Epididymal maturation and the acrosome reaction in mouse sperm: response to zona pellucida develops coincident with modification of M42 antigen

Development of the sperm's capacity to interact with the zona pellucida was investigated at the stage when the acrosome reaction (AR) is induced. The response of epididymal sperm to agents that affect the occurrence of the AR was used to monitor maturational changes. Despite the finding that sperm from the three main epididymal regions were competent to undergo ARs induced by the divalent cation ionophore A23187 (56% AR, 74% AR, and 83% AR in caput, corpus, and cauda, respectively), the cells' responses to solubilized zonae pellucidae were different. When challenged with 5 zonae equivalents/microliter, both corpus and cauda sperm shed their acrosomes in high numbers (75% AR and 86% AR, respectively), whereas caput sperm did not (23% AR). Previous work has shown that the presence of M42 monoclonal antibody (mAb) during in vitro and in vivo fertilization inhibits sperm penetration through the zona pellucida by specific interference with zonae pellucidae-induced ARs. In this study, presence of the M42 mAb did not affect the incidence of A23187-induced ARs, whereas the zona-induced ARs that occurred in both corpus and cauda sperm were inhibited fully with M42 immunoglobulin (Ig) G. In addition, the antigen recognized by M42 mAb on sperm, termed M42 Ag, was examined during epididymal maturation. Although antigen localization appeared indistinguishable by immunofluorescence on sperm taken from the caput, corpus, and cauda regions of the epididymis, modification of this antigen during epididymal transit was detected. Equilibrium-binding studies using 125I-M42 IgG demonstrated a progressive increase during epididymal transit in the amount of M42 mAb that bound to fixed cells. Corpus and cauda sperm bound 185% and 240%, respectively, of the 125I-M42 IgG detected on caput sperm. These changes in expression of M42 Ag paralleled a structural change: the Mr of the antigen decreased from a 195,000/210,000 doublet in caput sperm to a 185,000/200,000 doublet in corpus and cauda sperm, as determined by immunoblot analysis of sodium dodecyl sulfate (SDS)-extracted sperm. Results presented here demonstrate that mouse sperm develop the capacity to undergo a zona-induced AR during epididymal maturation. The M42 antigen, which is involved in the zona-induced AR, is modified during epididymal transit coincident with development of the sperm's responsiveness to zonae. Our working hypothesis, based on these results, is that development of the sperm's capacity to undergo a physiological AR is related to modification of M42 Ag.     

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.     

Evidence that aggregation of mouse sperm receptors by ZP3 triggers the acrosome reaction

In the mouse, considerable evidence indicates that initial sperm binding to the zona pellucida (ZP) is mediated by ZP3. In addition, this same glycoprotein is also responsible for inducing the acrosome reaction (AR). Whereas the O-linked oligosaccharides of ZP3 appear to mediate sperm-ZP binding, the portion of ZP3 bearing AR activity has not been defined. To try to understand the bifunctional role of ZP3 (binding and AR inducing activities), we have examined the hypothesis that ZP3 aggregates sperm receptor molecules. By analogy with findings in a variety of other extracellular signal transducing systems, including receptors for growth factors and insulin, this aggregation event could initiate the cascade resulting in the AR. To test this hypothesis, we have generated monospecific polyclonal antibodies against ZP2 and against ZP3, and examined the effects of these probes on capacitated sperm incubated in the absence or presence of various ZP protein preparations. For some experiments, we have used proteolytic fragments of ZP3, a preparation known to retain specific binding, but not AR-inducing, activity. We show here that capacitated mouse sperm, incubated with ZP glycopeptides, displayed ARs when incubated subsequently with anti-ZP3 IgG; ARs did not occur when parallel sperm samples were incubated with anti-ZP2 IgG or with anti-ZP3 Fab fragments. When capacitated sperm were treated successively, with (a) ZP3 glycopeptides, (b) anti-ZP3 Fab fragments, and (c) goat anti-rabbit IgG, ARs occurred in the majority of sperm. An alternative approach to examine this hypothesis used ZP proteins obtained from tubal eggs treated previously with bioactive phorbol diester (12-O-tetradecanoyl phorbol-13-acetate [TPA]). This preparation arrests capacitated sperm in an intermediate state of the AR. We demonstrate here that these sperm can be induced to undergo a complete AR by subsequent treatment with anti-ZP3 IgG. Together, these findings are consistent with the hypothesis under examination, and suggest that the aggregation of sperm molecules recognized by ZP3 glycopeptides or by TPA-treated ZP is sufficient to trigger the events that occur during acrosomal exocytosis.     

Acrosome reaction induced by immunoaggregation of a proteinase inhibitor bound to the murine sperm head.

ZP3, a glycoprotein of the murine zona pellucida, functions both to bind acrosome intact sperm and to induce the acrosome reaction. Solubilized whole zonae as well as purified ZP3 are able to induce acrosome reactions in capacitated sperm. Pronase digests of whole zonae yield glycopeptides that bind to sperm but are unable to induce acrosome reactions. However, immunoaggregation of these glycopeptides results in the exocytosis of the acrosome in the majority of treated sperm. The data suggest that ZP3 triggers the acrosome reaction by the aggregation of ZP3 binding sites on the sperm head. If aggregation of ZP3 binding sites is important in the induction of the acrosome reaction, then it may be possible to induce the acrosome reaction in the absence of zona by immunoaggregation of the sites. This presentation deals with the immunoaggregation of a proteinase inhibitor of seminal vesicle origin (SVI) that binds to a site on the sperm head known to participate in zona binding. We show that capacitated murine sperm, pretreated with the SVI, will acrosome react, as determined by Coomassie brilliant blue staining, when incubated with rabbit antiinhibitor antiserum (anti-SVI). The percentage of SVI-treated sperm displaying an acrosome reaction is dependent on the concentration of the immune serum. Sperm stain positive for intact acrosomes when anti-SVI Fab fragments or normal rabbit serum is substituted for the immune serum. However, when capacitated sperm, treated with both SVI and anti-SVI Fab fragments, are incubated with goat antirabbit IgG, the majority of sperm acrosome react. The data suggest that the aggregation of SVI bound to the sperm surface, in the absence of zona glycoproteins, is sufficient to induce the acrosome reaction.     

Zona pellucida-mediated acrosomal exocytosis in mouse spermatozoa: characterization of an intermediate stage prior to the completion of the acrosome reaction

The zona pellucida (ZP)-induced acrosome reaction in mouse sperm proceeds in two steps, identified by three sperm fluorescence patterns observed sequentially with the fluorescent probe chlortetracycline. Capacitated, acrosome-intact sperm displaying a B pattern proceed to an intermediate S pattern, and then progress from the S pattern to the fully acrosome-reacted AR pattern. Previously, it was not feasible to characterize the nature of the transient intermediate S pattern. Recently, it was demonstrated that sperm bind to the ZP of eggs treated with 12-O-tetradecanoyl phorbol-13-acetate (TPA) and undergo a B to S transition, but do not complete the acrosome reaction. These cells accumulate in the S pattern and fail to undergo the S to AR transition (Endo, Y., Schultz, R. M., and Kopf, G. S. 1987a. Dev. Biol. 119, 119-209). The present study utilized ZP from TPA-treated eggs to assess the state of S pattern sperm. The kinetics of the B to S transition of sperm incubated with either structurally intact or solubilized ZP from untreated or TPA-treated eggs are identical. Addition of either solubilized ZP from untreated eggs or A-23187 to S pattern sperm bound to intact or solubilized ZP from TPA-treated eggs induces the S to AR transition, while ZP from TPA-treated or fertilized eggs does not. Loss of the transmembrane pH gradient in the anterior portion of the sperm head, monitored by the fluorescent pH probe 9-N-dodecyl aminoacridine, follows the B to S transition in sperm incubated with ZP from unfertilized eggs, but no loss is observed when the B to S transition is induced using ZP from TPA-treated eggs. Subsequent addition of solubilized ZP from untreated eggs or A-23187 results in the loss of the transmembrane pH gradient of these S pattern sperm. Addition of nigericin to S pattern sperm bound to ZP from TPA-treated eggs discharges the transmembrane pH gradient and causes the S to AR transition. In contrast, nigericin added to B pattern sperm discharges the pH gradient but does not induce a B to S transition. Electron microscopic evaluation of S pattern-arrested sperm using ZP from TPA-treated eggs reveals intact plasma and outer acrosomal membranes. These results suggest that ZP from TPA-treated and fertilized eggs are modified such that the ZP ligands inducing the S to AR transition are lost or are inactivated.(ABSTRACT TRUNCATED AT 400 WORDS)     

A guanine nucleotide-binding regulatory protein in human sperm mediates acrosomal exocytosis induced by the human zona pellucida.

Guanine nucleotide-binding regulatory proteins play key intermediary roles in regulating zona pellucida-mediated acrosomal exocytosis in mouse and bull sperm. Since human sperm possess a Gi-like protein and undergo the acrosome reaction in response to the human zona pellucida, we investigated whether this G protein plays a regulatory role in this exocytotic process. Zonae pellucidae isolated from eggs that had been inseminated but had shown no signs of fertilization after retrieval for in vitro fertilization and embryo transfer were pooled into groups of greater than or equal to 50 in order to reduce variability in biological responses due to the possible presence of ZP that had undergone modifications associated with the polyspermy block. Acid-solubilized zonae pellucidae were incubated with capacitated sperm, and the sperm then assessed for the acrosome reaction using both the P. sativum agglutinin and chlortetracycline fluorescence assays; both assays gave similar results. Sperm incubated with solubilized zonae pellucidae at a final concentration of 2, 4, or 6 ZP/microliter underwent acrosomal exocytosis to a similar extent as compared with A-23187. Sperm were incubated with 1 microgram/ml pertussis toxin during capacitation to functionally inactivate the Gi-like protein. Pertussis toxin treatment of sperm did not affect sperm motility and the ability of the cells to bind to structurally intact zonae pellucidae. Pertussis toxin, however, completely inhibited the percentage acrosome reactions induced by solubilized zonae pellucidae. By contrast, the A-23187-induced acrosome reaction was insensitive to PT treatment. Pertussis toxin inhibition of the zona pellucida-induced acrosome reaction occurred in a concentration-dependent manner with maximal effects observed at 100 ng/ml PT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein dynamics in sperm membranes: implications for sperm function during gamete interaction.

A number of mammalian sperm plasma membrane antigens have been implicated as playing a functional role in sperm-egg interaction, by virtue of the fact that antibodies against these antigens interfere with fertilization. Two such mouse sperm plasma membrane antigens are M42, a 200/220 kD glycoprotein doublet, and M5, a 150-160 kD glycoprotein. We show that both of these antigens are concentrated on the posterior region of caudal epididymal and capacitated mouse sperm heads and are relatively diffusible, as determined by fluorescence recovery after photobleaching measurements (D = 3-8 x 10(-9) cm2/s with approximately 23% diffusing). Crosslinking of these antigens with bivalent antibodies causes them to redistribute into the anterior region (acrosomal crescent) of the sperm head. In contrast, we describe a third antigen, P220, which is also localized to the posterior region of the sperm head on caudal epididymal sperm but which exhibits very little diffusion and does not redistribute upon crosslinking. Bivalent anti-M42 blocks the ZP3-induced acrosome reaction. We have found that monovalent Fab fragments of anti-M42 do not block the ZP3-induced acrosome reaction, but that inhibition is restored by addition of a second antibody which crosslinks the Fabs. Thus, crosslinking is required for both inhibition of the acrosome reaction and redistribution. This suggests that redistribution of antigen away from the posterior region of the head may be part of the mechanism of inhibition of the ZP3-induced acrosome reaction.     

Evidence suggesting that the mouse sperm acrosome reaction initiated by the zona pellucida involves an alpha7 nicotinic acetylcholine receptor

The mammalian sperm acrosome reaction (AR) is essential to fertilization and is believed to be initiated in vivo by ZP3, a glycoprotein component of the egg zona pellucida (ZP). Recently, we reported the results of antagonist studies suggesting that a nicotinic acetylcholine receptor (nAChR) containing an alpha7 subunit (alpha7nAChR) plays a role in the human sperm AR initiated by recombinant human ZP3 or by acetylcholine (ACh). Here, we show that ACh can initiate the mouse sperm AR and that antagonists of the nAChR inhibit the AR initiated by ACh or by ZP obtained from ovarian oocytes (isolated heat-solubilized mouse ZP). Preincubation with three antagonists of the nAChR, alpha-bungarotoxin (100 nM), alpha-conotoxin IMI (100 nM), and methyllycaconitine (100 nM), significantly blocked AR initiation by ACh or by isolated heat-solubilized mouse ZP (P 相似文献   

Structural conservation of mouse and rat zona pellucida glycoproteins. Probing the native rat zona pellucida proteome by mass spectrometry

The mammalian zona pellucida is an egg extracellular matrix to which sperm bind. Mouse zonae are composed of three glycoproteins (ZP1, ZP2, and ZP3), while rat zonae contain four (ZP1, ZP2, ZP3, and ZP4/ZPB). Mouse sperm bind to zonae comprised solely of mouse ZP2 and ZP3. In this report, we show that rat sperm also bind to these zonae, indicating that ZP2 and ZP3 contain a "minimum structure(s)" to which rodent sperm can bind, and ZP1 and ZP4/ZPB are dispensable in these two rodents. These data are consistent with our mass spectrometric analysis of the native rat zona pellucida proteome (defined as the fraction of the total rat proteome to which the zonae glycoproteins contribute) demonstrating that the rat zonae glycoproteins share a high degree of conservation of structural features with respect to their mouse counterparts. The primary sequences of the rat zonae proteins have been deduced from cDNA. Each zona protein undergoes extensive co- and post-translational modification prior to its secretion and incorporation into an extracellular zona matrix. Each has a predicted N-terminal signal peptide that is cleaved off once protein translation begins and an anchoring C-terminal transmembrane domain from which the mature protein is released. Mass spectrometric analysis with a limited amount of native material allowed determination of the mature N-termini of rat ZP1 and ZP3, both of which are characterized by cyclization of glutamine to pyroglutamate; the N-terminus of ZP2 was identified by Edman degradation. The mature C-termini of ZP1 and ZP3 end two amino acids upstream of a conserved dibasic residue that is part of, but distinct from, the consensus furin cleavage sequence, while the C-terminus of ZP2 was not determined. Each zona protein contains a "zona domain" with eight conserved cysteine residues that is thought to play a role in the polymerization of the zona proteins into matrix filaments. Partial disulfide bond assignment indicates that the intramolecular disulfide patterns in rat ZP1, ZP2, and ZP3 are identical to those of their corresponding mouse counterparts. Last, nearly all potential N-glycosylation sites are occupied in the rat zonae glycoproteins (three of three for ZP1, six or seven of seven for ZP2, and four or five of six for ZP3). In comparison, potential O-glycosylation sites are numerous (59-83 Ser/Thr residues), but only two regions were observed to carry O-glycans in rat ZP3.     

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.     

Structural and functional relationships between mouse and hamster zona pellucida glycoproteins

The hamster egg's extracellular coat, or zona pellucida, consists of three glycoproteins, designated hZP1, hZP2, and hZP3, that exhibit extensive heterogeneity on SDS-PAGE. hZP1 is a relatively minor component of hamster zonae pellucidae, as compared with hZP2 and hZP3. In the presence of reducing agents, hZP1, 200,000 apparent Mr, migrates on SDS-PAGE with an apparent Mr of 103,000. This suggests that hZP1, like mouse ZP1, is composed of two polypeptides held together by intermolecular disulfides. When purified hamster ZP glycoproteins were tested at relatively low concentrations in an in vitro competition assay, employing either hamster or mouse gametes, only hZP3 (56,000 apparent Mr) exhibited sperm receptor activity (i.e., inhibited binding of sperm to eggs). Thus, apparently hZP3 is the hamster counterpart of mouse ZP3, the mouse egg receptor for sperm. Furthermore, at relatively high concentrations, solubilized hamster egg ZP preparations induced both hamster and mouse sperm to undergo the acrosome reaction in vitro. hZP3 is encoded by a relatively abundant ovarian mRNA that is detected by a mouse ZP3 cDNA probe and is the same size, about 1.5 kb, as mRNA encoding the mouse sperm receptor, ZP3 (83,000 apparent Mr). Like mouse ZP2, hZP2 undergoes limited proteolysis following artificial activation of hamster eggs in vitro. Results of in vitro assays employing intact eggs and isolated zonae pellucidae demonstrate that hamster eggs possess a ZP2-proteinase which has a substrate specificity similar to that of the mouse enzyme. These observations are discussed in terms of structural and functional relationships that may exist between hamster and mouse zona pellucida glycoproteins.     

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.     

Assessment of acrosomal status in rat spermatozoa: studies on carbohydrate and non-carbohydrate agonists

In the mouse and several other species, including man, capacitated acrosome-intact spermatozoa interact with natural [soluble zona pellucida (ZP) and progesterone (P4)] and synthetic [neoglycoproteins (ngps) and calcium (Ca(2+)) ionophore] agonists, prior to the initiation of a Ca(2+)-dependent signal transduction cascade. The net result is the fusion of the sperm plasma membrane overlying the outer acrosomal membrane at multiple sites and exocytosis of acrosomal contents [i.e., induction of the acrosome reaction (AR)]. This step is believed to be a prerequisite that enables the acrosome-reacted spermatozoon to penetrate the ZP and fertilize the egg. Although the rat is one of the most commonly used laboratory animals, very little is known about the chemical nature of agonists that induce the AR in this species. The lack of this information is primarily due to the fact that the rat sperm acrosome is a relatively thin structure. Thus, it is difficult to assess the status of the sperm acrosome in this species. In this report, we describe the use of a Coomassie brilliant blue dye staining procedure to assess the status of the rat sperm acrosome by light microscopy. The procedure is highly reproducible and has allowed us to determine the effects of carbohydrate (ngps and mouse ZP) and noncarbohydrate (P4 and Ca(2+) ionophore) agonists on capacitated spermatozoa. In addition, we have used a pharmacological approach to examine the functional significance of calmodulin (CaM), a Ca(2+)-binding protein, in induction of the AR in spermatozoa. Data presented in this report demonstrate that several ngps, solubilized mZP, P4, and Ca(2+) ionophores induce the AR in rat spermatozoa. Furthermore, we demonstrate that, whereas CaM antagonists blocked P4-induced AR, most of the inhibitors used had no significant effect on the Ca(2+) ionophore-induced (nonphysiological) AR.     

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.     

Effects of phorbol esters and a diacylglycerol on the mouse sperm acrosome reaction induced by the zona pellucida

Capacitated mouse sperm undergo the spontaneous acrosome reaction in suspension and the zona-induced acrosome reaction when bound to isolated, intact zonae pellucidae. The zona-induced acrosome reaction in the mouse resembles, in part, ligand-receptor-mediated exocytotic processes that occur in some somatic cells. Since such processes have been shown to be mediated in part by protein kinase C-catalyzed protein phosphorylation, the effects of phorbol esters, which are potent activators of this kinase, on both the spontaneous and the zona-induced acrosome reaction were examined. At concentrations up to 10 microM, 12-tetradecanoyl phorbol-13-acetate (TPA) had no effect on the time course of the spontaneous acrosome reaction as scored by the chlortetracycline (CTC) fluorescence assay. Capacitated, acrosome-intact sperm display Pattern B in the CTC assay with fluorescence on the anterior head; fully acrosome-reacted sperm display Pattern AR with no fluorescence on the head. The time course of the loss of Pattern B in the zona-induced acrosome reaction was markedly accelerated by 65 nM TPA as compared to controls, whereas the appearance of Pattern AR was retarded. The appearance of Pattern S, which is characterized by punctate fluorescence on the head and which marks an intermediate state between Pattern B and Pattern AR in the controls, was accelerated by 65 nM TPA to the same extent as the loss of Pattern B at early times post-binding to zonae. The disappearance of Pattern S at later times post-binding to zonae was retarded by 65 nM TPA to the same extent as the appearance of Pattern AR. The transitions between the fluorescence patterns, designated the B-to-S and the S-to-AR transitions, therefore define two stages of the zona-induced acrosome reaction, which are affected in opposite directions by TPA. The effects of 65 nM TPA are mimicked by 60 nM 4-beta-phorbol-12,13-didecanoate (4-beta-PDD) while the 4-alpha isomer is without effect. Such stereospecificity is similar to that reported for the activation of protein kinase C. The diacylglycerol, 1-oleyl-2-acetylglycerol, which is also known to activate protein kinase C, mimicked the effects of TPA and 4-beta-PDD on the time courses of the B-to-S and S-to-AR transitions. These results suggest that protein kinase C may play an intermediary role in the zona-induced mouse sperm acrosome reaction.     

Sperm epidermal growth factor receptor (EGFR) mediates α7 acetylcholine receptor (AChR) activation to promote fertilization

To attain fertilization the spermatozoon binds to the egg zona pellucida (ZP) via sperm receptor(s) and undergoes an acrosome reaction (AR). Several sperm receptors have been described in the literature; however, the identity of this receptor is not yet certain. In this study, we suggest that the α7 nicotinic acetylcholine receptor (α7nAChR) might be a sperm receptor activated by ZP to induce epidermal growth factor receptor (EGFR)-mediated AR. We found that isolated ZP or α7 agonists induced the AR in sperm from WT but not α7-null spermatozoa, and the induced AR was inhibited by α7 or EGFR antagonists. Moreover, α7-null sperm showed very little binding to the egg, and microfluidic affinity in vitro assay clearly showed that α7nAChR, as well as EGFR, interacted with ZP3. Induction of EGFR activation and the AR by an α7 agonist was inhibited by a Src family kinase (SFK) inhibitor. In conclusion we suggest that activation of α7 by ZP leads to SFK-dependent EGFR activation, Ca(2+) influx, and the acrosome reaction.     

Studies of sperm from mutant mice suggesting that two neurotransmitter receptors are important to the zona pellucida-initiated acrosome reaction

Two sperm neurotransmitter receptor/channels, the glycine receptor (GlyR) and a nicotinic acetylcholine receptor containing an alpha7 subunit (alpha7nAChR) were previously shown to be important to the mouse acrosome reaction (AR) initiated by solubilized egg zona pellucida (ZP). Here, we investigated whether sperm from homozygous mutant mice with a single amino acid mutation in the alpha subunit of their GlyR and sperm from homozygous mutant mice with an engineered disruption of the gene for the nicotinic acetylcholine receptor alpha7 subunit could undergo the AR on ZP-intact eggs. Wild-type and mutant sperm were treated with 3-quinuclidinyl benzilate (QNB), known to be an inhibitor of the ZP-initiated AR (but shown in the present work not to inhibit the acetylcholine-initiated AR). The ZP-initiated AR on ZP-intact eggs should occur only in sperm not treated with QNB. The absence of such an increase in the untreated mutant sperm would demonstrate that such sperm were unable to respond to the intact ZP. The results demonstrated for the first time that GlyR mutant sperm do not undergo the AR on ZP-intact mouse eggs, and that their ability to fertilize is inhibited by 63% in vitro. Moreover, we found that GlyR mutant sperm exhibited normal capacitation and confirmed that they not undergo the AR initiated by solubilized mouse ZP. Our studies demonstrated for the first time that sperm from mutant alpha7nAChR mice exhibit normal capacitation, do not undergo the AR in response to acetylcholine, solubilized ZP or on ZP-intact eggs, and display a 25% reduction in fertilization in vitro. This is the first genetic evidence for the importance of the alpha7nAChR in the ZP-initiated AR. While defects in either the GlyR or the alpha7nAChR completely inhibit the ZP-initiated AR, fertilization by these mutant sperm can still occur in vitro, probably due to sperm that complete spontaneous AR on the ZP.     

Calmodulin signals capacitation and triggers the agonist-induced acrosome reaction in mouse spermatozoa

Capacitated acrosome-intact spermatozoa interact with specific sugar residues on neoglycoproteins (ngps) or solubilized zona pellucida (ZP), the egg's extracellular glycocalyx, prior to the initiation of a signal transduction cascade that results in the fenestration and fusion of the sperm plasma membrane and the outer acrosomal membrane at multiple sites and exocytosis of acrosomal contents (i.e., induction of the acrosome reaction (AR)). The AR releases acrosomal contents at the site of sperm-zona binding and is thought to be a prerequisite event that allows spermatozoa to penetrate the ZP and fertilize the egg. Since Ca(2+)/calmodulin (CaM) plays a significant role in several cell signaling pathways and membrane fusion events, we have used a pharmacological approach to examine the role of CaM, a calcium-binding protein, in sperm capacitation and agonist-induced AR. Inclusion of CaM antagonists (calmodulin binding domain, calmidazolium, compound 48/80, ophiobolin A, W5, W7, and W13), either in in vitro capacitation medium or after sperm capacitation blocked the npg-/ZP-induced AR. Purified CaM largely reversed the AR blocking effects of antagonists during capacitation. Our results demonstrate that CaM plays an important role in priming (i.e., capacitation) of mouse spermatozoa as well as in the agonist-induced AR. These data allow us to propose that CaM regulates these events by modulating sperm membrane component(s).     

Acrosome reaction in the cumulus oophorus revisited: involvement of a novel sperm-released factor NYD-SP8

Fertilization is a process involving multiple steps that lead to the final fusion of one sperm and the oocyte to form the zygote. One of the steps, acrosome reaction (AR), is an exocytosis process, during which the outer acrosome membrane fuses with the inner sperm membrane, leading to the release of acrosome enzymes that facilitate sperm penetration of the egg investments. Though AR has been investigated for decades, the initial steps of AR in vivo, however, remain largely unknown. A well elucidated model holds the view that AR occurs on the surface of the zona pellucida (ZP), which is triggered by binding of sperm with one of the ZP glycosylated protein, ZP3. However, this model fails to explain the large number of ‘falsely’ acrosome-reacted sperms found within the cumulus layer in many species examined. With the emerging evidence of cross-talk between sperm and cumulus cells, the potential significance of AR in the cumulus oophorus, the outer layer of the egg, has been gradually revealed. Here we review the acrosome status within the cumulus layer, the cross-talk between sperm and cumulus cells with the involvement of a novel sperm-released factor, NYD-SP8, and re-evaluate the importance and physiological significance of the AR in the cumulus in fertilization.