Fast polyspermy block and activation potential : Correlated changes during oocyte maturation of a starfish

Sperm entry into the oocyte of the starfish, Asterina pectinifera, was prevented when the membrane potential of the oocyte was held more positive than −10 to −5 mV, and multiple sperm entries were induced when the potential was held more negative. Based on this potential-dependent fertilization block mechanism, it was demonstrated that an activation potential (AVP) which is induced immediately after the attachment of the first sperm to the egg surface plays the role of a fast polyspermy block. The AVP-mediated polyspermy block mechanism develops as the oocyte matures and deteriorates as it ages. AVPs of mature oocytes exceeded −5 mV (the critical potential level for fertilization block) within 1 sec, and the potential stayed at +12 mV even after the initiation of fertilization membrane elevation. Consequently, the entry of a second sperm is prevented. In contrast, AVPs of overripe oocytes took about 15 sec to attain −5 mV, or they did not attain −5 mV at all. In overripe oocytes multiple sperm entries were associated with “step depolarization(s)” in the rising phase of the AVPs before membrane elevation took place. Immature oocytes generated AVPs associated with sperm entries, but without membrane elevation. AVPs in immature oocytes were characterized by the step depolarization(s) in the rising phase, and an AVP could be evoked again by a second insemination 20 min after the first insemination. These findings indicate that immature oocytes lack both fast and slow polyspermy block mechanisms.     

Membrane depolarization facilitates sperm entry, large fertilization cone formation, and prolonged current responses in sea urchin oocytes

Depolarization of the sea urchin egg's membrane is required for two processes during fertilization: the entry of the fertilizing sperm and the block to polyspermy which prevents the entry of supernumerary sperm. In an immature sea urchin oocyte, the depolarization is very small in response to the attachment of a sperm. The purpose of this study was to determine whether the depolarization evoked by sperm attaching to an oocyte can facilitate sperm entry or induce the block to polyspermy. Individual oocytes of the sea urchin with diameters which ranged from 86 to 102% that of the average diameter for mature eggs from the same female were examined. The oocytes have a membrane potential of -73 +/- 6 mV (SD, n = 80) and a very low input resistance compared to that of mature eggs. Single sperm, following attachment to an oocyte, elicit a brief, small depolarization with a maximum amplitude of 8 +/- 1.4 mV (SE, n = 15), frequently followed by the formation of tiny filament-like fertilization cones, but the sperm fail to enter. If oocytes are voltage-clamped at membrane potentials more negative than -20 mV, following attachment of the sperm small transient inward currents occur, similar filament-like cones form, and the sperm do not enter. When many sperm attach to an oocyte which is not voltage clamped, the depolarizations sum to create a large depolarization with an amplitude of 60 to 80 mV, which shifts the oocyte's membrane potential to a value between -10 and +5 mV; more positive values are not attained. At such membrane potentials, whether the potential is maintained by the summed depolarizations of many attached sperm or by voltage clamp, large fertilization cones form, the sperm enter, and the oocytes can become highly polyspermic. In oocytes voltage clamped at +20 mV, however, both sperm entry and fertilization cone formation are suppressed. Therefore, both types of voltage-dependence for sperm entry are present in oocytes, although the depolarization caused by a single sperm is not large enough to permit its entry, nor is the depolarization caused by many sperm sufficient to prevent the entry of supernumerary sperm.     

Monospermy and polyspermy after partial zona dissection of reinseminated human oocytes

Partial zona dissection (PZD), a zona drilling method that uses mechanical force to open the zona pcllucida while the oocyte is shrunken in a sucrose solution, was applied to 121 unfertilized 1-day-old mature human oocytes prior to reinsemination. The 115 surviving oocytes were divided into three groups in which the duration between sucrose addition and reinsemination was varied: I) Less than 20 minutes, II) 21 to 45 minutes, and III) longer than 45 minutes. There was a trend toward a reduced fertilization and polyspermy rate as the time between sucrose exposure and insemination in sucrose-free medium increased. Moreover, there was a statistically significant reduction in the number of oocytes penetrated by more than four sperm in group III (0/41) versus group I (7/34), and in group III, parthcnogcnctic development was observed. The incidence of polyspermy was also increased in oocytes manipulated more than 25 hours after retrieval compared with those manipulated 21-24 hours after recovery, supporting the idea that aged oocytes have a reduced ability to block polyspermy. Oocyte contraction in sucrose occurred in three different patterns: spherical, pear-shaped, and crenated. Both the fertilization and polyspermy rates were significantly higher in the crenated group. These results indicate that changes resembling activation occur following sucrose exposure and that sucrose activation can be used to reduce the risk of polyspermic fertilization in zona drilling procedures. In addition, the pattern of shrinkage in sucrose can be used as an indicator of oocyte receptivity to sperm penetration.     

Insemination of rabbit eggs is associated with slow depolarization and repetitive diphasic membrane potentials

The plasma membrane of the rabbit egg allows only one sperm to enter the egg during fertilization, but the mechanism of this block to polyspermy is unknown. Electrophysiology and in vitro fertilization techniques were employed in this study to investigate the possibility that a voltage block to polyspermy exists in rabbit eggs. Ovulated zona-intact eggs had a mean membrane potential of -71 +/- 2.1 mV (interior negative). A stereotypic response occurred 12-135 min following in vitro insemination in 19 of 40 eggs. Association of this stereotypic response with the appearance of pronuclei suggested that the electrical response was related to some interaction of gametes. This response consisted of a slow transient 8 +/- 1.5 mV depolarization upon which were superimposed up to 36 repetitive diphasic insemination potentials. Each potential consisted of a brief 2.0 +/- 0.44 mV hyperpolarization followed by a slow 2.5 +/- 0.45 mV depolarization. The small amplitude of the stereotypic response when compared with the large variation of resting potentials suggested that the response was insufficient to block polyspermy by a mechanism dependent upon the magnitude of the rabbit egg membrane potential.     

Fertilization in crabs: IV. The fertilization potential consists of a sustained egg membrane hyperpolarization

The electrophysiological properties of immature and mature oocytes of two crabs were analyzed. Growing immature oocytes of Carcinus maenas and fully grown immature oocytes of Maia squinado had essentially K⁺ dependent resting potentials, Em, of ?61 ? 1 mV, n=19, and ?67.3 ± 0.5 mV, n=68, respectively. Fully grown immature oocytes of Carcinus maenas showed an Em of ?40 ± 1.5 mV, n=19, that was k⁺ and Cl^? dependent. In mature oocytes of both species, the plasma membrane became exclusively permeable to cl^? and the Em attained–41 ± 1 mV, n=49 and ?34 ± 1.5 mV, n=27 for Carcinus maenas and Maia squinado, respectively. After in vitro insemination, a dramatic increase in egg membrane permeability to K⁺ was observed. This instantaneously caused a sustained hyperpolarization constituting, for these crabs, the fertilization potential. We observed that concurrently with this electrical response to fertilization, sperm reinitiated the oocyte meiotic maturation previously arrested at the first metaphase. The triggering mechanism of the fertilization potential as well as the possible occurrence of a physiological polyspermy are discussed.     

Measurement of plasma membrane and mitochondrial potentials in sea urchin sperm. Changes upon activation and induction of the acrosome reaction

The relationship between the plasma membrane potential and activation of sperm motility and respiration, or induction of the acrosome reaction, was explored in sperm of the sea urchin Strongylocentrotus purpuratus. Plasma and mitochondrial membrane potentials were estimated by measuring the uptake of [14C]thiocyanate ( [14C]SCN-) and [3H]tetraphenylphosphonium ( [3H]TPP+) in intact sperm and sperm made permeant with digitonin. Mitochondrial potentials up to-185 mV were found, consistent with data for TPP+ uptake into mitochondria from other cell types. Values for TPP+ uptake corrected for mitochondrial accumulation and estimates of SCN- uptake both indicated that the plasma membrane potential was about -30 mV for actively respiring sperm in seawater and about -60 mV for quiescent sperm in Na+-free seawater. Activation of sperm motility and respiration induced by Na+ increased the intracellular pH and caused a depolarization of both the plasma membrane and mitochondrial potentials. However, membrane potential depolarization did not occur when the activation was induced by increased extracellular pH or by the peptide speract, although activation was always linked to increased intracellular pH. The acrosome reaction, on the other hand, was always associated with sperm plasma membrane potential depolarization, whether it was induced by the physiological effector from the egg surface or by several artificial triggering regimens. Thus, activation of respiration and motility is primarily controlled by increased intracellular pH (Christen, R., Schackmann, R. W., and Shapiro, B. M. (1982) J. Biol. Chem. 257, 14881-14890), whereas the acrosome reaction also requires depolarization of the plasma membrane potential.     

Electrical response to fertilization in ascidian oocytes

The fertilization potential of the ascidian oocyte has been studied using two intracellular electrodes. Two classes of oocyte were observed; low resting potential (RP) oocytes of ?20 to ?35 mV and high RP oocytes of ?80 to ?90 mV. The two types have comparable membrane resistance, falling in the range of 100–300 MΩ, and both may be fertilized and develop normally, although the fertilization potential (FP) is different in the two cases. High RP oocytes give rise to step-like regenerative potentials which attain positive values, whereas low RP oocytes give rise to slower depolarizations that reach zero level. In both cases the FP was sometimes preceded by a small-step depolarization as normally observed in the sea urchin. Irrespective of the original RP, the membrane resistance always decreased to 1–10% of its initial value during the first few minutes of the FP plateau. In contrast when the membrane was depolarized to a comparable potential by current injection the membrane resistance did not decrease. Polyspermic fertilization was induced by removing the extracellular coats, aging the oocytes and using high densities of sperm. The FP in monospermic and polyspermic oocytes were comparable and we could not correlate additional sperm interactions with additional electrical events. Our results suggest that the plasma membrane in ascidian oocytes lacks intrinsic mechanisms, electrical or otherwise, to prevent polyspermic fertilization.     

An electrophysiological study of parthenogenetic activation in mammalian oocytes

Using conventional electrophysiological techniques, we have investigated the electrical responses of mouse and hamster oocytes in metaphase of the second meiotic division to agents which induce parthenogenetic activation. Oocytes from MF1 mice responded to 8.7% ethanol and to 0.3% benzyl alcohol by a depolarization (sometimes preceded by a brief hyperpolarization). The response to ethanol did not "desensitize," and the membrane potential recovered completely when the exposure to ethanol was interrupted. The response was accompanied by a decrease in membrane input resistance (Rin) and had an equilibrium potential of about +5 mV in standard medium and of -10mV in Na-free medium. The oocytes responded to A23187 and to La3+ by an increased Rin, and usually lysed during or after treatment. Multiphasic responses were elicited by ethanol and by Ca-ionophore in metaphase II hamster oocytes; an early hyperpolarization accompanied by a decreased Rin was a common feature of the response to both activating agents. The early hyperpolarization was no longer elicited when the cells were exposed for a second time to ethanol or A23187. K+ and Cl- were the ions mainly involved in the hyperpolarizing potential elicited by A23187, and K+ (but not Cl-) was the ionic species mainly involved in ethanol response. The above responses were peculiar to metaphase II oocytes since mouse and hamster ovarian oocytes (in prophase I) and fertilized eggs either failed to respond to the activating agents, or responded by increasing Rin. The variety of electrical responses to parthenogenetic agents indicates that in mammalian oocytes parthenogenetic activation is not triggered by a "classical" activation potential.     

NAADP triggers the fertilization potential in starfish oocytes

In invertebrates oocytes or eggs, the fertilization or activation potential establishes the fast electrical block to polyspermy and, in some species, provides the Ca2+ influx which contributes to the following intracellular Ca2+ wave. In echinoderms, the molecule triggering the activation potential is still unknown. The aim of this study was to assess whether nicotinic acid-adenine dinucleotide phosphate (NAADP) elicited the fertilization potential in starfish oocytes. The changes in membrane potential induced by the sperm were measured in oocytes held at a low resting potential, so that the Ca2+-action potential was inactivated and only the initial slower depolarization caused by the sperm could be studied. Decreasing extracellular Na+ concentration did not prevent the onset of the fertilization potential, while removal of external Ca2+ abolished it. The pre-incubation with SK&F 96365 and verapamil and the pre-injection of BAPTA inhibited the fertilization potential, while the injection of heparin only reduced its duration. The biophysical and pharmacological properties of the sperm-elicited depolarization were similar to those displayed by the NAADP-activated Ca2+-mediated current recently described in starfish oocytes. Indeed, the desensitization of NAADP-receptors prevented the onset of the fertilization potential. Taken together, these data suggest that NAADP could trigger the fertilization potential in starfish oocytes.     

Effects of glucocorticoids on maturation of pig oocytes and their subsequent fertilizing capacity in vitro

The aim of this study was to assess the possible role of glucocorticoids in the maturation of pig oocytes and their subsequent fertilizing capacity in vitro. Pig cumulus-enclosed oocytes collected from prepubertal gilts were cultured in Waymouth MB752/1 medium supplemented with sodium pyruvate (50 microg/ml), LH (0.5 microg/ml), FSH (0.5 microg/ml), and estradiol-17beta (1 microg/ml) in the presence or absence of cortisol or dexamethasone (DEX) for 24 h; they then were cultured without hormonal supplements in the presence or absence of cortisol or DEX for an additional 16-24 h. Treatment of cumulus-enclosed or denuded oocytes with increasing concentrations of cortisol or DEX for 48 h resulted in a dose-response inhibition of germinal vesicle breakdown (GVB). Increasing duration (12-48 h) of treatment with DEX (10 microg/ml) led to a time-dependent inhibition of GVB, which achieved statistical significance by 12 h. The addition of DEX (10 microg/ml) to maturation medium immediately after culture or at 12 h, 24 h, or 36 h after culture also decreased the percentage of oocytes with GVB. When oocytes were exposed to DEX for 48 h, the maturation rate was reduced. The degree of this reduction was dependent on DEX, and a concentration of DEX higher than 0.1 microg/ml was needed. The inhibitory effect of DEX on the maturation of oocytes was prevented by the glucocorticoid receptor antagonist RU-486. Exposure of oocytes to DEX for 40 h did not prevent sperm penetration, affect the incidence of polyspermy, or decrease the ability of oocytes to form a male pronucleus. The intracellular concentration of glutathione (GSH) in cumulus-enclosed oocytes was 4.4 mM per oocyte. Exposure of oocytes to DEX (0.01-10 microg/ml) had no effect on GSH concentration. These results demonstrate that glucocorticoids directly inhibit the meiotic but not cytoplasmic maturation of pig oocytes in vitro. This inhibitory effect is not mediated through a decrease in the level of intracellular GSH.     

Cortical granule exocytosis in Bufo arenarum oocytes matured in vitro

Denuded Bufo arenarum oocytes matured in vitro by progesterone treatment exhibited abnormal segmentation due to the penetration of more than one sperm. These oocytes were able to respond to activation stimuli and exhibited the external signs characteristic of activation. However, the prevention of polyspermy was not effective in these oocytes, which exhibited numerous sperm in their cytoplasm. The aim of this work was to analyse the cortical reaction in polyspermic Bufo arenarum oocytes matured in vitro. The result indicate that the cortical reaction of these oocytes seems to occur with a chronological sequence similar to that described for ovoposited oocytes of this species. In addition, when, 1 min after pricking, cortical granule exocytosis occurred, the oocytes became refractory to sperm entry, suggesting that they are able to establish a slow block to polyspermy.     

去甲肾上腺素引起蟾蜍背根神经节神经细胞膜电位变化的离子机制

本文应用细胞内记录方法,对去甲肾上腺素(NA)引起蟾蜍背根神经节(DRG)神经细胞膜电位去极化或超极化反应时的膜电导及翻转电位值进行了测量,并观察了钾和钙离子通道阻断剂灌流DRG对NA引起膜电位反应的影响。当NA引起去极化反应时,15个细胞的膜电导减小32.6％。少数细胞膜电导开始增加,继而减小(n=4)。NA超极化反应时膜电导增加13.2％(n=8)。NA去极化反应的翻转电位值为-88.5±0.9mV((?)±SE,n=4),NA超极化反应在膜电位处于-89至-92mV时消失。 钾通道阻断剂四乙铵可使NA去极化幅值增加73.7±11.9％((?)±SE,n=7),并使NA超极化幅值减小40.5％(n=4)。细胞内注入氯化铯使苯肾上腺素去极化幅值增加34.5％(n=4)。钙通道阻断剂氯化锰使NA去极化及超极化反应分别减小50.5±9.9％((?)±SE,n=10)和89.5±4.9％((?)±SE,n=7)。结果提示,NA引起DRG神经细胞膜电位的去极化或超极化反应,可能与膜的钾及钙通道活动的改变有关。     

Maturation of Xenopus oocytes: II. Observations on membrane potential

Large, progesterone-responsive oocytes within their follicles have an average resting potential of about ?25 mV. When manually dissected out of their follicles, most of these oocytes undergo a hyperpolarization over the next 30–60 min to values of about ?60 to ?80 mV. The relatively high negative membrane potentials previously recorded on dissected amphibian oocytes may thus be an artifact in the sense that such measurements do not reflect the electrical characteristics of the oocyte within the follicle. The available evidence indicates that the hyperpolarization reflects the activation of an electrogenic Na⁺,K⁺-transport process. One of the terminal events of oogenesis appears to be a suppression of the generation of the Na⁺,K⁺-transport process when oocytes are ovulated artificially (by dissection) or naturally. Continuous, long-term recordings on dissected oocytes reveal that a rather pronounced depolarization of the membrane potential together with an inflection in the recorded potential around the time of germinal vesicle breakdown takes place in the presence of progesterone. Recordings of oocytes within the follicle reveal similar changes, although reduced in absolute magnitude. In both cases, final membrane potentials of ?10 to ?15 mV are achieved. The electrophysiological changes which accompany the normal maturation process thus do not appear to be as pronounced as previously indicated.     

An electrical block is required to prevent polyspermy in eggs fertilized by natural mating of Xenopus laevis

In 27% DeBoer's saline (DBS), which yields maximum fertility rates, Xenopus eggs fertilized in vitro are monospermic, regardless of sperm concentration. One block to polyspermy (the “slow” block), described previously, occurs at the fertilization envelope that is elevated in response to the cortical reaction. This paper describes properties of an earlier, “fast” block at the plasma membrane and evaluates the functional significance of the two blocks at physiological sperm concentrations in natural mating conditions. Unfertilized eggs have a resting membrane potential of ?19 mV in 27% DBS. Fertilization triggers a rapid depolarization to +8 mV (the fertilization potential, FP); the potential remains positive for ca. 15 min. Activation of eggs with the ionophore, A23187, produces a slower but similar depolarization (the activation potential, AP). As in other amphibian eggs, the FP appears to result from a net efflux of Cl^?, since the peak of the FP (or the AP in ionophore-activated eggs) decreases as the concentration of chloride salts in the medium is increased. In 67% DBS no FP or AP is observed; eggs fertilized in 67% DBS become polyspermic and average 2 sperm entry sites per egg. In the 5–37 mM range, I^? and Br^?, but not F^?, are more effective than Cl^? in producing polyspermy. In 20 mM NaI the plasma membrane hyperpolarizes in response to sperm or ionophore; 100% levels of polyspermy and an average of 14 sperm entry sites per egg are observed. NaI does not inhibit or retard elevation of the fertilization envelope; the cortical reaction and fertilization envelope are normal in transmission electron micrographs. In 67% DBS, which also inhibits the fast block, the slow block was estimated to become functional 6–8 min after insemination. Eggs fertilized by natural mating in 20 mM NaI exhibit polyspermy levels of 50–90% and average 5 sperm entry sites per egg. Since eggs become polyspermic when fertilized by natural mating under conditions that inhibit the fast, but not the slow, block to polyspermy, we conclude that the fast block is essential to the prevention of polyspermy at the sperm concentrations normally encountered by the egg.     

Induction of a sodium-dependent depolarization by external calcium removal in human sperm

Removal of external calcium with EGTA (from 2.5 mm to nanomolar levels) caused a remarkable depolarization in human sperm. This depolarization was initially fast. It was followed by a slow phase that brought the Vm to values of over 0 mV in 1-2 min. The slow and sustained phase correlated with a sustained decrease in intracellular calcium. However, calcium removal still induced depolarization in sperm with enhanced intracellular calcium (induced by progesterone), indicating that the sustained depolarization was not caused by a sustained intracellular calcium decrease. The depolarization was reduced as the external sodium content was substituted with choline, indicating that it was due to a sodium current, and was observed in lithium but not in tetramethylammonium-containing medium. In low sodium medium, the addition of sodium after calcium removal induced depolarization to the extent of which slightly increased in 2 min. The depolarization was completely inhibited by external magnesium (Ki = 1.16 mm). The addition of calcium or magnesium to calcium removal-induced depolarized sperm induced hyperpolarization that was inhibited by ouabain and was also prevented in medium without potassium, suggesting that the activity of the electrogenic Na+,K+-ATPase was involved. The conductance activated by calcium removal might unveil the presence of a calcium channel that in the absence of external calcium allows sodium permeation and that in normal conditions might contribute to the resting intracellular calcium concentration.     

An electrically mediated block to polyspermy in the primitive urodele Hynobius nebulosus and phylogenetic comparison with other amphibians

At fertilization, the egg of the primitive urodele, Hynobius nebulosus, produced a fertilization potential which rose from -12 to +47 mV. A similar activation potential was elicited by pricking with a needle, by applying A23187, or by electric shock. The potential change was mediated by an increased permeability to Cl-. Clamping the egg's membrane potential at +40 mV blocked fertilization, while clamping at +20 mV induced polyspermy. These results indicated the occurrence of an electrical polyspermy block, typical of anurans, but atypical of urodeles. Furthermore, Hynobius eggs fertilized by natural mating incorporated only one sperm nucleus, and experimentally polyspermic eggs underwent multipolar division. Accessory sperm did not degenerate in the egg cytoplasm, indicating lack of an intracellular polyspermy block. By comparison, fertilization of Bufo japonicus (anuran) was also voltage dependent, whereas that of Cynops pyrrhogaster (urodele) was voltage independent. Thus polyspermy prevention mechanisms in Hynobius closely resemble those of anuran amphibians and differ from those of higher urodeles.     

The spike component of the fertilization potential in the toad, Bufo japonicus: changes during meiotic maturation and absence during cross-fertilization

Immature oocytes of the toad, Bufo japonicus, inseminated between first- and second-meiotic metaphase, exhibited polyspermy. Monospermy occurred when the oocytes had reached second-meiotic metaphase. Electrical recording during insemination of the immature oocyte showed fast-rising and slow-rising spikes followed by a gradual shift to a positive membrane potential. The number of fast spikes in each oocyte corresponded well with the number of sperm observed in cytological sections. Mature oocytes elicited one fast spike followed by a rapid rise to a positive plateau. Ion-substitution experiments indicated that, like the plateau, the initial fast spike is mediated mainly by increased permeability of the oocyte plasma membrane to halides such as Cl- or I-. When inseminated with sperm of the newt, Cynops pyrrhogaster, mature Bufo oocytes exhibited polyspermy accompanied by a gradual hyperpolarization and a slowly developing positive plateau, without the fast spike that occurs in self-species fertilization. These results indicated that the spike component of the fertilization potential can be dissociated from the plateau component, and may be elicited by different mechanisms.     

The Program of and Mechanisms of Fertilization in the Echinoderm Egg

Current research on the mechanisms of sperm-egg fusion, theblock to polyspermy, and metabolic activation are described.A cinemicrographic analysis of fertilization reveals that fusionof sperm and egg occurs between non-motile gametes, indicatingthat the flagellar motion of sperm is not required. The blockto polyspermy is reviewed, emphasizing recent work on the roleof cortical granule protease in altering sperm receptors ofthe vitelline layer. Metabolic activation or derepression at fertilization is highlyregulated and occurs in a definite sequence. The primary eventappears to be release of intracellular Ca²⁺. The timing of metabolicderepression is different in starfish oocytes. Here, a partof the derepression occurs during maturation and another partat fertilization.     

Mouse Sperm Membrane Potential Hyperpolarization Is Necessary and Sufficient to Prepare Sperm for the Acrosome Reaction

Mammalian sperm are unable to fertilize the egg immediately after ejaculation; they acquire this capacity during migration in the female reproductive tract. This maturational process is called capacitation and in mouse sperm it involves a plasma membrane reorganization, extensive changes in the state of protein phosphorylation, increases in intracellular pH (pH_i) and Ca²⁺ ([Ca²⁺]_i), and the appearance of hyperactivated motility. In addition, mouse sperm capacitation is associated with the hyperpolarization of the cell membrane potential. However, the functional role of this process is not known. In this work, to dissect the role of this membrane potential change, hyperpolarization was induced in noncapacitated sperm using either the ENaC inhibitor amiloride, the CFTR agonist genistein or the K⁺ ionophore valinomycin. In this experimental setting, other capacitation-associated processes such as activation of a cAMP-dependent pathway and the consequent increase in protein tyrosine phosphorylation were not observed. However, hyperpolarization was sufficient to prepare sperm for the acrosome reaction induced either by depolarization with high K⁺ or by addition of solubilized zona pellucida (sZP). Moreover, K⁺ and sZP were also able to increase [Ca²⁺]_i in non-capacitated sperm treated with these hyperpolarizing agents but not in untreated cells. On the other hand, in conditions that support capacitation-associated processes blocking hyperpolarization by adding valinomycin and increasing K⁺ concentrations inhibited the agonist-induced acrosome reaction as well as the increase in [Ca²⁺]_i. Altogether, these results suggest that sperm hyperpolarization by itself is key to enabling mice sperm to undergo the acrosome reaction.