Artificial induction of the acrosome reaction in human spermatozoa

This study investigated the use of human follicular fluid andpentoxifylline as inducers of the human sperm acrosome reactionin vitro. Motile sperm suspensions were prepared using a discontinuousPercoll gradient, preincubated for 3 h, divided into aliquotsand exposed to various concentrations of non-heart-inactivatedfollicular fluid for 1 and 24 h and pentoxifylline for 30 min.Detection of the acrosome reaction involved the combined useof a fluorescent vital stain, H33258 [GenBank] , and fluorescein isothiocyanate-conjugatedpeanut agglutinin (FITC-PNA). A short (1 h) exposure to follicularfluid at concentrations of 50% or more, did not compromise spermmotility and significantly increased the proportion of spermatozoahaving completed the acrosome reaction. Similarly, a 30 minexposure to pentoxifylline also significantly increased theproportion of spermatozoa having completed the acrosome reaction.     

The spontaneous acrosome reaction of human spermatozoa incubated in vitro

Motile human sperm populations were prepared from liquefiedsemen (10 donors x 3 replicates) using Percoll density gradientsat 30–60 min post-ejaculation. Sperm suspensions wereincubated in a complex ’synthetic tubal fluid‘ culturemedium (STF) at 37°C under 5% CO₂ in air for up to 36 h.Parallel aliquots were incubated with 50 µM A23187 toinduce maximum acrosome loss (AR_max). Acrosome reactions wereassessed using both the triple-stain (TS) technique and fluorescentpeanut agglutinin (PNA) lectin-labelling. During incubation,the proportion of TS acrosome reacted spermatozoa increasedfrom 9.1 to 54.3% with AR_max being 68.3%. Spermatozoa showingintact acrosomes by PNA labelling decreased from 68.4 to 26.1%over 36 h of incubation (AR_max = 13.8%). Simultaneously, spermatozoashowing complete acrosomal loss (no PNA labelling) increasedfrom 8.1 to 27.0% (AR_max= 46.3%). Therefore, while only 23.5%of cells were actually undergoing acrosomal changes at the startof incubation, this had increased to 46.9% after 36 h (AR_max=40.7%). These experiments clearly show that even in selectedpopulations, not all human spermatozoa are capable of undergoingan acrosome reaction. However, the incidence of acrosomal changesafter 36 h of incubation did approach the AR_max. These levelsof spontaneous occurrence of the human sperm acrosome reactionwere higher than those reported in many other in-vitro incubationstudies: an improvement that may be attributable to the morephysiological nature of the STF culture medium     

The acrosome reaction in boar spermatozoa

In order to gain more insight into the molecular alterationsof the acrosome, boar spermatozoa were incubated in a calcium-containingmedium in the presence of the lonophore A23187. The time-courseof the acrosome reaction was assessed by phase-contrast microscopy.Different stages of the acrosome reaction were studied by immunoelectronmicroscopy using a specific antibody directed against the completeouter acrosomal membrane. The introduction of monoclonal antibodiesgenerated by immunization of Balb/c mice with the isolated outeracrosomal membrane permitted a study of the topography of distinctmembrane proteins during the acrosome reaction. The exposureof activation of a fucose-binding protein important for sperm—zonaattachment was studied using neo-glycoproteins labelled withcolloidal gold for transmission electron microscopy.     

gamma-Aminobutyric acid (GABA) induces the acrosome reaction in human spermatozoa

The sperm acrosome reaction takes place in response to progesterone and zona pellucida. Progesterone may act on more than one type of surface receptor, of which one is a gamma-aminobutyric acid (GABA) type A-like receptor. Although there is direct evidence of GABA initiation of mouse sperm acrosome reaction, there are conflicting results regarding GABA- induced exocytosis in human spermatozoa. We have examined whether GABA would initiate exocytosis in human spermatozoa using the chlortetracycline assay and a zona-free hamster oocyte test. Human spermatozoa preincubated for > or = 3 h in Biggers-Whitten-Whittingham medium with 0.35% bovine serum albumin underwent acrosome reactions in response to GABA, with maximal responses in spermatozoa preincubated for 9 h. The effect was concentration-dependent. Preincubated spermatozoa treated with GABA were able to fertilize a higher proportion of zona-free oocytes, with a higher number of spermatozoa penetrating each oocyte. Exposure of preincubated spermatozoa to GABA and progesterone together resulted in a higher proportion of acrosome reactions than when each agonist was used alone. The effect of GABA was mediated by the influx of extracellular Ca2+ because inclusion of EGTA or the Ca2+ channel antagonist La3+ prevented GABA-induced acrosome reactions. These results indicate that GABA can initiate exocytosis in capacitated human spermatozoa and open up possibilities for studies of signalling mechanisms activated upon occupancy of the GABAA receptor present on the sperm surface.      

Effect of platelet-activating factor on motility and acrosome reaction of human spermatozoa

The effect of platelet-activating factor (PAF) on motility parametersand induction of the acrosome reaction in human spermatozoawas investigated in 36 unselected men with different degreesof initial sperm motility. The characteristics of sperm movementwere assessed by computer-assisted sperm analysis (Hamilton-ThornMotility Analyser) and the percentage of acrosome-reacted spermatozoawas evaluated after 1 h incubation with PAF (10 nM) and stainingwith fluorescent peanut lectin. We found that short-term (4h max) incubation with PAF significantly enhanced total andprogressive sperm motility as well as acrosome reaction. Anincrease of sperm motility in response to PAF was present in16 out of the 25 subjects studied (defined as responders) andwas inversely correlated with basal motility. In the 11 samples(six responders and five non-responders) where the incubationwith PAF was prolonged overnight, an increase of sperm motilitywas present in all the subjects studied. Similarly, an increasein numbers of acrosome reactions in response to 10 nM PAF waspresent in 20 out of the 26 subjects examined, and was inhibitedby the PAF receptor antagonist L659 989. Our results indicatea possible physiological role for PAF in fertilization and suggesta potential use of PAF in in-vitro fertilization techniquesin cases of reduced sperm motility.     

A comparison of three methods for detecting the acrosome reaction in human spermatozoa

This study was designed to compare three different fluorescentprobes to assay the acrosome reaction in human spermatozoa:chlortetracycline (CTC), mannosylated bovine serum albumin (BSA)labelled with fluorescein (MAF), and quinacrine (QN)- Normalhuman sperm ejaculates were washed and allowed to swim up for30–60 min. Samples were examined under epifluorescencefor the percentage of the acrosome reacted spermatozoa, as detectedby the three probes. There was no significant difference betweensamples of fresh, uncapadtated spermatozoa evaluated with CTC,MAF or QN; all gave <10% reacted. Following capacitationfor 3 h, the percentage of spontaneously reacted spermatozoawas higher than in fresh spermatozoa; CTC and MAF gave the samepercentage (12%), while QN indicated a higher percentage (18%)of reacted spermatozoa (P < 0.001). Following exposure toionophore A23187 at 1 h, the percentage of acrosome reactionsincreased to a mean of 31% as detected with CTC or MAF; themean percentage (45%) was significantly higher with QN (P <0.0001). Further incubation up to 2 h with A23187 did not changethese percentages. These results suggest that the QN probe detectsthe onset stage of the acrosome reaction, whereas the CTC andMAF probes detect the later stages in which the acrosomal capis lost. Use of the two types of probe provides a means forfiner resolution of the time course of the acrosome reactionin the human spermatozoa.     

Cryopreservation of human spermatozoa with pentoxifylline improves the post-thaw agonist-induced acrosome reaction rate

Cryopreservation causes extensive damage to spermatozoa, thereby impairing their fertilizing ability. The purpose of this study was to determine if the direct addition of pentoxifylline to the seminal plasma before cryopreservation improved sperm motility and acrosome reaction. Semen specimens from 15 healthy volunteers were divided into two aliquots. One aliquot was treated by adding 5 mM pentoxifylline directly to the seminal plasma (treatment group) and the other aliquot received no treatment (control group). Both aliquots were then cryopreserved by using the liquid nitrogen freezing method. The percentage of motile spermatozoa and various motion characteristics were then evaluated by performing computer-assisted semen analysis. The sperm viability was determined with a supra-vital dye, Hoechst-33258, and the acrosome reaction (spontaneous and calcium ionophore-induced) was monitored using fluorescein isothiocyanate-conjugated peanut lectin (FITC-PNA) binding assays. Pentoxifylline treatment significantly increased the sperm motility, the amplitude of lateral head displacement, the hyperactivation status, and the frequency of spontaneous acrosome reactions before freezing (P < 0.05). After post- thaw, no difference in motion characteristics (except percentage motility) between treated and control groups were observed. Acrosome loss due to the freeze-thaw process was less in the pentoxifylline- treated group (P = 0.0003). In addition, the percentage of cryopreserved acrosome-intact spermatozoa that underwent further acrosome reactions in response to calcium-ionophore challenge was significantly higher in the treated group (P = 0.03). Pentoxifylline treatment before freezing improved the acrosome reaction to ionophore challenge in cryopreserved spermatozoa. Treatment with pentoxifylline appears to minimize sperm damage during the freeze-thaw process and may improve fertilization rates with assisted reproductive procedures such as intrauterine insemination or in-vitro fertilization.      

Fertilization and early embryology: Determination of the acrosome reaction in human spermatozoa is predictive of fertilization in vitro

The acrosome reaction was determined in aliquots from ejaculatesof 74 patients undergoing in-vitro fertilization at the Universityof Giessen, Germany, by means of the triple-stain technique.The percentage of acrosome-reacted spermatozoa after low-temperatureinduction of the acrosome reaction was not significantly relatedwith the fertilization rate (H test, P = 0.693, SJ test, P =0.366). However, all patients showing < 13.0% acrosome-reactedspermatozoa had poor fertilization rates. Highly significantdifferences between patients could be detected by correlatingthe inducibility of the acrosome reaction with the fertilizationrate (H test, P = 0.018; SJ test, P = 0.004); patients withhigh fertilization rates showed a corresponding high inducibilityof acrosome reactions. From our results, it is evident thatpercentages of acrosome-reacted spermatozoa < 13.0% or aninducibility of the acrosome reaction of <7.5% are indicativeof subfertility.     

Effects of cryopreservation on progesterone-induced ion fluxes and acrosome reaction in human spermatozoa

The present study evaluated the effects of cryopreservation on progesterone-induced variations of calcium ion concentration [Ca(2+)](i), plasma membrane potential and acrosome reaction in human spermatozoa. Spermatozoa from 10 fertile donors were divided in two equivalent aliquots, one used as control (fresh spermatozoa) and the other used after freezing-thawing. Measurement of spermatozoa [Ca(2+)](i) before and after freezing-thawing showed a significant reduction of basal [Ca(2+)](i) in thawed spermatozoa (P < 0.01). Progesterone induced a rise of [Ca(2+)](i) both in fresh and thawed spermatozoa with a significant reduction after freezing-thawing (P < 0.01). The monitoring of sperm plasma membrane potential demonstrated that progesterone induced plasma membrane depolarization in fresh spermatozoa that was absent in thawed spermatozoa. The inhibitory effects of freezing-thawing on progesterone induced [Ca(2+)](i) and plasma membrane potential variations in human spermatozoa were closely related to the inhibition of the acrosome reaction. In conclusion the present study demonstrates that freezing-thawing procedures reduce the responsiveness of human spermatozoa to progesterone in terms of [Ca(2+)](i) rise and completely inhibit its effects on plasma membrane potential variations, thus supporting the hypothesis that freezing-thawing procedures may differently modify the plasma membrane receptors for progesterone in human spermatozoa which are known to express at least two receptors for this steroid in their plasma membrane.     

Recombinant human zona pellucida glycoprotein 3 induces calcium influx and acrosome reaction in human spermatozoa

Recombinant human ZP3 (rhuZP3) generated by Chinese hamsterovary cells transfected with a plasmid containing human ZP3cDNA was used to study the acrosome reaction (AR) and intracellularcalcium fluxes in capacitated human spermatozoa. Conditionedmedium containing rhuZP3 significantly induced the AR (P <0.005)in 59.4 ± 4.7% of spermatozoa (control = 8.5 ±3.1%) and caused complete acrosomal loss in a further 17.2 ±3.8% of cells (control = 3.7 ± 0.7%; mean ± SEM,n = 5). Sperm motility was not affected and acrosomal exocytosisin response to rhuZP3 was also shown to be time-dependent. Basalconcentrations of sperm intracellular calcium were measured(82 ± 7 nM; mean ± SEM, n = 9). A transient increasein intracellular calcium (typically up to 400–450 nM)occurred within 1 min of rhuZP3 addition and was followed bysustained lower values of calcium (200–400 nM). Theseresponses were dependent on the amount of rhuZP3. This is thefirst report of zona protein-induced changes in intracellularcalcium levels in human spermatozoa. The results support thepremise that ZP3 is an aganist of the human sperm AR and thatrhuZP3 generated in a eukaryotic cell is effective in this respect. acrosome reaction/calcium/human/spermatozoa/ZP3     

Induction of acrosome reaction in human spermatozoa used for subzonal insemination.

Human spermatozoa were injected into the perivitelline space of oocytes from 43 couples (44 cycles) in whom fertilization had failed in conventional in-vitro fertilization (IVF). The spermatozoa were treated to enhance the percentage of acrosome-free spermatozoa either by incubation for 24 h in T6 medium with 50% follicular fluid (v/v) or by incubation for 24 h in T6 medium followed by electroporation and incubation for a few hours in T6 medium with 3.5 mM pentoxifylline. After these two procedures, the mean percentage of acrosome-free spermatozoa increased to 35.5 and 53.9% respectively. Up to three spermatozoa were injected into the perivitelline space of metaphase II oocytes; few oocytes were damaged during the injection procedure. The overall fertilization rate was 30.9% of the 433 oocytes that were intact after subzonal insemination. Only 3% of the injected oocytes had more than two pronuclei. The cleavage rate of the fertilized oocytes was 80%. There was no difference in the fertilization and cleavage rates between the two sperm treatment procedures. One, two or three embryos were replaced in 34 cycles and seven patients became pregnant. In three of the four ongoing pregnancies, prenatal diagnosis by amniocentesis indicated a normal karyotype.     

Selection and characterization of human acrosome reacted spermatozoa

In order to study the characteristics of human acrosome reactedspermatozoa, we developed a method to select them. Spermatozoa,obtained from 20 fertile volunteers, were selected by usingGB24 antibody fixed on magnetic immunobeads, after acrosomereaction induced by 50% follicular fluid. Bead-bound spermatozoawere then detached using sheep anti-mouse IGG F(ab')2 antibody.This method allowed recovery of 170 ± 48x10³ spermatozoa(n = 20), free of GB24 antibody, as assessed by incubation withFITC-rabbit anti-mouse antibody. The percentage of acrosomereacted spermatozoa in the selected population was 88 ±3% versus 32 ± 6% in the whole sperm population. Concerningsperm morphology, the percentage of head abnormalities was lowered(15 ± 3% versus 20 ± 3%). The motility of selectedspermatozoa was dramatically reduced (7 ± 3% versus 53± 7% in the whole population) despite no difference inviability (84 ± 3% versus 80 ± 4%). However, theviability after an 18 h incubation was very low (1 ±0.5% versus 46 ± 5%). These results show that acrosomereaction occurs in the most morphologically normal spermatozoaand is followed by a loss in motility and a decrease in longevity.     

The incidence of spontaneous acrosome reaction in homogeneous populations of hyperactivated human spermatozoa.

The incidence of spontaneous acrosome reaction occurring in 1314 individually selected hyperactivated (HA) human spermatozoa was compared to that occurring in 8226 individually selected non-hyperactivated spermatozoa (non-HA) sampled over an incubation time course to allow for capacitation. Two-way analysis of variance showed a significant difference between HA and non-HA spermatozoa for the mean percent acrosome reacted (R), partially acrosome reacted (PR) and combined total (R+PR) (P < 0.001). One-way analysis showed that among the HA spermatozoa there were marked differences among the proportion showing R+PR at the various time points (P = 0.005). Using the same end point, there was no significant evidence of change with time for the non-HA spermatozoa. The overall data indicated that HA human spermatozoa have a greater propensity for spontaneous acrosomal loss than non-HA spermatozoa during incubation in synthetic culture media.     

The influence of human follicular fluid on the acrosome reaction, fertilizing capacity and proteinase activity of human spermatozoa

The present study was designed to assess physiological and enzymatic changes in human spermatozoa following incubation in human follicular fluid (HFF). Initially, it was determined that infertility patients (n = 29) scored dramatically higher on the hamster egg penetration test (HEPT) when spermatozoa were incubated with HFF (22.9 +/- 4.4%) rather than the standard swim-up alone (4.6 +/- 1.1%). To further evaluate this effect, in experiment I, spermatozoa were obtained from proven fertile individuals and evaluated following exposure to three different HFF samples as well as control treatments (medium, cul de sac fluid and heparin). There were no significant differences in HEPT scores following sperm incubation in the three different follicular fluids although incubation in all the fluids significantly (P less than 0.01) enhanced sperm penetration (%PEN) when compared to the standard swim-up and other control treatments. The absence of an effect of cul de sac fluid on spermatozoa indicates that not all body fluids contain factors which stimulate sperm fertilizing capacity. The effect of HFF was demonstrated in a infertile patient population as well as in donors of proven fertility. In experiment II, the effect of HFF on the acrosome reaction (%AR), sperm fertilizing capacity and changes in sperm proteolytic enzymes were determined. There was no significant difference in the %AR between freshly ejaculated (7.9 +/- 3.1) and medium incubated (9.4 +/- 1.6) spermatozoa; however, in both of these treatments the %AR was less (P less than 0.01) than for spermatozoa treated with HFF (45.6 +/- 4.7). The %PEN following incubation of spermatozoa in HFF (52.2 +/- 6.8) was greatly increased (P +/- 0.01) compared to the standard swim-up (19 +/- 3.9).     

The role of follicular fluid in inducing the acrosome reaction of human spermatozoa incubated in vitro

Motile human sperm populations were prepared from liquefiedsemen (5 donors x 3 replicates) using Percoll gradients at 30–60 min post-ejaculation and preincubated in a complex ‘syntheitictubal fluid’ culture medium (STF) at 37° C under 5%CO₂ in air for 6h. Aliquots of these suspensions were then incubatedfor a further 2 h in STF containing 0, 5, 25, 50, 75 and 100%(v/v) pooled human follicular fluid (FF). Another aliquot wastreated with 10 µm A23187 in STF for 20min and then incubatedin fresh STF medium for a futher 2h to induce maximal acrosomeloss. Acrosome reactions were assessed using both the triple-staintechnique and fluorescent peanut agglutinin lectin-labelling.Sperm motility and movement characteristics were assessed fromvideorecordings using digital image analysis (CellSoft). Exposureto FF caused only relatively small proportions of the preincubatedspermatozoa to undergo acrosome reactions. The size of theseresponsive sub-populations was smaller than that capable ofresponding to a Ca²⁺ influx generated by A23187. Increased FFconcentrations induced a progressive loss of motility and trendsfor changes in movement characteristics that may have been relatedto reduced intracellular Ca²⁺. This interpretation of theseobervations is that while FF may act to stimulate or promotethe human sperm acrosome reaction it does not appear to be aspecific inducer of it. Consequently, a precise role for FFat the relatively low concentrations that would be expectedto be present in the tubal ampulla in the physiological regulationof human fertilization remains unproven     

Enhancement of motility and acrosome reaction in human spermatozoa: differential activation by type-specific phosphodiesterase inhibitors

Inhibition of sperm phosphodiesterase (PDE) has been shown to increase cAMP concentrations and stimulate motility and the acrosome reaction. While several PDE genes exist in mammals, little is known about the physiological role of PDE forms expressed in human spermatozoa. Using type-selective inhibitors, we identified two of the PDE forms expressed in human spermatozoa and studied their involvement in sperm function. Selective inhibitors of calcium-calmodulin-regulated PDE1 (8-methoxy- isobutyl-methylxanthine) and cAMP-specific PDE4 (RS-25344, Rolipram) were used to study PDE forms in human sperm extracts. 8-MeIBMX and Rolipram/RS-25344 inhibited sperm PDE activity by 35-40 and 25-30% respectively. Subcellular fractionation of the sperm homogenate suggests these pharmacologically distinct forms may be located in separate cellular regions. To evaluate the functional significance of different PDE forms, the effect of type-specific PDE inhibition on sperm motility and the acrosome reaction was examined. PDE4 inhibitors enhanced sperm motility over controls without affecting the acrosome reaction, while PDE1 inhibitors selectively stimulated the acrosome reaction. These data indicate at least two distinct PDE types exist in human spermatozoa. Our findings also support the hypothesis that PDE subtypes affect sperm function by regulating separate pools of cAMP and may ultimately offer novel treatments to infertile couples with abnormal semen parameters.      

A simple method for assessment of the human acrosome reaction of spermatozoa bound to the zona spellucida: lack of relationship with ionophore A23187-induced acrosome reaction

Acrosome reactions induced by the calcium ionophore A23187 [GenBank] andzona pellucid a (ZP) were studied. Sperm samples were obtainedfrom fertile men or men with normal semen analysis and normalsperm-ZP binding. Oocytes were obtained, with the consent ofthe patients, after the failure of fertilization in vitro. Motilespermatozoa selected by a swim-up technique were incubated with10 µM A23187 [GenBank] for 1 h, four oocytes for 2 h or solubilizedZP (4 ZP/µl) for 2 h. Spermatozoa bound to the ZP weredislodged and collected in a small volume of phosphate-bufferedsaline by aspirating the oocytes with a glass pipette with aninner diameter (120 µm) slightly smaller than the diameterof the oocyte. The acrosome status of the spermatozoa was determinedusing fluorescein-labelled Pisum sativum agglutinin. The proportionof spermatozoa undergoing the acrosome reaction on the ZP at2 h varied over a wide range (5–99%), but the agreementbetween results for the same semen sample exposed to differentgroups of oocytes was good: the standard deviations of the differencesbeing 9%. Pre-incubation of spermatozoa for 2 h did not increasethe ZP-induced acrosome reaction. Re-incubation of ZP with thesame sperm suspension for 2 h after removing ZP-bound spermatozoafrom the first 2 h incubation produced a significantly lowerZP-induced acrosome reaction in the second incubation (22 ±16%) than in the first incubation (30 ± 14%; P < 0.001,n = 20). There was no significant difference in the ZP-inducedacrosome reaction with oocytes with ZP which had or had notbeen penetrated by spermatozoa during the in-vitro fertilizationinsemination. Pre-incubation of spermatozoa with solubilizedZP blocked sperm-ZP binding. However, the acrosome reactioninduced by solubilized ZP (4 ZP/µl) was significantlylower than the acrosome reaction induced by intact ZP (10 ±5 and 30 ± 13% respectively, n = 11, P < 0.001), butthere was a high correlation (Spearman r = 0.822, P < 0.01)between the results. On the other hand, although the averageof the acrosome reaction was similar for A23187 [GenBank] (42%) and forZP (43%), there was no significant correlation between the resultsfor the two stimuli (n = 60). In conclusion, a useful methodfor assessing the ZP-induced acrosome reaction has been developedusing oocytes which failed to fertilize in vitro. The lack ofa relationship between the results of the chemical (A23187 [GenBank] )and physiological (ZP) stimali for the acrosome reaction inthe same subjects questions the biological basis of using A23187 [GenBank] for tests of sperm function. Solubilized human ZP in a concentrationthat blocks sperm-ZP binding is a less efficient inducer ofthe acrosome reaction than is intact ZP. It is possible thatthe three-dimensional structure of the ZP is important for inductionof the acrosome reaction or that spermatozoa which bind to theZP are more likely to acrosome react Assessment of the physiologicalacrosome reaction for diagnosis of sperm defects which interferewith the fertilization process should be concentrated on thespermatozoa which are capable of binding to the ZP.     

Intracellular calcium store depletion and acrosome reaction in human spermatozoa: role of calcium and plasma membrane potential

We evaluated the presence and role of internal calcium stores in human uncapacitated spermatozoa by determining the effects of two inhibitors of Ca2+ ATPase of the sarco-endoplasmic reticulum (SERCA-ATPase), thapsigargin and cyclopiazonic acid (CPA) on intracellular calcium concentrations, [Ca2+](i), plasma membrane potential and acrosome reaction. Using a fluorescent conjugate of thapsigargin, we localized internal Ca2+ stores on the acrosome, post-acrosomal region and sperm midpiece. SERCA-ATPase inhibitors induced a rise in [Ca2+](i) both in Ca2+ and Ca2+-free media but under these latter conditions it was reduced with a progressive decline to baseline values; the re-addition of Ca2+-stimulated a rise in [Ca2+](i). This demonstrated that internal Ca2+ store depletion can evoke the opening of Ca2+-channels on sperm plasma membrane, thus showing the existence of "capacitative" Ca2+ entry into these specialized cells. The addition of thapsigargin to human spematozoa induced a dose-dependent increase in acrosome reaction percentages, but only when Ca2+ was present in the external medium. Plasma membrane potential monitoring showed that these inhibitors induced a depolarization dependent on Ca2+ influx from external medium and that this was preceded by a transient hyperpolarization caused by activation of Ca2+-dependent K+ channels. When K+-dependent plasma membrane hyperpolarization was inhibited, the thapsigargin- and CPA-stimulated rise in [Ca2+](i) plasma membrane depolarization and acrosome reaction were abolished. In conclusion, the present study demonstrates that human spermatozoa possess internal Ca2+ stores and that the capacitative Ca2+ entry pathway present in these cells regulates important biological processes that are fundamental for the acrosome reaction.