Inhibition of fertilization of the rabbit ova in vitro by the antibody to the inner acrosomal membrane of rabbit spermatozoa

The antibody to the rabbit sperm inner acrosomal membrane, raised in guinea pig, completely inhibited the fertilization of rabbit ova in vitro. The F(ab')2 of the antibody was equally effective in inhibiting fertilization. The antibody appeared to exert its inhibitory effect by binding to the inner acrosomal membrane of acrosome-reacted sperm. The antibody-treated sperm did not attach to or penetrate the zona pellucida. Thus, anti-IAM offers a great potential as a contraceptive agent.     

The regulation of acrosomal exocytosis. I. Sperm capacitation is required for the induction of acrosome reactions by the bovine zona pellucida in vitro

The regulation of acrosomal exocytosis in capacitated bovine spermatozoa by soluble extracts of zonae pellucidae was examined. Kinetic studies demonstrated that zonae pellucidae stimulated synchronous acrosome reactions. The t1/2 of this process was 5-10 min and response was maximal at 20 min. The apparent initial rate of exocytosis in sperm populations was dependent upon the concentration of zona pellucida protein, with an ED50 and a maximally effective dosage of 20 and 50 ng protein/microliter, respectively. Zonae pellucidae caused up to a 48-fold increase in the apparent initial rate and a 3- to 4-fold stimulation in the net occurrence of exocytosis. In contrast, solubilized zonae pellucidae did not induce acrosome reactions in uncapacitated sperm. The development of a capacitated state, as assayed by the ability of sperm to fertilize eggs in vitro, was compared to the expression of zona pellucida-regulated acrosome reactions in a series of kinetic experiments. Both activities were manifest with similar kinetics and displayed identical dependencies toward stimulatory and inhibitory agents in vitro. It is concluded that capacitation is an essential prerequisite for the induction of acrosomal exocytosis in bovine sperm by the zona pellucida.     

Proteolytic activity of rabbit perivitelline spermatozoa.

Acrosin, an acrosomal serine protease, has been associated with binding of spermatozoa and their penetration through the zona pellucida. This study was aimed at determining whether the remaining proacrosin/acrosin system on rabbit perivitelline spermatozoa still has proteolytic activity and whether this activity is involved in further penetration of unfertilised rabbit eggs. Eight hundred and sixty-five rabbit perivitelline spermatozoa were evaluated by the gelatin-substrate film technique for the detection of acrosin on individual spermatozoan. Fifteen per cent of the studied spermatozoa showed small digestion halos on the gelatin film. The proteolytic activity of rabbit perivitelline spermatozoa was inhibited in the presence of 1 mg/ml of soybean trypsin inhibitor (SBTI) or with 20 micrograms/ml of a mixture of the monoclonal anti-proacrosin/acrosin antibody. In vitro fertilisation occurred in 21.8% of rabbit oocytes co-incubated with perivitelline spermatozoa and was completely inhibited when oocytes were incubated with 600 micrograms/ml of a mixture of three anti-acrosin monoclonal antibodies (ACRO-A8C10, ACRO-C2B10 and ACRO-C5F10). Inseminations in the presence of anti-cholera monoclonal antibody (irrelevant to spermatozoa) resulted in 17.6% fertilisation. These results support the idea that the residual proacrosin/acrosin system in perivitelline spermatozoa might be involved in spermatozoal binding and/or second penetration through the zona pellucida.     

Tubulin and microtubule-like structures in mammalian acrosomes.

Immunofluorescence and electron microscopy were used to demonstrate the presence of tubulin and microtubule-like structures in rabbit and rhesus monkey sperm acrosomes. The distribution of tubulin within the acrosome appeared similar to the distribution of proteolytic activity, suggesting an association of proteinase with these structures. Electron microscopy of rhesus monkey sperm incubated in vitro demonstrated a progressive condensation of the acrosomal matrix into microtubule-like structure. A system of this type could explain the linear patterns of silver-proteinate staining in the pentration tunnel through the zona pellucida.     

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.     

The status of acrosomal caps of hamster spermatozoa immediately before fertilization in vivo

Adult female golden hamsters were induced to superovulate. When they were mated several hours prior to ovulation or artificially inseminated about the time of ovulation, nearly 100% of their eggs were subsequently fertilized monospermically. During the progression of fertilization when the eggs were still surrounded by compact cumulus oophorus, the contents of the ampullary region of the oviducts were collected and spermatozoa moving in the ampullary fluid, within the cumulus and on/in the zonae pellucidae of unfertilized eggs, were examined by light and electron microscopy to evaluate the status of their acrosomal caps. Most spermatozoa swimming in the ampullary fluid had apparently intact acrosomal caps, while the vast majority moving within the cumulus had distinctly modified acrosomal caps. Most spermatozoa that had passed through the cumulus and reached the zona surfaces had remnants of their acrosomal caps (“acrosomal ghosts”). When the ghosts were present around the sperm heads on the zona, the heads pivoted about a point roughly corresponding to the places where the ghosts were located. The ghosts seemed to firmly attach to the zona surfaces, then were split open by the sperm heads and left behind as the sperm heads advanced into the zona. A few spermatozoa on the zona surfaces had no acrosomal ghosts (at least not detectable by light microscopy). In this case, the sperm head pivoted about either the inner acrosomal membrane or the equatorial segment of the acrosome. In no instance were spermatozoa with intact acrosomal caps found on zona surfaces. We infer from these observations that most spermatozoa in vivo initiate their acrosome reactions while they are advancing through the cumulus. When they arrive at the zona surfaces, acrosomal ghosts are generally present on the sperm heads. These ghosts appear to hold sperm heads to zona surfaces as well as to restrict the direction of advancement of sperm head through the zona. In a minority of cases, ghostless spermatozoa reach the zona surfaces. As these spermatozoa appear to be able to penetrate the zona successfully, structures other than the acrosomal ghost (ie, the inner acrosomal membrane and the plasma membrane over the equatorial segment of the acrosome) may also attach to zona surfaces before spermatozoa penetrate into the zona.     

Phosphatidylcholine and phosphatidylinositol-specific phospholipases C of bull and rabbit spermatozoa.

Acrosomal reaction is an essential prerequisite to fertilization. The changes in lipid composition of sperm membranes cause fusion of the plasma and outer acrosomal membranes that results in the exocytosis of acrosomal contents. We report that both bull and rabbit spermatozoa contain a phosphatidylcholine-specific phospholipase C (PC-PLC) that hydrolyzes L-alpha-dipalmitoyl-(choline-methyl-14C-153.0 Ci/mmol and a phosphatidylinositol-specific phospholipase C (PI-PLC) that hydrolyzes L-alpha-(Myo-Inositol-2-3H (N)-5.2 Ci mmol. PI-PLC from bull sperm acrosome has been purified 568 x fold with a specific activity 6.25 +/- 0.6 nmol/min/mg protein, km 0.004 mM, and Vmax 12 nmol/min/mg protein. Both enzymes had optimum at pH 7.5. The activity of PC-PLC remained unaffected by varying concentrations of Ca2+, whereas PI-PLC activity was significantly increased. The bulk of PI-PLC was found to be associated with inner acrosomal membrane of bull and rabbit sperm, while PC-PLC was found in the outer acrosomal membranes in the bull sperm and the plasma membrane of the rabbit sperm. Both enzymes are compartmentalized in sperm cell.     

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.     

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.     

First attempt to characterize 3H-fucose acrosomal label in spermatoza

Acrosomes of rabbit spermatozoa were labelled by tritiated fucose introduced into the cells during spermatogenesis. The labelling was analysed simultaneously by autoradiography and biochemically. In compact intact acrosomes the labelling was confined strictly to the acrosomal region of the sperm head. In swollen and detached acrosomes the autoradiographic grains were associated mostly with the acrosomal cap. Only in some cells a small proportion of radioactivity was detected to be associated with denuded sperm heads. In acrosomal extracts a considerable share of radioactivity coincided with gel filtration fractions showing esterase activity (BAEE-N-alpha-benzoyl-L-agrinine ethyl ester splitting), akin to that exhibited by acrosin.     

First attempt to characterize 3H-fucose acrosomal label in spermatozoa

Summary Acrosomes of rabbit spermatozoa were labelled by tritiated fucose introduced into the cells during spermatogenesis. The labelling was analysed simultaneously by autoradiography and biochemically. In compact intact acrosomes the labelling was confined strictly to the acrosomal region of the sperm head. In swollen and detached acrosomes the autoradiographic grains were associated mostly with the acrosomal cap. Only in some cells a small proportion of radioactivity was detected to be associated with denuded sperm heads. In acrosomal extracts a considerable share of radioactivity coincided with gel filtration fractions showing esterase activity (BAEE-Nalpha-benzoyl-l-arginine ethyl ester splitting), akin to that exhibited by acrosin.     

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.     

Localization of antigens on the inner-acrosomal membrane of the rabbit sperm by immunofluorescence

The acrosome-reacted spermatozoa interact with the zona pellucida through their limiting inner acrosomal membrane (IAM). The antigenic properties of IAM were determined using the antibody to IAM raised in a male guinea pig. The antisera was incubated with the acetone powder of rabbit lung, liver, kidney, heart, muscle, and blood cells. The unabsorbed antibodies specifically interacted with antigens on the IAM as determined by the immunofluorescence technique.     

Identification and characterization of RING-finger ubiquitin ligase UBR7 in mammalian spermatozoa

The ubiquitin-proteasome system (UPS) controls intracellular protein turnover in a substrate-specific manner via E3-type ubiquitin ligases. Mammalian fertilization and particularly sperm penetration through the oocyte vitelline coat, the zona pellucida (ZP), is regulated by UPS. We use an extrinsic substrate of the proteasome-dependent ubiquitin-fusion degradation pathway, the mutant ubiquitin UBB⁺¹, to provide evidence that an E3-type ligase activity exists in sperm-acrosomal fractions. Protein electrophoresis gels from such de novo ubiquitination experiments contained a unique protein band identified by tandem mass spectrometry as being similar to ubiquitin ligase UBR7 (alternative name: C14ORF130). Corresponding mRNA was amplified from boar testis and several variants of the UBR7 protein were detected in boar, mouse and human sperm extracts by Western blotting. Genomic analysis indicated a high degree of evolutionary conservation, remarkably constant purifying selection and conserved testis expression of the UBR7 gene. By immunofluorescence, UBR7 was localized to the spermatid acrosomal cap and sperm acrosome, in addition to hotspots of proteasomal activity in spermatids, such as the cytoplasmic lobe, caudal manchette, nucleus and centrosome. During fertilization, UBR7 remained with the ZP-bound acrosomal shroud following acrosomal exocytosis. Thus, UBR7 is present in the acrosomal cap of round spermatids and within the acrosomal matrix of mature boar spermatozoa. These data provide the first evidence of ubiquitin ligase activity in mammalian spermatozoa and indicate UBR7 involvement in spermiogenesis.     

Fucoidin inhibits attachment of guinea pig spermatozoa to the zona pellucida through binding to the inner acrosomal membrane and equatorial domains

Our previous study has shown that fucoidin, an algal heteropolysaccharide, is a potent inhibitor of sperm-zona binding in the guinea pig, hamster and human. To visualize the surface site of fucoidin binding, a biotinated derivative (B-Fuc) of the native fucoidin was prepared. B-Fuc retained the inhibitory activity and was used in conjunction with FITC-avidin to localize its binding sites on guinea pig spermatozoa using fluorescence microscopy. In living acrosome-reacted spermatozoa, B-Fuc bound predominantly to the inner acrosomal membrane and equatorial segment domains. The binding was effectively competed by a 10-fold excess of native fucoidin, but not by a 10-fold excess of heparin or a 20-fold excess of biotinated normal rabbit serum IgG. B-Fuc binding patterns on dead spermatozoa were quite different from that of living spermatozoa. The post-acrosomal region, rather than the inner acrosomal membrane and equatorial domains, was intensely labeled. This indicates the importance of using living cells in assessing true surface binding sites whenever possible. We conclude that the inner acrosomal membrane and/or equatorial domains are critical for zona binding in the guinea pig.     

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.     

Zonadhesin: characterization, localization, and zona pellucida binding.

Zonadhesin is a multiple-domain transmembrane protein that is believed to function as a sperm-zona pellucida binding protein. In this study we sequenced zonadhesin from rabbit testis and analyzed its processing, expression, localization, and zona pellucida binding. We show that the precursor protein occurs exclusively in the testis and that proteolytic processing results in the formation of three fragments: p43 (D1 domain), p97 (D2-D4 domains), and p58 (D4 domain-C-terminal). In mature spermatozoa the p43 and p97 fragments exist as disulfide-bonded dimers. During spermatogenesis, synthesis of zonadhesin mRNA chiefly occurs in primary spermatocytes, whereas the protein is abundant in both Sertoli cells and spermatids. In spermatozoa the protein is localized exclusively to the anterior acrosome but is not available for binding antibody on live spermatozoa. Once the acrosome reaction is induced, zonadhesin is lost from the spermatozoon, but remains with the acrosomal shroud. We show that recombinant D4 domain can bind zona pellucida, and we propose that zonadhesin functions after the acrosome reaction has been initiated to bind the acrosomal shroud to the zona pellucida.     

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.     

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.     

Identification of a zona-binding protein from boar spermatozoa as proacrosin

The initial stages of fertilization in vertebrates and invertebrates are thought to involve complementary recognition molecules on spermatozoa and eggs. In a previous work (C. R. Brown and R. Jones, 1987, Development) we described one such putative molecule (a protein of approximate molecular weight 53 kDa) in detergent extracts of boar spermatozoa that has affinity for glycoproteins from the zona pellucida of pig eggs. This molecule has now been identified as proacrosin, the zymogen form of the acrosomal protease acrosin, on the basis of its electrophoretic behavior, the ability of zona glycoproteins to recognize and bind to proacrosin on Western blots, and the cross-reactivity of specific antisera to the 53-kDa molecule and proacrosin. A role is proposed for this enzyme in binding the sperm head to the zona pellucida during the initial stages of sperm-egg interaction.