Simulations of intracellular calcium release dynamics in response to a high-intensity, ultrashort electric pulse.

Numerical simulations for electrically induced, intracellular calcium release from the endoplasmic reticulum are reported. A two-step model is used for self-consistency. Distributed electrical circuit representation coupled with the Smoluchowski equation yields the ER membrane nanoporation for calcium outflow based on a numerical simulation. This is combined with the continuum Li-Rinzel model and drift diffusion for calcium dynamics. Our results are shown to be in agreement with reported calcium release data. A modest increase (rough doubling) of the cellular calcium is predicted in the absence of extra-cellular calcium. In particular, the applied field of 15 kV/cm with 60 ns pulse duration makes for a strong comparison. No oscillations are predicted and the net recovery period of about 5 min are both in agreement with published experimental results. A quantitative explanation for the lack of such oscillatory behavior, based on the density dependent calcium fluxes, is also provided.

Plasma membrane voltage changes during nanosecond pulsed electric field exposure.

The change in the membrane potential of Jurkat cells in response to nanosecond pulsed electric fields was studied for pulses with a duration of 60 ns and maximum field strengths of approximately 100 kV/cm (100 V/cell diameter). Membranes of Jurkat cells were stained with a fast voltage-sensitive dye, ANNINE-6, which has a subnanosecond voltage response time. A temporal resolution of 5 ns was achieved by the excitation of this dye with a tunable laser pulse. The laser pulse was synchronized with the applied electric field to record images at times before, during, and after exposure. When exposing the Jurkat cells to a pulse, the voltage across the membrane at the anodic pole of the cell reached values of 1.6 V after 15 ns, almost twice the voltage level generally required for electroporation. Voltages across the membrane on the side facing the cathode reached values of only 0.6 V in the same time period, indicating a strong asymmetry in conduction mechanisms in the membranes of the two opposite cell hemispheres. This small voltage drop of 0.6-1.6 V across the plasma membrane demonstrates that nearly the entire imposed electric field of 10 V/mum penetrates into the interior of the cell and every organelle.

Microneedle array for measuring wound generated electric fields.

A microneedle array has been fabricated and applied to the measurement of transdermal skin potentials in human subjects. Potential changes were recorded in the vicinity of superficial wounds, confirming the generation of a lateral electric field in human skin. The measured electric field decays with distance from the wound edge, and is directed towards the wound. The measurement of endogenous fields in skin is a prelude to the study of the therapeutic efficacy of applied electric fields to chronic non-healing wounds.

Endogenous electric fields in embryos during development, regeneration and wound healing.

All embryos that have been investigated drive ionic currents through themselves and these currents will generate internal electric fields. Here, those examples in which such fields have been measured directly are discussed. The first such measurements were made in chick embryos and about 20 mV mm(-1) was measured near the posterior intestinal portal in 2-4 day-old embryos. This electric field is important for the development of tail structures because reducing its magnitude results in abnormal tail development. The second embryonic electric field measured directly was in the axolotl, where a rostral-caudal field of about the same magnitude was detected. Modification of this field during neurulation but not gastrulation caused developmental abnormalities. Most recently, the development of left-right asymmetry in frog and chick embryos was found to require a voltage difference between blastomeres at a very early developmental stage. This field was measured in the chick embryo to be 10-20 mV mm(-1) across the primitive streak. Mammalian skin wounds generate 150 mV mm(-1) fields lateral to the wound and corneal epidermal wounds exhibit lateral fields of 40 mV mm(-1). The presence of these endogenous fields would suggest that exposures to external electric fields should be limited to magnitudes of less than 0.1 V m(-1).

Sperm factor initiates capacitance and conductance changes in mouse eggs that are more similar to fertilization than IP(3)- or Ca(2+)-induced changes.

We used patch clamp electrophysiology and concurrent imaging with the Ca(2+)-sensitive dye, fura-2, to study the temporal relationship between membrane capacitance and conductance and intracellular free Ca(2+) concentration ([Ca(2+)](i)) during mouse egg fertilization. We found an approximately 2 pF step increase in egg membrane capacitance and a minor increase in conductance with no change in [Ca(2+)](i) at sperm fusion. This was followed approximately 1 min later by a rise in [Ca(2+)](i) that led to larger changes in capacitance and conductance. The most common pattern for these later capacitance changes was an initial capacitance decrease, followed by a larger increase and eventual return to the approximate starting value. There was some variation in this pattern, and sub-microM peak [Ca(2+)](i) favored capacitance decrease, while higher [Ca(2+)](i) favored capacitance increase. The magnitude of accompanying conductance increases was variable and did not correlate well with peak [Ca(2+)](i). The intracellular introduction of porcine sperm factor reproduced the postfusion capacitance and conductance changes with a similar [Ca(2+)](i) dependence. Raising [Ca(2+)](i) by the intracellular introduction of IP(3) initiated fertilization-like capacitance changes, but the conductance changes were slower to activate. Capacitance decrease could be induced when [Ca(2+)](i) was increased modestly by activation of an endogenous Ca(2+) current, with little effect on resting conductance. These results suggest that net turnover of the mouse egg surface membrane is sensitive to [Ca(2+)](i) and that sperm and the active component of sperm factor may be doing more than initiating the IP(3)-mediated release of intracellular Ca(2+).

Chemokine receptor expression by human syncytiotrophoblast.

Despite their potential importance in placental HIV infection and placental immune function, nothing is known about the expression of chemokine receptors by human syncytiotrophoblast cells. Immunocytochemical analysis revealed that primary cultures of term syncytiotrophoblast cells express CCR1, CCR3, CXCR4, and CCR6. Immunohistochemical examination of cryosections of term placental villous tissue confirmed the expression of CCR3, CXCR4, and CCR6 by trophoblast cells. The primary syncytiotrophoblast cultures showed no reactivity with antibodies against CCR5. In the villous tissue sections, CCR5 was detected in stromal cells and blood vessel walls but was not found in trophoblast cells. RT-PCR analysis of RNA extracted from cultured syncytiotrophoblast cells confirmed that the cells express message for CCR1, CCR3, CXCR4, CCR6 and CCR10. No transcripts corresponding to CCR2b, CCR5, or CCR8 were detected. Other experiments showed that exposure of syncytiotrophoblast cells to soluble SDF-1alpha elicited a calcium mobilization response, consistent with the expression of functional CXCR4. Thus, human syncytiotrophoblast cells express CXCR4, a known co-receptor for TCL-tropic HIV-1 isolates but do not express CCR5, the major co-receptor for M-tropic isolates. In addition to implications for the maternal-fetal transmission of HIV, the expression of chemokine receptors by syncytiotrophoblast cells could be important in other aspects of placental immune function.

Cyclic AMP-dependent protein kinase A plays a role in the directed migration of human keratinocytes in a DC electric field.

Skin wound healing requires epithelial cell migration for re-epithelialization, wound closure, and re-establishment of normal function. We believe that one of the earliest signals to initiate wound healing is the lateral electric field generated by the wound current. Normal human epidermal keratinocytes migrate towards the negative pole, representing the center of the wound, in direct currents of a physiological strength, 100 mV/mm. Virtually nothing is known about the signal transduction mechanisms used by these cells to sense the endogenous electric field. To elucidate possible protein kinase (PK) involvement in the process, PK inhibitors were utilized. Two important findings have been described. Firstly, addition of 50 nM KT5720, an inhibitor of PKA, resulted in a 53% percent reduction in the directional response of keratinocytes in the electric field, while not significantly affecting general cell motility. The reduction was dose-dependent, there was a gradual decrease in the directional response from 5 to 50 nM. Secondly, addition of 1 microM ML-7, a myosin light chain kinase inhibitor, resulted in an approximate 31% decrease in the distance the cells migrated without affecting directional migration. The PKC inhibitors GF109203X at 4 microM and H-7 at 20 microM and W-7, a CaM kinase inhibitor, did not significantly alter either directed migration or cell migration, although they all resulted in a slight reduction in directional migration. D-erythro-sphingosine at 15 microM, a PKC inhibitor, had virtually no effect on either migration distance or directed migration. These findings demonstrate that divergent kinase signaling pathways regulate general cell motility and sustained directional migration and highlight the complexity of the signal transduction mechanisms involved. The inhibitor studies described in this paper implicate a role for PKA in the regulation of the directional migratory response to applied electric fields, galvanotaxis.

DC electric fields induce rapid directional migration in cultured human corneal epithelial cells.

After an epithelium is wounded, multiple soluble and extracellular matrix-associated signals induce a repair response. An often-overlooked signal is the endogenous electrical field established in the vicinity of the wound immediately upon disruption of epithelial integrity. Previous studies have detected lateral electric fields of approximately 42 mV mm-1 near bovine corneal wounds. In addition, electric fields on the order of 100-200 mV mm-1 have been measured lateral to wounds in mammalian epidermis. Here we report the migratory response of human corneal epithelial cells to DC electric fields of similar, physiologic magnitude. Our findings demonstrate that in a 100 mV mm-1 DC field, corneal epithelial cells demonstrate directed migration towards the cathode. The migratory speed and distances traversed by cultured human corneal epithelial cells is remarkably similar to those of cultured skin-derived keratinocytes under similar conditions; however, corneal epithelial cells demonstrate a more rapid directional response to the field than keratinocytes. These findings suggest that endogenous, wound-induced electric fields present in the cornea play an important role in human corneal wound healing, by orienting the directional response of migratory cells so that they efficiently re-epithelialize the wounded area.

The galvanotaxis response mechanism of keratinocytes can be modeled as a proportional controller.

Human keratinocytes actively crawl in vitro when plated onto a collagen-coated glass substrate, and their direction of migration is totally random. In response to an imposed DC electric field, they migrate asymmetrically, moving mostly toward the negative pole of the field. The authors have analyzed experimental data reported by others to determine the basic characteristics of the cellular response machinery in these keratinocytes. This movement can be completely described mathematically using two independent variables: the speed, V, and the angle of migration, phi. The authors propose a model in which a steerer (controller without feedback) is responsible for determining the speed, and an automatic controller (controller with feedback) is responsible for determining the angle of migration. The torque to rotate is induced by a deterministic cellular signal and a stochastic cellular signal. The cellular machine characteristics are determined as follows: The angular dependence of the detection unit is sin phi; the detection unit detects the guiding field in a linear fashion; the cellular reaction unit can be described by a constant; the chemical amplifier, as well as the cellular motor work, is linear; the cellular characteristic time, which quantifies the cellular stochastic signal, is 50 min.

Involucrin-positive keratinocytes demonstrate decreased migration speed but sustained directional migration in a DC electric field.

When skin is wounded, keratinocytes from the cut edges of the epidermis migrate over the wounded area to re-epithelialize the wound. It is not clear which cells of the epidermis have the capacity to migrate and contribute to this re-epithelialization: the less differentiated cells of the basal layer, or the more differentiated, involucrin-positive suprabasilar cells. Here we demonstrate that both involucrin-negative and involucrin-positive cells are able to respond to a directional cue for migration with sustained directional migration. When cultured keratinocytes are exposed to a physiologic DC electric field of 100 mV per mm as a cue to guide migration (galvanotaxis) they migrate toward the cathode with equivalent directionality. The involucrin-positive cells, however, display mean migration speeds approximately one half (23.6 microm per h) of the mean rate achieved by involucrin-negative cells (46.5 microm per h). Despite their decreased migration rates, involucrin-positive cells appear to possess an intact mechanism for sensing a directional signal, transducing that signal, and responding with sustained directional migration. Because electric fields are endogenous in skin wounds, it is likely that both the basal, involucrin-negative cells and the involucrin-positive suprabasilar cells respond to this cue with directional migration. The new observation that involucrin-positive cells can indeed migrate suggests that these cells may also contribute to wound re-epithelialization in vivo.

Epidermal growth factor receptor relocalization and kinase activity are necessary for directional migration of keratinocytes in DC electric fields.

Human keratinocytes migrate towards the negative pole in DC electric fields of physiological strength. This directional migration is promoted by epidermal growth factor (EGF). To investigate how EGF and its receptor (EGFR) regulate this directionality, we first examined the effect of protein tyrosine kinase inhibitors, including PD158780, a specific inhibitor for EGFR, on this response. At low concentrations, PD158780 inhibited keratinocyte migration directionality, but not the rate of migration; at higher concentrations, it reduced the migration rate as well. The less specific inhibitors, genistein, lavendustin A and tyrphostin B46, reduced the migration rate, but did not affect migration directionality. These data suggest that inhibition of EGFR kinase activity alone reduces directed motility, and inhibition of multiple tyrosine kinases, including EGFR, reduces the cell migration rate. EGFR redistribution also correlates with directional migration. EGFR concentrated on the cathodal face of the cell as early as 5 minutes after exposure to electric fields. PD158780 abolished EGFR localization to the cathodal face. These data suggest that EGFR kinase activity and redistribution in the plasma membrane are required for the directional migration of keratinocytes in DC electric fields. This study provides the first insights into the mechanisms of directed cell migration in electric fields.

Calcium influx factor is synthesized by yeast and mammalian cells depleted of organellar calcium stores.

Depletion of endoplasmic reticulum Ca2+ stores leads to the entry of extracellular Ca2+ into the cytoplasm, a process termed capacitative or store-operated Ca2+ entry. Partially purified extracts were prepared from the human Jurkat T lymphocyte cell line and yeast in which Ca2+ stores were depleted by chemical and genetic means, respectively. After microinjection into Xenopus laevis oocytes, the extracts elicited a wave of increased cytoplasmic free Ca2+ ([Ca2+]i) that spread from the point of injection across the oocyte. Extracts from cells with replete organellar Ca2+ stores were inactive. The increases depended on extracellular Ca2+, were unaffected by the inositol 1,4,5-trisphosphate (IP3) inhibitor heparin or an anti-IP3 receptor antibody and were unchanged when the endoplasmic reticulum was segregated to the hemisphere opposite the injection site by centrifugation. Confocal microscopy revealed that [Ca2+]i increases were most pronounced at the periphery of the oocyte. The patterns of [Ca2+]i increases were replicated by computer simulations based on a diffusible messenger of about 700 Da that directly activates Ca2+ influx. In addition, ICRAC, a Ca2+ release-activated Ca2+ current monitored in Jurkat cells by whole-cell patch clamp recordings, was more rapidly activated when active extracts were included in the patch pipette than by the inclusion of a Ca2+ chelator or IP3. These data support the existence in yeast and mammalian cells depleted of Ca2+ stores of a functionally conserved diffusible calcium influx factor that directly activates Ca2+ influx.

Tyrosine kinase inhibitors block sperm-induced egg activation in Xenopus laevis.

Fertilization of Xenopus laevis eggs triggers a wave of increased [Ca2+]i. The exact signal transduction pathway culminating in this Ca2+ wave remains unknown. To determine whether increases in tyrosine kinase activity are part of this pathway, we microinjected tyrosine kinase inhibitors into unfertilized eggs. Upon fertilization, signs of activation were monitored, such as fertilization envelope liftoff and the Ca2+ wave (for eggs microinjected with lavendustin A). Various concentrations of lavendustin A and tyrphostin B46 were microinjected, as well as inactive forms of these compounds (lavendustin B and tyrphostin A1) to provide negative controls. Peptide A, a 20-amino-acid peptide derived from the SH2 region of pp60(v-src) tyrosine kinase, was also microinjected. Peptide A inhibits tyrosine kinase activity but not PKA or PKG activity. Dose-response curves for lavendustin A, tyrphostin B46, and peptide A show clear inhibition of vitelline envelope liftoff by these three compounds. Confocal imaging of eggs coinjected with lavendustin A and Oregon Green-dextran showed that the Ca2+ wave was inhibited under normal insemination conditions but that the block of the Ca2+ wave could be overcome with very high sperm densities. A phenomenon of small local Ca2+ increases termed "hot spots" seen in lavendustin A containing eggs is also described. Since this inhibition of egg activation by tyrosine kinase inhibitors can be overcome by Ca2+ microinjection, the inhibitors must act on a step in the signal transduction cascade that is upstream of the Ca2+ wave.

Migration of human keratinocytes in electric fields requires growth factors and extracellular calcium.

Currents that leak out of wounds generate electric fields lateral to the wound. These fields induce directional locomotion of human keratinocytes in vitro and may promote wound healing in vivo. We have examined the effects of growth factors and calcium, normally present in culture medium and the wound fluid, on the directional migration of human keratinocytes in culture. In electric fields of physiologic strength (100 mV per mm), keratinocytes migrated directionally towards the cathode at a rate of about 1 microm per min. This directional migration requires several growth factors. In the absence of these growth factors, the cell migration rate decreased but directionality was maintained. Epidermal growth factor alone restored cell migration rates at concentrations as low as 0.2 ng per ml. Insulin at 5-100 microg per ml or bovine pituitary extract at 0.2%-2% vol/vol also stimulated keratinocyte motility but was not sufficient to fully restore the migration rate. Keratinocyte migration in electric fields requires extracellular calcium. Changes in calcium concentrations from 3 microM to 3.3 mM did not significantly change keratinocyte migration rate nor directionality in electric fields; however, addition of the chelator ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to migration medium reduced, and eventually abolished, keratinocyte motility. Our results show that (i) growth factors and extracellular calcium are required for electric field-induced directional migration of human keratinocytes, and (ii) keratinocytes migrate equally well in low and high calcium media.

Voltage-dependent activation of frog eggs by a sperm surface disintegrin peptide.

Fertilin, a sperm protein of the metalloprotease/disintegrin/cysteine-rich (MDC) family, plays a critical role in sperm-egg binding in mammals. Peptides corresponding to the disintegrin domain of fertilin and antibodies against fertilin have been shown to inhibit mammalian sperm-egg binding and fusion. A protein from the same family, xMDC16, was recently cloned from frog (Xenopus laevis) testis and was found to be involved in frog sperm-egg binding. Here we report that xMDC16 is localized predominantly on the posterior surface of egg jelly-activated sperm, and peptides from the disintegrin domain of this protein activate eggs when applied near the egg surface. Egg activation was dependent on (1) specific amino acid residues (KTX); (2) the presence of divalent cations, but not external Ca2+ alone; and (3) voltage across the egg plasma membrane. This is the first demonstration of egg activation in vertebrates by the surface application of a peptide derived from a sperm surface protein, supporting a model for egg activation that involves a signal transducing receptor for sperm in the egg's plasma membrane.

Characterization of the sperm-induced calcium wave in Xenopus eggs using confocal microscopy.

We have used confocal microscopy to examine the [Ca2+]i increase in the albino eggs of the frog Xenopus laevis after fertilization. Eggs were placed in agar wells with their animal poles downward so that fertilization occurred preferentially in the equatorial plane, and confocal microscopy was used to provide a two-dimensional optical section through the three-dimensional Ca2+ wave. These data indicate that the wave of increased [Ca2+]i traverses the entire egg and converges uniformly on the antipode. We show that ratioing two different fluorescent dyes to correct for variations in cell thickness is not a reliable technique for this very thick cell due to differential absorption with depth. Indo-1-dextran proves to be a more reliable Ca2+ indicator in this respect. Indo-1-dextran measurements indicate that the resting [Ca2+]i is not uniform throughout the egg but exhibits a 15% higher [Ca2+]i in the cortex than deep in the cytoplasm. This difference is accentuated during wave propagation and is not dependent on extracellular Ca2+. The average peak [Ca2+]i in the center of the egg as the wave propagates through it is 0.7 microM, approximately 60% of the peak cortical [Ca2+]i. The wave velocity through the center of the egg (5.7 micron/s) is slower than that in the cortex (8.9 micron/s), and both velocities vary slightly during transit. The cortical wave speed is particularly high at the beginning (15.7 micron/s) and end (17.2 micron/s) of the wave. Eggs injected with 30-80 microM of 3 kD heparin to compete with inositol-1,4,5,-trisphosphate for binding to its receptor exhibited multiple localized spots of elevated [Ca2+]i, and many of these did not initiate a wave. For those that did lead to a wave, it was usually slow moving and exhibited a reduced (60% reduction) amplitude compared with controls.

HDL causes mesangial cell mitogenesis through a tyrosine kinase-dependent receptor mechanism.

Hypercholesterolemia and mesangial cell proliferation have been proposed to play a role in the progression of glomerulosclerosis in diabetic nephropathy and other renal diseases. Although LDL is mitogenic for and cytotoxic to mesangial cells, the effect of HDL on these cells is unknown. HDL stimulates fibroblast mitogenesis and is the principal cholesterol-bearing lipoprotein in the rat, the experimental model for studying the effect of hyperlipidemia on renal disease. Insulin is mitogenic in several cell systems, and its levels are increased in serum in non-insulin-dependent diabetes mellitus. This study investigates whether HDL acts as a growth factor in mesangial cells and whether it functions in parallel with insulin. It was found that HDL at protein concentrations between 10 and 500 microg/ml, both alone and in the presence of 100 nM insulin, increased DNA synthesis in mesangial cells (129 to 165% of control for HDL alone; 140 to 235% for HDL plus insulin), whereas HDL at 1000 microg/ml and greater inhibited mesangial cell proliferation. Insulin alone at 100 nM stimulated [3H]thymidine incorporation in the same cell system (145% of control); the mitogenic effect of insulin was additive to that of HDL. Purified apo A-I had a similar effect, but at significantly lower concentrations. Specific binding of HDL to mesangial cells was demonstrated (B(max) [binding constant] of 5.19 +/- 0.70 x 10(-7) micromol of HDL bound/mg cell protein and K(b) of 2.83 +/- 0.22 nM). Tetranitromethane alters apo A-I, preventing binding to its cognate receptor. Tetranitromethane-modified HDL did not bind to mesangial cells and had no effect on [3H]thymidine incorporation. Addition of HDL to mesangial cells caused an immediate transient increase in free intracellular calcium in several representative mesangial cells, similar to the response seen with platelet-derived growth factor. The mitogenic effect of HDL was not altered after attenuation of cellular protein kinase C activity, but the stimulatory effect of HDL alone and in combination with insulin on DNA synthesis was completely eliminated after inhibition of cellular tyrosine kinases by 24-h pretreatment with 0.25 microM herbimycin A. Thus, HDL binds to a specific apo A-I-dependent receptor, promotes DNA synthesis, and initiates second-messenger events by a tyrosine kinase-dependent and protein kinase C-independent mechanism.

Identification of metalloprotease/disintegrins in Xenopus laevis testis with a potential role in fertilization.

Proteins containing a membrane-anchored metalloprotease domain, a disintegrin domain, and a cysteine-rich region (MDC proteins) are thought to play an important role in mammalian fertilization, as well as in somatic cell-cell interactions. We have identified PCR sequence tags encoding the disintegrin domain of five distinct MDC proteins from Xenopus laevis testis cDNA. Four of these sequence tags (xMDC9, xMDC11.1, xMDC11.2, and xMDC13) showed strong similarity to known mammalian MDC proteins, whereas the fifth (xMDC16) apparently represents a novel family member. Northern blot analysis revealed that the mRNA for xMDC16 was only expressed in testis, and not in heart, muscle, liver, ovaries, or eggs, whereas the mRNAs corresponding to the four other PCR products were expressed in testis and in some or all somatic tissues tested. The xMDC16 protein sequence, as predicted from the full-length cDNA, contains a metalloprotease domain with the active-site sequence HEXXH, a disintegrin domain, a cysteine-rich region, an EGF repeat, a transmembrane domain, and a short cytoplasmic tail. To study a potential role for these xMDC proteins in fertilization, peptides corresponding to the predicted integrin-binding domain of each protein were tested for their ability to inhibit X. laevis fertilization. Cyclic and linear xMDC16 peptides inhibited fertilization in a concentration-dependent manner, whereas xMDC16 peptides that were scrambled or had certain amino acid replacements in the predicted integrin-binding domain did not affect fertilization. Cyclic and linear xMDC9 peptides and linear xMDC13 peptides also inhibited fertilization similarly to xMDC16 peptides, whereas peptides corresponding to the predicted integrin-binding site of xMDC11.1 and xMDC11.2 did not. These results are discussed in the context of a model in which multiple MDC protein-receptor interactions are necessary for fertilization to occur.

Progesterone triggers a wave of increased free calcium during the human sperm acrosome reaction.

The mammalian sperm acrosome reaction (AR), an essential fertilization event, requires an influx of Ca2+. The Ca2+ increase occurring in the human sperm head during the AR initiated by progesterone, a putative in vivo AR initiator, was investigated using video-image analysis with fura-2, a fluorescent Ca2+ probe. Progesterone treatment of capacitated human sperm resulted in a wave-like increase in sperm head cytosolic [Ca2+]i that appears to increase fastest in a region near the equatorial segment and then spreads throughout the rest of the head. The progesterone-mediated Ca2+ increase in the sperm head was strongly inhibited and the wave eliminated by picrotoxin, a blocker of GABAA receptor/Cl- channels and an inhibitor of the progesterone-mediated Cl- efflux and progesterone-initiated AR of human sperm. These results are the first to detect a ligand-mediated Ca2+ wave in sperm and to suggest that Cl- efflux influences Ca2+ influx during the AR.