levin LR, and Buck J. Soluble adenylyl cyclase as an evolutionarily conserved bicarbonate sensor. Science 289: 625628, 2000.
9. Cross NL and Razy-Faulkner P. Control of human sperm intracellular pH by cholesterol and its relationship to the response of the acrosome to progesterone. Biol Reprod 56: 11691174, 1997.[Abstract]
10. Darszon A, Beltran C, Felix R, Nishigaki T, and Trevino CL. Ion transport in sperm signaling. Dev Biol 240: 114, 2001.[CrossRef][ISI][Medline]
11. González-Martínez MT. Induction of a sodium-dependent depolarization by external calcium removal in human sperm. J Biol Chem 278: 3630436310, 2003.
12. González-Martínez MT, Bonilla-Hernández MA, and Guzmán-Grenfell AM. Stimulation of voltage dependent calcium channels during capacitation and by progesterone in human sperm. Arch Biochem Biophys 408: 205210, 2002.[CrossRef][ISI][Medline]
13. Guzmán-Grenfell AM, Bonilla-Hernandez MA, and González-Martínez MT. Glucose induces a Na+,K+-ATPase-dependent transient hyperpolarization in human sperm. I. Induction of changes in plasma membrane potential by the proton ionophore CCCP. Biochim Biophys Acta 1464: 188198, 2000.[ISI][Medline]
14. Guzman-Grenfell AM and Gonzalez-Martinez MT. Lack of voltage-dependent calcium channel opening during the calcium influx induced by progesterone in human sperm. Effect of calcium channel deactivation and inactivation. J Androl 25: 117122, 2004.
15. Hamamah S, Magnoux E, Royere D, Barthelemy C, Dacheux JL, and Gatti JL. Internal pH of human spermatozoa: effect of ions, human follicular fluid and progesterone. Mol Hum Reprod 2: 219224, 1996.[Abstract]
16. Ho HC and Suarez SS. Hyperactivation of mammalian spermatozoa: function and regulation. Reproduction 122: 519526, 2001.
17. Hong CY, Chiang BN, and Wei YH. Mitochondrial respiration inhibitors and human sperm motility: implication in the development of spermicides. Br J Clin Pharmacol 16: 487490, 1983.[ISI][Medline]
18. Jaiswal BS and Conti M. Calcium regulation of the soluble adenylyl cyclase expressed in mammalian spermatozoa. Proc Natl Acad Sci USA 100: 1067610681, 2003.
19. Jones JM and Bavister BD. Acidification of intracellular pH in bovine spermatozoa suppresses motility and extends viable life. J Androl 21: 616624, 2000.
20. Kaufman DS, Goligorsky MS, Nord EP, and Graber ML. Perturbation of cell pH regulation by H2O2 in renal epithelial cells. Arch Biochem Biophys 302: 245254, 1993.[CrossRef][ISI][Medline]
21. Kiss L and Korn SJ. Modulation of N-type Ca2+ channels by intracellular pH in chick sensory neurons. J Neurophysiol 81: 18391847, 1999.
22. Laver DR, Eager KR, Taoube L, and Lamb GD. Effects of cytoplasmic and luminal pH on Ca2+ release channels from rabbit skeletal muscle. Biophys J 78: 18351851, 2000.
23. Linares-Hernández L, Guzman-Grenfell AM, Hicks-Gomez JJ, and González-Martínez MT. Voltage dependent calcium influx in human sperm assessed by simultaneous detection of intracellular calcium and membrane potential. Biochim Biophys Acta 1372: 112, 1998.[CrossRef][ISI][Medline]
24. Makler A, Makler-Shiran E, Stoller J, Lissak A, Abramovici H, and Blumenfeld Z. Use of a sealed mini-chamber to investigate human sperm motility in real time under aerobic and anaerobic conditions. Arch Androl 29: 255261, 1992.[ISI][Medline]
25. Osheroff JE, Visconti PE, Valenzuela JP, Travis AJ, Alvarez J, and Kopf GS. Regulation of human sperm capacitation by a cholesterol efflux-stimulated signal transduction pathway leading to protein kinase A-mediated up-regulation of protein tyrosine phosphorylation. Mol Hum Reprod 5: 10171026, 1999.
26. Peng HL, Jensen PE, Nilsson H, and Aalkjær C. Effect of acidosis on tension and [Ca2+]i in rat cerebral arteries: is there a role for membrane potential? Am J Physiol Heart Circ Physiol 274: H655H662, 1998.
27. Quill TA, Ren D, Clapham DE, and Garbers DL. A voltage-gated ion channel expressed specifically in spermatozoa. Proc Natl Acad Sci USA 98: 1252712531, 2001.
28. Quill TA, Sugden SA, Rossi KL, Doolittle LK, Hammer RE, and Garbers DL. Hyperactivated sperm motility driven by CatSper2 is required for fertilization. Proc Natl Acad Sci USA 100: 1486914874, 2003.
29. Rizzuto R. Calcium mobilization from mitochondria in synaptic transmitter release. J Cell Biol 163: 441443, 2003.
30. Rodriguez E and Darszon A. Intracellular sodium changes during the speract response and the acrosome reaction in sea urchin sperm. J Physiol 546: 89100, 2003.
31. Santi CM, Santos T, Hernandez-Cruz A, and Darszon A. Properties of a novel pH-dependent Ca2+ permeation pathway present in male germ cells with possible roles in spermatogenesis and mature sperm function. J Gen Physiol 112: 3353, 1998.
32. Schuhmann K, Voelker C, Hofer GF, Pflugelmeier H, Klugbauer N, Hofmann F, Romanin C, and Groschner K. Essential role of the beta subunit in modulation of C-class L-type Ca2+ channels by intracellular pH. FEBS Lett 408: 7580, 1997.[CrossRef][ISI][Medline]
33. Suarez SS, Wolf DP, and Meizel S. Induction of the acrosome reaction in human spermatozoa by a fraction of human follicular fluid. Gamete Res 14: 107121, 1986.[ISI]
34. Tombaugh GC and Somjen GG. Differential sensitivity to intracellular pH among high- and low-threshold Ca2+ currents in isolated rat CA1 neurons. J Neurophysiol 77: 639653, 1997.
35. Wang D, King SM, Quill TA, Doolittle LK, and Garbers DL. A new sperm-specific Na+/H+ exchanger required for sperm motility and fertility. Nat Cell Biol 5: 11171122, 2003.[CrossRef][ISI][Medline]
36. Wennemuth G, Babcock DF, and Hille B. Calcium clearance mechanisms of mouse sperm. J Gen Physiol 122: 115128, 2003.
37. Wennemuth G, Carlson AE, Harper AJ, and Babcock DF. Bicarbonate actions on flagellar and Ca2+-channel responses: initial events in sperm activation. Development 130: 13171326, 2003.
38. Wennemuth G, Westenbroek RE, Xu T, Hille B, and Babcock DF. CaV2.2 and CaV2.3 (N- and R-type) Ca2+ channels in depolarization-evoked entry of Ca2+ into mouse sperm. J Biol Chem 275: 2121021217, 2000.
39. Yamakage M, Lindeman KS, Hirshman CA, and Croxton TL. Intracellular pH regulates voltage-dependent Ca2+ channels in porcine tracheal smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 268: L642L646, 1995.