Mouse gamete interactions: The zona pellucida is the site of the acrosome reaction leading to fertilization in vitro

The question of whether the acrosome reaction, which leads to fertilization, occurs in intact sperm bound to the zona pellucida of the egg or in intact sperm before contact with the egg, was addressed by assessing the effect of 3-quinuclidinyl benzilate (QNB) on the two types of acrosome reaction. QNB is a specific inhibitor of the fertilization of zona-intact mouse eggs by mouse sperm. Mouse spermatozoa in suspension underwent acrosome reactions at a low rate, which could be accelerated by addition of 5 μM divalent cation ionophore A23187; the occurrence of such acrosome reactions was not inhibited by QNB. The rate at which acrosome reactions occurred in sperm bound to the zona pellucida of cumulus-free eggs, bound to isolated zonae, or exposed to acid-solubilized zona components, was greatly accelerated relative to that observed in the absence of zonae. These acrosome reactions were strongly inhibited by QNB at concentrations which inhibit the fertilization of zona-intact mouse eggs in vitro. These data suggest that the zona pellucida can induce acrosome reactions in mouse spermatozoa and that these acrosome reactions are the ones which lead to the fertilization of zona-intact eggs. In contrast, the acrosome rection in sperm which are not in contact with the zona is not associated with fertilization of zona-intact eggs.     

Mouse gamete interactions during fertilization in vitro. Chlortetracycline as a fluorescent probe for the mouse sperm acrosome reaction

We have developed an assay for detecting the acrosome reaction in mouse sperm using chlortetracycline (CTC) as a fluorescent probe. Sperm known to be intact with nonreacted acrosomes show CTC fluorescence in the presence of Ca2+ over the anterior portion of the sperm head on the plasma membrane covering the acrosome. Sperm which have undergone the acrosome reaction do not show fluorescence on the sperm head. Mouse sperm bind to zonae pellucidae of cumulus-free eggs in vitro in a Ca2+-dependent reaction; these sperm are intact by the CTC assay. Intact sperm bind to mechanically isolated zonae under the same conditions: the egg is apparently unnecessary for this inital reaction. Sperm suspensions, in which greater than 50% of the motile population had completed the acrosome reaction, were prepared by incubation in hyperosmolal medium followed by treatment with the divalent cation ionophore, A23187. Cumulus-free eggs challenged with such sperm suspensions preferentially bind intact sperm; acrosome-reacted sperm do not bind. We conclude that the plasma membrane of the mouse sperm is responsible for recognition of the egg's zona pellucida and that the obligatory sequence of reactions leading to fusion of mouse gametes is binding of the intact sperm to the zona pellucida, followed by the acrosome reaction at the zona surface, followed in turn by sperm penetration of the zona.     

Kinetics of onset of mouse sperm acrosome reaction induced by solubilized zona pellucida: fluorimetric determination of loss of pH gradient between acrosomal lumen and medium monitored by dapoxyl (2-aminoethyl) sulfonamide and of intracellular Ca(2+) changes monitored by fluo-3

The onset of the zona pellucida-induced acrosome reaction in mouse sperm is marked by loss of the pH gradient existing in acrosome-intact sperm between the acidic acrosomal lumen and the suspending medium, due to pore formation between outer acrosomal and plasma membranes. In earlier work, it was shown that this pH gradient loss occurred in single sperm bound to structurally intact zonae pellucidae with a half-time of 2.1 min; the extended kinetics of this loss determined in a sperm population bound to intact zonae was due to a 180-min range of variable lag times. We hypothesized that this lag time range was due to steric constraints imposed by the three-dimensional structure of the structurally intact zona pellucida, and that this constraint should be removed in solubilized zonae. The fluorescent probe, Dapoxyl(TM) (2-aminoethyl)sulfonamide (DAES) allowed a test of this hypothesis in a population of sperm cells. It is a weak base that is non-fluorescent in aqueous solution, but which accumulates in the acidic acrosomal compartment due to the pH gradient with highly enhanced fluorescence; loss of the pH gradient leads to a decrease in fluorescence. The half-time for DAES fluorescence loss in a population of capacitated, acrosome-intact sperm in response to solubilized zona pellucida protein was 2.13 +/- 0.10 min (SEM, n = 9). The agreement between single cell and cell population kinetics validates the hypothesis of steric constraint in the structurally intact zona pellucida. The change in intracellular Ca(2+) concentration in response to solubilized zona pellucida, as monitored with fluo-3, was a rapid increase, followed by a decrease, with a half-time of 0.85 +/- 0.09 min (SEM, n = 6) to a steady state level higher than the initial level, indicating this Ca(2+) transient as the precursor reaction to onset of the zona-induced acrosome reaction.     

Calcium influx into mouse spermatozoa activated by solubilized mouse zona pellucida,monitored with the calcium fluorescent indicator,fluo-3. inhibition of the influx by three inhibitors of the zona pellucida induced acrosome reaction: Tyrphostin A48, pertussis toxin,and 3-quinuclidinyl benzilate

The fluorescent calcium indicator, fluo-3, was loaded as the membrane permeant tetraacetoxymethyl (AM) ester into cauda epididymal mouse sperm at 25°C for 20 min in the absence of bovine serum albumin (BSA) and presence of the dispersant, Pluronic F-127. Excess indicator was removed by two centrifugation washes at 100g for 10 min, a procedure that did not impair sperm motility. Upon resuspension in medium containing 20 mg/ml BSA to promote capacitation, the sperm cells exhibited readily detectable fluorescence uniformly distributed in the cytoplasm. Cell fluorescence was stable over the time of the experiments and was responsive to changes in intracellular calcium concentration, [Ca²⁺]_j. Initial [Ca²⁺]_j was 231 ± 58 nM (±SE, n = 43). Addition of heat-solubilized mouse zonae pellucidae to capacitated sperm increased [Ca²⁺]_j by 106 ± 19 nM (±SE, n = 18), the higher steady-state concentration being reached after 30 min. Subsequent addition of the non-fluorescent calcium ionophore Br-A23187 resulted in a further increase of 114 ± 18 nM (± SE, n = 18), the higher steady-state concentration being reached after 6 min. The increase in [Ca²⁺]_j induced by solubilized zonae pellucidae was largely blocked by 3-quinuclidinyl benzilate (QNB) an antagonist of muscarinic receptors that was earlier shown to block the zona pellucida induced acrosome reaction in mouse sperm (Florman and Storey, 1982: Dev Biol 91:121–130). This [Ca²⁺]_j increase was completely blocked by the tyrosine kinase inhibitor, tyrphostin A48, and by the inactivator of G₁ proteins, pertussis toxin. At the concentrations at which they blocked the zona pellucida-induced increase in [Ca²⁺]_j all three inhibitors also blocked the zona pellucidainduced acrosome reaction. These results indicate that [Ca²⁺]_j increase in is an early, if not the initial, reaction in the sequence leading to zona pellucida induced acrosomal exocytosis in mouse sperm. The observation that the three inhibitors, each having a different mode of action, all block the zona pellucida induced [Ca²⁺]_j suggests that the sperm plasma membrane receptors mediating the zona pellucida induced acrosome reaction may function as a complex, whose formation is activated by zona pellucida ligand binding. © 1994 Wiley-Liss, Inc.     

Evidence for plasma membrane impermeability to small ions in acrosome-intact mouse spermatozoa bound to mouse zonae pellucidae, using an aminoacridine fluorescent pH probe: time course of the zona-induced acrosome reaction monitored by both chlortetracycline and pH probe fluorescence

Previous studies have shown that capacitated mouse spermatozoa bind to zonae pellucidae of mouse eggs with acrosomes apparently intact. The question addressed in this study was the following: are the membrane permeability barriers of the apparently acrosome-intact sperm still retained or is there a preliminary stage of the acrosome reaction in which these barriers are lost and the intracellular space becomes accessible to extracellular substrates? The experimental approach was to use the fluorescent pH probe 9-amino-3-chloro-7-methoxyacridine, which accumulates in intracellular spaces of lower pH than the suspending medium with the result that the cells become fluorescent. Freshly capacitated mouse spermatozoa bound to isolated zonae showed uniform fluorescence over the head and midpiece with this fluorescent probe at early times of binding. The fluorescence was abolished by NH4+ and nigericin, agents that equilibrate H+ across cell membranes. At these early times of binding, the acrosomes were fully intact as judged by chlortetracycline fluorescence pattern, which itself was unaffected by either N4+ or nigericin. The time course of the loss of this chlortetracycline pattern characteristic of acrosome-intactness was closely paralleled by loss of fluorescence of 9-amino-3-chloro-6-methoxyacridine over the first 90 min; thereafter, loss of the chlortetracycline pattern was somewhat more rapid. This result shows that acrosome-intact sperm bound to zonae pellucidae retain the permeability barriers of the plasma membrane to small cations; no evidence was found for an early stage of membrane "leakiness" preceding the acrosome reaction. The ionophore A23187 induced a very rapid acrosome reaction in sperm bound to isolated zonae, as judged with both fluorescence probes. This rapid reaction was partially inhibited by 3-quinuclidinyl benzilate, which, in the absence of ionophore, completely blocks the occurrence of the acrosome reaction in sperm bound to zonae. This suggests involvement of a specific calcium entry mechanism in the acrosome reaction of mouse sperm induced by mouse zonae pellucidae.     

Phospholipase Cdelta4 is required for Ca2+ mobilization essential for acrosome reaction in sperm

Zona pellucida (ZP)-induced acrosome reaction in sperm is a required step for mammalian fertilization. However, the precise mechanism of the acrosome reaction remains unclear. We previously reported that PLCdelta4 is involved in the ZP-induced acrosome reaction in mouse sperm. Here we have monitored Ca2+ responses in single sperm, and we report that the [Ca2+]i increase in response to ZP, which is essential for driving the acrosome reaction in vivo, is absent in PLCdelta4-/- sperm. Progesterone, another physiological inducer of the acrosome reaction, failed to induce sustained [Ca2+]i increases in PLCdelta4-/- sperm, and consequently the acrosome reaction was partially inhibited. In addition, we observed oscillatory [Ca2+]i increases in wild-type sperm in response to these acrosome inducers. Calcium imaging studies revealed that the [Ca2+]i increases induced by exposure to ZP and progesterone started at different sites within the sperm head, indicating that these agonists induce the acrosome reaction via different Ca2+ mechanisms. Furthermore, store-operated channel (SOC) activity was severely impaired in PLCdelta4-/- sperm. These results indicate that PLCdelta4 is an important enzyme for intracellular [Ca2+]i mobilization in the ZP-induced acrosome reaction and for sustained [Ca2+]i increases through SOC induced by ZP and progesterone in sperm.     

The acrosomal vesicle of mouse sperm is a calcium store

Subsequent to binding to the zona pellucida, mammalian sperm undergo a regulated sequence of events that ultimately lead to acrosomal exocytosis. Like most regulated exocytotic processes, a rise in intracellular calcium is sufficient to trigger this event although the precise mechanism of how this is achieved is still unclear. Numerous studies on mouse sperm have indicated that a voltage-operated Ca2+ channel plays some immediate role following sperm binding to the zona pellucida glycoprotein ZP3. However, there is also evidence that the mammalian sperm acrosome contains a high density of IP3 receptors, suggesting that the exocytotic event involves the release of Ca2+ from the acrosome. The release of Ca2+ from the acrosome may directly trigger exocytosis or may activate store-operated Ca2+ channels in the plasma membrane. To test the hypothesis that the acrosome is an intracellular store we loaded mammalian sperm with the membrane permeant forms of three Ca2+-sensitive fluorescent indicator dyes: fura-2, indo-1, and Calcium Green-5N. Fluorescence microscopy revealed that the sperm were labeled in all intracellular compartments. When fura-2 labeled sperm were treated with 150 microM MnCl2 to quench all fluorescence in the cytosol, or when the sperm were labeled with the low affinity dye Calcium Green-5N, there was a large Ca2+ signal in the acrosome. Consistent with the acrosome serving as an intracellular Ca2+ reservoir, the addition of 20 microM thapsigargin, a potent inhibitor of the smooth endoplasmic reticular Ca2+-ATPase (SERCA), to populations of capacitated sperm resulted in nearly 100% acrosomal exocytosis within 60 min (tau1/2 approximately 10 min), in the absence of extracellular Ca2+. Additionally, treatment of sperm with 100 microM thimerosal, an IP3 receptor agonist, also resulted in acrosomal exocytosis. Taken together, these data suggest that the mouse sperm acrosome is a Ca2+ store that regulates its own exocytosis through an IP3 Ca2+ mobilization pathway.     

Maitotoxin potently promotes Ca2+ influx in mouse spermatogenic cells and sperm, and induces the acrosome reaction

Maitotoxin (MTX), a potent marine toxin, activates Ca2+ entry via nonselective cation channels in a wide variety of cells. The identity of the channels involved in MTX action remains unknown. In mammalian sperm, Ca2+ entry through store-operated channels regulates a number of physiological events including the acrosome reaction (AR). Here we report that MTX produced an increase in the intracellular concentration of Ca2+ ([Ca2+]i) in spermatogenic cells that depended on extracellular Ca2+. Ni2+ and SKF96365 diminished the MTX-activated Ca2+ uptake, at concentrations they inhibit store-operated channels, and in a similar manner as they inhibit the Ca2+ influx activated following depletion of intracellular stores by thapsigargin (Tpg). In addition, MTX significantly increased [Ca2+]i in single mature sperm and effectively induced the AR with a half-maximal concentration (ED50) of approximately 1.1 nM. Notably, SKF96365 similarly inhibited the MTX-induced increase in sperm [Ca2+]i and the AR triggered by the toxin, Tpg and zona pellucida. These results suggest that putative MTX-activated channels may be involved in the Ca2+ influx required for mouse sperm AR.     

Aggregation of beta-1,4-galactosyltransferase on mouse sperm induces the acrosome reaction

beta-1,4-Galactosyltransferase (GalTase) is present on the surface of mouse sperm, where it functions during fertilization by binding to oligosaccharide residues in the egg zona pellucida. The specific oligosaccharide substrates for sperm GalTase reside on the glycoprotein ZP3, which possesses both sperm-binding and acrosome reaction-inducing activity. A variety of reagents that perturb sperm GalTase activity inhibit sperm binding to the zona pellucida, including UDP-galactose, N-acetylglucosamine, alpha-lactalbumin, and anti-GalTase Fab fragments. However, none of these reagents are able to cross-link GalTase within the membrane nor are they able to induce the acrosome reaction. On the other hand, intact anti-GalTase IgG blocks sperm-zona binding as well as induces the acrosome reaction. Anti-GalTase IgG induces the acrosome reaction by aggregating GalTase on the sperm plasma membrane, as shown by the inability of anti-Gal-Tase Fab fragments to induce the acrosome reaction unless cross-linked with goat anti-rabbit IgG. These data suggest that zona pellucida oligosaccharides induce the acrosome reaction by clustering GalTase on the sperm surface.     

Ca(2+) entry through store-operated channels in mouse sperm is initiated by egg ZP3 and drives the acrosome reaction

Fertilization occurs after the completion of the sperm acrosome reaction, a secretory event that is triggered during gamete adhesion. ZP3, an egg zona pellucida glycoprotein, produces a sustained increase of the internal Ca(2+) concentration in mouse sperm, leading to acrosome reactions. Here we show that the sustained Ca(2+) concentration increase is due to the persistent activation of a Ca(2+) influx mechanism during the late stages of ZP3 signal transduction. These cells also possess a Ca(2+) store depletion-activated Ca(2+) entry pathway that is open after treatment with thapsigargin. Thapsigargin and ZP3 activate the same Ca(2+) permeation mechanism, as demonstrated by fluorescence quenching experiments and by channel antagonists. These studies show that ZP3 generates a sustained Ca(2+) influx through a store depletion-operated pathway and that this drives the exocytotic acrosome reaction.     

An ultrastructural study of epididymal mouse spermatozoa binding to zonae pellucidae in vitro: sequential relationship to the acrosome reaction

Mouse sperm bind to the zona pellucida of the egg prior to penetration of the zona and entry into the perivitelline space. The question then arises: when does the acrosome reaction occur relative to these processes? An ultrastructural study of mouse epididymal sperm bound to the surface of the zona and in the privitelline space was undertaken to clarify this point. Cumulus-free mouse eggs were inseminated in either a complete defined culture medium capable of supporting in vitro fertilization or in Tris/NaCl buffer containing Ca+2. Both media support sperm binding to the zona to the same extent; binding is complete in 15 minutes. Unbound sperm were removed by a step gradient density centrifugation to yield a preparation of eggs with sperm firmly bound. All sperm in the perivitelline space had undergone the acrosome reaction. Sperm bound at the surface of the zonae pellucidae of eggs recovered at ten minutes after insemination all had intact acrosomes. At 40 minutes after insemination, half of the sperm were intact; the other half were in the initial stages of the acrosome reaction. At 90 minutes after insemination, 12% of the sperm had undergone the full acrosome reaction and were starting to penetrate the zona; of the balance, half were in various stages of the acrosome reaction, while half were still intact. These findings support the hypothesis that the sequence of the early reactions leading to fertilization in the mouse is: intact sperm binding to zona; acrosome reaction at the zona surface; penetration of the zona.     

In vitro studies of the golden hamster sperm acrosome reaction: completion on the zona pellucida and induction by homologous soluble zonae pellucidae

We have studied the occurrence of the golden hamster sperm acrosome reaction (AR) in vitro during interaction with the oocyte investments: the cumulus cell matrix and the zona pellucida. Hamster sperm were capacitated in a defined medium that does not induce the AR. These spermatozoa were allowed to interact with the ovum vestments, the events of which were recorded using high-speed videomicrography. Frame-by-frame analysis revealed that sperm did not complete the AR in the cumulus cell matrix, but did so on the zona pellucida. Furthermore, a higher percentage of sperm completed the AR on the zona pellucida of cumulus-invested than on cumulus-free eggs. We also investigated the effect of solubilized hamster and mouse zonae pellucidae on the hamster sperm AR. Addition of solubilized hamster zonae to capacitated sperm elicited the AR within 15 min. Solubilized mouse zonae were significantly less effective, indicating that the zona-induced AR in hamster sperm may be species specific. These results suggest that the hamster zona pellucida is an inducer of the AR in the intact or soluble form, and that the majority of spermatozoa traverse the cumulus cell matrix without completing the AR in our in vitro system.     

Regulating the acrosome reaction

The acrosome reaction is a secretory event that must be completed by the sperm of many animal species prior to fusion with eggs. In mammals, exocytosis in triggered by ZP3, a glycoprotein component of the egg pellucida, following gamete contact. ZP3 promotes a sustained influx of Ca2+ into sperm that is necessary for the acrosome reaction. Here, we discuss the mechanism by which ZP3 generates Ca2+ entry, as well as the upstream events leading to this influx and downstream processes that couple it with exocytosis.     

Differential sensitivity of progesterone- and zona pellucida-induced acrosome reactions to pertussis toxin

Pertussis toxin-sensitive guanine nucleotide-binding regulatory proteins (G proteins) have previously been shown to mediate the zona pellucida-induced acrosome reaction in mammalian sperm. In this study we compared the inhibitory effect of pertussis toxin on the zona-induced acrosome reaction in human spermatozoa with that on the reaction induced by progesterone, another physiological acrosome reaction-promoting stimulus associated with the ovulated oocyte. Up to the concentration of 1 μg/ml, pertussis toxin did not produce any direct effects on the acrosome reaction frequency nor did it influence sperm movement and viability. However, preincubation of spermatozoa with the toxin at a concentration of 100 ng/ml completely abolished the increase in the acrosome reaction frequency upon subsequent exposure to solubilized zona pellucida material. In contrast, the same treatment did not impair the ability of spermatozoa to initiate the acrosome reaction in response to progesterone. Moreover, the preincubation with pertussis toxin did not modify the changes in the intracellular concentration of calcium ions occurring after progesterone addition. These data suggest that different physiological stimuli may utilize different signal transduction pathways to induce the human sperm acrosome reaction. © 1993 Wiley-Liss, Inc.     

Mouse sperm antigens that participate in fertilization. IV. A monoclonal antibody prevents zona penetration by inhibition of the acrosome reaction

To investigate the molecular basis of gamete interaction in mammals, monoclonal antibodies (mAbs) have been generated by syngeneic immunization with mouse testis. Previous work has described two particular mAbs, M41 and M42, which localize indistinguishably to the plasma membrane overlying a restricted portion of the acrosome, but recognize different antigens. One of the mAbs, M42, inhibits mouse fertilization in vitro significantly, but only in the presence of the zona pellucida, whereas M41 has no apparent effect upon any assayable event in the fertilization process. The experiments described here were performed to identify the precise event of sperm-zona interaction (sperm-zona binding, induction of the acrosome reaction, or penetration through the zona) that is affected by M42 mAb. Capacitated mouse sperm binding to the zona pellucida was undiminished following pretreatment with M42 mAb, when compared to levels achieved using either no mAb- or to M41 mAb-treated control sperm. When the effect of mAbs on the zona-induced AR was examined, the percentage of acrosome reacted (AR) sperm at the zona surface increased with time, plateauing at approximately 90 min post-insemination, with 78% of the bound cells AR in the control and the M41 mAb-treated groups. M42-treated sperm never achieved greater than 23% AR cells over the 120-min interval assayed. To quantitate this effect, capacitated sperm were exposed to increasing concentrations of acid-solubilized zonae. Increased proportions of AR sperm were found in the control and M41 mAb-treated groups, up to a maximum of 70-76% AR cells with 8 or 12 zonae/microliter. In contrast, M42-treated sperm displayed only 21-28% AR cells over the entire range of zonae concentrations tested. An entirely different result emerged when acrosome reactions were induced with A23187: M42 was no longer able to prevent the AR. This ability of A23187 to override M42 mAb's inhibitory effect on the AR permitted specific examination of the possible effect of M42 mAb on sperm penetration through the zona pellucida. In the presence of A23187, zona penetration levels for M42 mAb-treated sperm were equivalent, both qualitatively and quantitatively, to control and to M41 mAb-treated sperm under the same conditions. It appears, therefore, that M42 mAb identifies a high molecular weight doublet (220-240 kDa) of mouse sperm that participates specifically in the induction of the sperm's acrosome reaction as it occurs under physiological conditions.     

Sperm-egg interactions in the mouse: sequence of events and induction of the acrosome reaction by a zona pellucida glycoprotein

In this investigation, the interaction of mouse sperm with unfertilized eggs and embryos, solubilized zonae pellucidae isolated from eggs and embryos, and purified zona pellucida glycoproteins ZP1, 2, and 3 (J. D. Bleil, and P. M. Wassarman, (1980b) Dev. Biol. 76, 185-202) has been examined in vitro by light and electron microscopy. The experiments described were carried out in order to determine the temporal sequence of events during sperm-egg interaction in vitro and to identify the component(s) of zonae pellucidae responsible for inducing mouse sperm to undergo the acrosome reaction. "Pulse-chase" analysis of the sequence of sperm-egg interactions revealed that mouse sperm first "attach" loosely and then "bind" tightly to the unfertilized egg's zona pellucida. Binding of sperm to egg zonae pellucidae is followed by induction of the acrosome reaction. Induction of the acrosome reaction can be mediated by the zona pellucida, since solubilized zonae pellucidae isolated from unfertilized eggs were found to be just as effective as the calcium ionophore A23187 in inducing the reaction in vitro. Furthermore, ZP3 purified from zonae pellucidae isolated from unfertilized eggs, but not from two-cell embryos, was also just as effective as either solubilized zonae pellucidae from eggs or ionophore A23187 in inducing the acrosome reaction. ZP1 and 2 from both eggs and embryos, and ZP3 from embryos, had little effect on the extent of the acrosome reaction as compared to control samples. The results of these and other experiments (J. D. Bleil, and P. M. Wassarman, (1980b) Cell 20, 873-882) strongly suggest that, at least in vitro, mouse sperm recognize and bind to ZP3 of egg zonae pellucidae, and that such binding leads to the induction of the acrosome reaction. Modification of ZP3 following fertilization eliminates sperm binding to zonae pellucidae and, consequently, induction of the acrosome reaction is precluded.     

Sperm binding to the zona pellucida is not sufficient to induce acrosome exocytosis

At fertilization, spermatozoa bind to the zona pellucida (ZP1, ZP2, ZP3) surrounding ovulated mouse eggs, undergo acrosome exocytosis and penetrate the zona matrix before gamete fusion. Following fertilization, ZP2 is proteolytically cleaved and sperm no longer bind to embryos. We assessed Acr3-EGFP sperm binding to wild-type and huZP2 rescue eggs in which human ZP2 replaces mouse ZP2 but remains uncleaved after fertilization. The observed de novo binding of Acr3-EGFP sperm to embryos derived from huZP2 rescue mice supports a ;zona scaffold' model of sperm-egg recognition in which intact ZP2 dictates a three-dimensional structure supportive of sperm binding, independent of fertilization and cortical granule exocytosis. Surprisingly, the acrosomes of the bound sperm remain intact for at least 24 hours in the presence of uncleaved human ZP2 regardless of whether sperm are added before or after fertilization. The persistence of intact acrosomes indicates that sperm binding to the zona pellucida is not sufficient to induce acrosome exocytosis. A filter penetration assay suggests an alternative mechanism in which penetration into the zona matrix initiates a mechanosensory signal transduction necessary to trigger the acrosome reaction.     

ZP3-dependent activation of sperm cation channels regulates acrosomal secretion during mammalian fertilization

The sperm acrosome reaction is a Ca(2+)-dependent secretory event required for fertilization. Adhesion to the egg's zona pellucida promotes Ca2+ influx through voltage-sensitive channels, thereby initiating secretion. We used potentiometric fluorescent probes to determine the role of sperm membrane potential in regulating Ca2+ entry. ZP3, the glycoprotein agonist of the zona pellucida, depolarizes sperm membranes by activating a pertussis toxin-insensitive mechanism with the characteristics of a poorly selective cation channel. ZP3 also activates a pertussis toxin-sensitive pathway that produces a transient rise in internal pH. The concerted effects of depolarization and alkalinization open voltage-sensitive Ca2+ channels. These observations suggest that mammalian sperm utilize membrane potential-dependent signal transduction mechanisms and that a depolarization pathway is an upstream transducing element coupling adhesion to secretion during fertilization.     

Characterization of rabbit testis beta-galactosidase and arylsulfatase A: purification and localization in spermatozoa during the acrosome reaction.

Examination of the role of carbohydrates in specific recognition between spermatozoa and zona pellucida has focussed on understanding the interaction of sperm hydrolases or lectin-like molecules with zona pellucida ligands. To elucidate the role of specific spermatozoan hydrolases in gamete interaction, rabbit testis beta-galactosidase and arylsulfatase A were purified, characterized, and localized in spermatozoa. beta-Galactosidase and arylsulfatase A co-purified after affinity, size, or reverse-phase chromatography. N-Terminal amino acid analysis and enzymatic characterization suggested that neither enzyme is a testis-specific isozyme. Size chromatography indicated that both enzymes aggregated into macromolecular complexes at pH 4.0, while both dissociated at pH 8.0. beta-Galactosidase and arylsulfatase A co-localized on the sperm surface and in the acrosome and postacrosomal regions of spermatozoa. Throughout the zona-induced acrosome reaction, both enzymes remained associated with the detached acrosomal cap and postacrosomal region of acrosome-reacted spermatozoa. Because the acrosome is an acidic subcellular compartment, internal beta-galactosidase and arylsulfatase A are probably aggregated in acrosome-intact spermatozoa and dissociate as they are exposed to pH increases during the acrosome reaction.