Ultrastructural mapping of a sperm plasma membrane autoantigen before and after the acrosome reaction

The rabbit sperm plasma membrane autoantigen, RSA, is a zona binding protein that binds the spermatozoon to the zona pellucida both before and after the acrosome reaction. In the present study rabbit spermatozoa undergoing the acrosome reaction in vitro are described and monospecific polyclonal mouse anti-RSA and protein A-gold label is used with the label-fracture technique (Pinto de Silva and Kan, J Cell Biol, 99:1156–1161, 1984) to map the location of RSA at the ultrastructural level before and after the acrosome reaction. RSA is most concentrated in the plasma membrane over the postacrosomal-equatorial region border. The label appears to cluster over the anterior aspects of the postacrosomal region's tooth-like projections. Following the acrosome reaction, RSA is still present in the postacrosomal region and often appears clustered in the medial aspects of the equatorial region.     

EARLY STEPS OF SPERM—EGG INTERACTIONS DURING MAMMALIAN FERTILIZATION

Mammalian eggs are surrounded by two egg coats: the cumulus oophorus and the zona pellucida, which is an extracellular matrix composed of sulfated glycoproteins. The first association of the spermatozoon with the zona pellucida occurs between the zona glycoprotein, ZP3 and sperm receptors, located at the sperm plasma membrane, such as the 95kDa tyrosine kinase-protein. This association induces the acrosome reaction and exposes the proacrosin/acrosin system. Proacrosin transforms itself, by autoactivation, into the proteolytical active form: acrosin. This is a serine protease that has been shown to be involved in secondary binding of spermatozoa to the zona pellucida and in the penetration of mammalian spermatozoa through it. The zona pellucida is a specific and natural substrate for acrosin and its hydrolysis and fertilization can be inhibited by antiacrosin monoclonal antibodies. Moreover, inin vitrofertilization experiments, trypsin inhibitors significantly inhibits fertilization. The use of the silver-enhanced immunogold technique has allowed immunolocalization of the proacrosin/acrosin system in spermatozoa after the occurrence of the acrosome reaction. This system remains associated to the surface of the inner acrosomal membrane for several hours in human, rabbit and guinea-pig spermatozoa while in the hamster it is rapidly lost. In the hamster, the loss of acrosin parallels the capability of the sperm to cross the zona pellucida. Rabbit perivitelline spermatozoa can fertilize freshly ovulated rabbit eggs and retain acrosin in the equatorial and postacrosomal region. These spermatozoa also show digestion halos on gelatin plates that can be inhibited by trypsin inhibitors. This evidence strongly suggests the involvement of acrosin in sperm penetration through the mammalian zona. Recently it was shown, however, that acrosin would not be essential for fertilization. It is likely, then, that such an important phenomenon in the mammalian reproductive cycle would be ensured though several alternative mechanisms.     

Localization of zona pellucida binding sites on rabbit spermatozoa and induction of the acrosome reaction by solubilized zonae

The binding of mammalian spermatozoa to the egg's extracellular coat, the zona pellucida, is a complex process which culminates in species-specific penetration of the sperm to the egg plasma membrane. To investigate where on the spermatozoon's surface the zona binding sites are located, whole rabbit zonae were labeled with FITC, heat solubilized and used to observe the surface binding patterns on live spermatozoa. Before the acrosome reaction the zona binding sites are located either over the entire head as well as the middle piece or alternatively in patches along the apical ridge of the head. After the acrosome reaction there is a 29% loss of fluorescence and the zona binding sites are present in the posterior aspect of the acrosomal region, the anterior postacrosomal region and the middle piece. These results demonstrate the presence of zona binding sites after the acrosome reaction which would account for the sperm's ability to remain bound to the zona after the acrosome reaction. Further, we report for the first time that solubilized rabbit zonae pellucidae will induce the acrosome reaction in in vitro capacitated rabbit sperm whereas solubilized pig zonae pellucidae will not. Since rabbit sperm bind pig zonae, the induction and specificity of the physiological acrosome reaction must reside in the affinity of the binding rather than the binding itself.     

Immunodetection of acrosin during the acrosome reaction of hamster, guinea-pig and human spermatozoa.

Mammalian sperm acrosomes contain a trypsin-like protease called acrosin which causes limited and specific hydrolysis of the extracellular matrix of the mammalian egg, the zona pellucida. Acrosin was localized on hamster, guinea-pig and human sperm using monoclonal and polyclonal antibodies to human acrosin labelled with colloidal gold. This was visualized directly with transmission electron microscopy, and with light and scanning microscopy after silver enhancement of the colloidal gold probe. Four distinct labelling patterns were found during capacitation and the acrosome reaction in hamster and guinea-pig spermatozoa, and three patterns were found in human spermatozoa. In the hamster, acrosin was not detected on the inner acrosomal surface after the completion of the acrosome reaction, thus correlating with the observation that hamster spermatozoa lose the ability to penetrate the zona after the acrosome reaction. With guinea-pig and human spermatozoa, acrosin was still detected after the completion of the acrosome reaction, thus correlating with the observation that acrosome reacted guinea-pig spermatozoa bind to and penetrate the zona pellucida.     

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.     

Role of intra-acrosomal antigenic molecules acrin 1 (MN7) and acrin 2 (MC41) in penetration of the zona pellucida in fertilization in mice

In this study the role of two intra-acrosomal molecules, acrin 1 (MN7) and acrin 2 (MC41), during in vitro fertilization (IVF) was examined. The pertinent monoclonal antibodies mMN7 and mMC41 specifically recognize a 90 kDa protein (acrin 1) localized to the entire acrosome and a 200 kDa protein (acrin 2) localized to the cortex region of the anterior acrosome, respectively. Experiments were designed to assess the effects of mMN7 and mMC41 on fertilization in mice using TYH medium containing mMN7 or mMC41 at 0.0, 0.025, 0.05 and 0.1 mg ml-1. Under these conditions, capacitated spermatozoa inseminated the cumulus-invested oocytes. Acrosome-reacted spermatozoa inseminated the zona pellucida-free oocytes. The antibodies had no effect on sperm motility and primary binding to the zona pellucida, but significantly inhibited the rate of fertilization of zona pellucida-intact oocytes in a dose-dependent manner. A significantly small number of spermatozoa remained attached to the zona pellucida at 5 h after insemination in the presence of mMC41. mMC41 and mMN7 antibodies did not affect the fertilization rate of zona pellucida-free oocytes. Confocal laser scanning microscopy with indirect immunofluorescence traced the effect of the monoclonal antibodies on the zona pellucida-induced acrosome reaction, and revealed that mMN7 prevented completion of acrosomal matrix dispersal, whereas mMC41 did not affect the acrosome reaction. mMC41 appeared to inhibit secondary binding or some biochemical steps on the zona pellucida after the acrosome reaction but before penetration of the zona pellucida. Thus, the intra-acrosomal antigenic molecules acrin 1 and acrin 2 are essential for distinct events before sperm penetration of the zona pellucida in mice.     

Status of the rat acrosome during sperm-zona pellucida interactions

Over the past 40 years evidence from many sources has indicated that the mammalian acrosome reaction occurs within or near the cumulus oophorus. Recently, however, workers investigating in vitro fertilization in the mouse have concluded that in this system the acrosome reaction takes place on the surface of the zona pellucida. We have investigated the interaction of rat spermatozoa and the zona pellucida by using the scanning electron microscope (SEM) and two monoclonal antibodies which are directed to antigens of the rat sperm acrosome. When in vitro inseminated eggs from which the cumulus has been removed are viewed with the SEM some sperm heads on the surface of the zona pellucida appear unaltered whereas others appear to be undergoing changes. In vivo, all displayed altered head morphology. Using immunogold labeling we found that the two antibodies employed, 2C4 and 5B1, were directed to acrosomal content and vesiculating acrosomal membranes. Immunofluoresence staining of zonae pellucidae in in vitro fertilization studies revealed numerous small positive regions. These were presumably acrosomal content and membranes which had been left on the zona surface by spermatozoa which had been associated with the zona surface. Our results suggest that the rat acrosome interacts with the zona pellucida. During this interaction some acrosomal content and membranes detach from the spermatozoon and remain on the surface of the zona pellucida.     

PH‐20 but not acrosin is involved in sperm penetration of the macaque zona pellucida

In this study, we investigated the functions of PH‐20 and acrosin during the interaction of macaque sperm with the zona pellucida. Both of these sperm enzymes have been reported to be present on the inner acrosomal membrane of acrosome reacted sperm, and have been suggested to play a role during secondary sperm‐zona binding in other species. Anti‐macaque PH‐20 IgG, anti‐pig acrosin IgG and soybean trypsin inhibitor (SBTI) were used as probes for immunolocalization of the two proteins at the ultrastructural level, and as reagents for blocking sperm penetration of the macaque zona pellucida in vitro. As a control, we performed similar studies with antibodies to CD‐46, which is also located on the inner acrosomal membrane, but has no known function in sperm‐zona pellucida interaction. After labeling with anti‐acrosin IgG, gold label was not present on the sperm surface before the acrosome reaction, but was detected over the entire head of sperm that were induced to acrosome react with calcium ionophore A23187. In contrast, when sperm were induced to acrosome react by binding to intact zona pellucida, acrosin was present in the acrosomal shroud but not on the inner acrosomal membrane. Similar results were obtained when SBTI was used as a probe for enzyme localization. PH‐20 and CD‐46 were demonstrated on the inner acrosomal membrane of sperm induced to acrosome react by ionophore treatment and by zona binding. Neither anti‐acrosin IgG nor anti‐CD‐46 IgG affected sperm penetration of the zona at concentrations up to 300 μg/ml, but zona penetration was blocked completely when anti‐PH‐20 IgG (100 μg/ml) was present during sperm‐oocyte interaction. Ultrastructural observations of oocytes incubated with anti‐PH‐20 IgG showed that acrosomal shrouds were present on the zona surface but no sperm had begun to penetrate into the zona substance. We conclude that anti‐PH‐20 IgG prevented sperm penetration of the macaque zona pellucida by interference with secondary sperm‐zona binding, rather than primary sperm‐zona binding or the zona‐induced acrosome reaction. Acrosin was not detected on the inner acrosomal membrane of sperm that are induced to acrosome react after zona binding, and acrosin does not appear to be critical for sperm penetration of the macaque zona pellucida. Mol. Reprod. Dev. 53:350–362, 1999. © 1999 Wiley‐Liss, Inc.     

Inhibition of the vacuolar H(+)-pump with bafilomycin A1 does not induce acrosome reaction or activate proacrosin in mouse spermatozoa

Acrosomal protease activation is regarded as an important event triggered by acrosomal reaction and leading to sperm passage through zona pellucida. Mammalian acrosome has an internal acid pH that probably helps to maintain inactive proenzymes that otherwise could be precociously activated and prevent normal fertilization. In this work, we have studied the effect of bafilomycin A1, a potent and specific inhibitor of vacuolar H(+)-pump (V-ATPase), on acrosome reaction and proacrosin activation. We used the pH-sensitive probe Lysotracker Green DND-26 to monitor qualitatively intra-acrosomal pH in cauda epididymal mouse spermatozoa. Our results showed that loss of Lysotracker label induced by bafilomycin A1 (acrosome alkalinization) did not induce acrosome reaction or proacrosin activation. We also developed a new technique for imaging the acrosome, and for evaluating the acrosome reaction, in live mouse spermatozoa using Lysotracker DND-26. These results showed that the V-ATPase is a key regulator of mammalian acrosome pH, and that acrosome alkalinization is not the only prerequisite to activate proacrosin under in vivo conditions. Our results suggest that acrosome alkalinization and acrosome reaction are two processes that could be independently regulated during mammalian sperm capacitation.     

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.     

Ultrastructural localization of labeled acrosomal glycoproteins during in vivo fertilization in the rabbit

Rabbit spermatozoa were labeled predominantely in their acrosomal glycoproteins by 1-³H-glucosamine during spermiogenesis. Ova fertilized in vivo by spermatozoa labeled 22 days earlier were analyzed by fine-structure autoradiography for the localization of the label. The latter was found associated with 1) the fused membranes of the acrosomal cap remaining on the zona pellucida surface, 2) the material released on the zona surface after the acrosome reaction and possibly detectable after tannic acid fixation, 3) the equatorial segment of the sperm head and the preequatorial swellings, and 4) other sperm components, eg, the sperm tail. No labeling, on the other hand, was detected on the denuded leading edge of spermatozoa found either in the penetration slit or in the perivitelline space. Our observations suggest the involvement of acrosomal glycoproteins in different mechanisms of sperm/zona pellucida interaction but are not in favor of a major role of (enzymatic) glycoproteins bound to the inner acrosomal membrane during the penetration of the zona pellucida.     

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.     

Role of the sperm proteasome during fertilization and gamete interaction in the mouse

In this work, we have investigated the role of the sperm proteasome during in vitro fertilization (IVF) and gamete interaction in the mouse. Proteasome activity was measured in extract and intact sperm using a specific substrate. In addition, sperm were treated with specific proteasome inhibitors and evaluated during IVF, binding to the zona pellucida, and progesterone- and zona pellucida-induced acrosome reactions. In other experiments, sperm membrane proteins were obtained resuspending them in Triton X-114, shaking vigorously and let standing by 4 hr. Soluble sperm proteins were partitioned in the aqueous phase and sperm membrane proteins in the detergent phase. In both phases, proteasome activity was measured. Labeling of cell surface sperm proteins was carried out with the cell-impermeable NHS-LC biotin, extracted with Triton X-114, and mixing with avidin-agarose beads. Nonpermeabilized sperm were incubated with an anti-proteasome monoclonal antibody and evaluated by indirect immunofluorescence. The results indicate that sperm extracts as well as intact sperm had proteasome activity; the sperm proteasome was involved in IVF, specifically during sperm-zona pellucida binding and the acrosome reaction; soluble sperm membrane proteins exhibited proteasome activity; biotin experiments indicated the presence of proteasomes on the sperm surface, which was corroborated by indirect immunofluorescence experiments. All these observations indicate that the mouse sperm proteasome participates in the binding to the zona pellucida and the acrosome reaction and that there is a pool of proteasomes located on the sperm head.     

Localization of rabbit sperm acrosin during the acrosome reaction induced by immobilized zona matrix

To better understand the loss of the acrosomal cap on the surface of the zona pellucida and the function of the equatorial-postacrosomal region after the acrosome reaction, we have constructed an in vitro system using heat-solubilized zonae pellucidae dried onto a coverslip and incubated with capacitated spermatozoa. This system allows good optical resolution of spermatozoonzona interaction. Induction of the acrosome reaction by zonae on coverslips (30%) is comparable to the induction of the reaction reported previously for rabbit spermatozoa using solubilized zonae in solution. Antiserum to rabbit proacrosin, antiserum to a porcine 49-kDa proacrosin fragment, and antiserum to a porcine 14-kDa C-terminal acrosin fragment were utilized to monitor the acrosome reaction. Rabbit proacrosin/acrosin is not present on the surface of live, acrosome-intact, swimming spermatozoa. After contact with zona, the acrosome reaction begins and proacrosin/acrosin becomes available to bind antibody, first as a crescent in the apical region and then more posteriorly until the entire anterior acrosome is labeled. Proacrosin/acrosin remains on the equatorial and postacrosomal regions of acrosome-reacted spermatozoa and also remains associated with the acrosomal cap even after the spermatozoon is no longer associated with it. Further studies using zona-coated coverslips should lead to a more detailed understanding of the mechanism of zona penetration.     

Molecular basis of fertilization in the mouse

Recent studies of mouse fertilization have identified two complementary gamete receptors that mediate sperm-egg binding. Sperm surface β1,4-galactosyltransferase (GalTase) binds to specific oligosaccharides of the egg coat (zona pellucida) glycoprotein ZP3. Evidence suggests that these same molecules may stimulate the acrosome reaction in sperm. After the acrosome reaction, it is thought that sperm remain adherent to the zona by binding another glycoprotein, ZP2. The acrosome-reacted sperm releases hydrolytic enzymes, including acrosin and N-acetylglucosaminidase, enabling it to penetrate the zona pellucida. After the penetrating sperm binds to the egg membrane and activates development, N-acetylglucosaminidase is exocytosed from egg cortical granules and, as part of the zona block to polyspermy, globally removes the sperm GalTase binding site from ZP3 oligosaccharides.     

Influence of capacitation and fluids from the male and female genital tract on the zona binding ability of bull spermatozoa.

Before fertilization, inseminated spermatozoa acquire the ability to fertilize an egg, a phenomenon called capacitation. Bovine sperm capacitation is influenced by factors originating from both the male and female genital tract, and results in intracellular and membrane changes of the spermatozoa that facilitate the induction of the acrosome reaction. However, the effects of reproductive tract secretions and capacitation on the binding of spermatozoa to the zona pellucida have not been investigated. In this study, a sperm-egg binding assay was used to determine whether the ability of bull spermatozoa to bind to the zona pellucida was altered during in vitro capacitation by heparin or oviductal fluid, or by treatment of spermatozoa from the cauda epididymidis with accessory sex gland fluid. In addition, biotinylated solubilized zona pellucida proteins were used to visualize zona binding on spermatozoa. The ability of bull spermatozoa to bind to the zona pellucida was increased after both heparin and oviductal fluid induced in vitro capacitation. Exposure of spermatozoa from the cauda epididymidis to accessory sex gland fluid resulted in a direct increase in zona binding ability, followed by a further increase during capacitation in vitro. Binding of solubilized zona proteins was restricted to the acrosomal cap of bull spermatozoa. It is suggested that the observed increased ability of bull spermatozoa to bind to the zona pellucida enables optimal sperm-egg attachment, which also relates to the induction of the acrosome reaction by the zona pellucida. Thus, increased zona binding ability is likely to be an essential part of the process of capacitation.     

Acrosomal status and motility of guinea pig spermatozoa during in vitro penetration of the cumulus oophorus

Previous studies have suggested that both acrosome-intact and acrosome-reacted guinea pig sperm are capable of binding to the zona pellucida of cumulus-free oocytes, but the acrosomal status of guinea pig sperm during penetration of the cumulus has not been reported. We made video recordings of the interaction between capacitated guinea pig sperm and cumulus-invested guinea pig oocytes. The videotapes were analysed to identify sperm with hyperactivated motility and to classify the acrosomal status of sperm during penetration of the cumulus and after binding to the zona pellucida. The resolution of the video recordings was not sufficient to recognise sperm with swollen acrosomes. However, sperm that had completed the acrosome reaction were easily identified. Acrosome-reacted sperm were found adherent to the outer boundary of the cumulus, but were never observed to penetrate the cumulus. The percentage of acrosome-intact, hyperactivated sperm was higher in the cumulus oophorus than in culture medium, suggesting that changes in motility were elicited in response to contact with the cumulus. Fully acrosome-reacted sperm were found adherent to the zona pellucida, and solubilised guinea pig zona pellucida was capable of inducing acrosome reactions in capacitated guinea pig sperm. Acrosome-intact sperm were also observed on the zona, but they were not tightly bound and did not have hyperactivated motility, suggesting that these sperm were not functionally capacitated. Our observations demonstrate that guinea pig sperm penetrate the cumulus matrix in an acrosome-intact state. Although we did not observe sperm undergoing the acrosome reaction, our observations and experimental data suggest that the acrosome reaction of guinea pig sperm is completed on or near the surface of the zona pellucida.     

Ultrastructural observations on gamete interactions using micromanipulated mouse oocytes

Cumulus-free mouse oocytes were subjected to zona opening by cracking with microhooks (ZC) or acid drilling (ZD) and fixed 30–90 min after insemination (10⁵ pre-capacitated motile sperms/ml). Ultrastructural observations were made on serially thin-sectioned oocytes: 15 ZC and 12 ZD. The zona lesion in ZC oocytes was a clean cut, whereas in ZD oocytes it formed a patchy area of partial zona loss, with reduced microvillar height on the underlying oocyte surface. Spermatozoa were observed within the perivitelline space and partially fusing with the oocyte after 30 min in both situations. Only acrosome-reacted sperm heads were observed to fuse: acrosome intact forms were generally in contact with the zona pellucida, either with the inner or outer surface. Acrosome-intact spermatozoa were also observed deeply embedded in the zona matrix, possibly indicating surface enzyme activity preceding the membrane fusion events of the acrosome reaction proper. The observations are consistent with the need for spermatozoa to make contact preferentially with the zona pellucida during the course of the acrosome reaction.     

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.     

Ultrastructure of opossum oocyte investing coats and their sensitivity to trypsin and hyaluronidase

Ovulated opossum oocytes are surrounded by a zona pellucida, but not by cumulus cells. Opossum sperm carry at least four acrosomal hydrolases (hyaluronidase, acrosin, N-acetylhexosaminidase, and arylsulfatase); the functions of these enzymes in opossum fertilization are uncertain. To identify possible substrates for these hydrolases, the ultrastructure of opossum oocytes was examined after fixation in the presence of ruthenium red which stabilizes extracellular matrices. This oocyte is unusual in having a wide perivitelline space containing a highly structured extracellular matrix (ECM). The ECM is comprised of granules and filaments, and it resembles matrices known to contain hyaluronic acid in other systems. Hydrolases, known to be present in opossum acrosomes, were tested for their effect on the ultrastructure of the zona pellucida and matrix of the perivitelline space. Trypsin dissolved the zona pellucida and decreased the size of the granules in the perivitelline space. Streptomyces hyaluronidase, which specifically attacks hyaluronic acid, removed only matrix filaments. Arylsulfatase, N-acetylhexosaminidase, and beta-glucuronidase did not affect the zona pellucida or ECM in our assay. These observations are consistent with the ideas that (1) opossum sperm must penetrate two oocyte investments, the zona pellucida and ECM of the perivitelline space; (2) the ECM contains hyaluronic acid (filaments) and protein (granules); (3) opossum sperm acrosin may function in penetration of the zona pellucida and ECM; and (4) opossum sperm hyaluronidase may function in penetration of the ECM by degrading hyaluronic acid (filaments). Dissolution of the granules and filaments from oocyte microvilli is probably necessary to permit close apposition and fusion of the sperm and oocyte membranes. The evolutionary significance of these results is discussed.