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a Laboratory of Embryology, Faculty of Biological Science, Pontifical Catholic University of Chile, Santiago, Chile
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Secondary binding involves ionic interactions between polysulfated groups on the ZPGPs and basic residues on proacrosin [5–7]. The fact that proacrosin and ß-acrosin have the same binding capacity to polysulfated groups on ZPGPs suggests that the carboxy terminal end of proacrosin is not involved in this binding [8, 9]. The density and orientation of sulfated groups on ZPGPs seem to be critical parameters mediating recognition and binding, since small changes in stoichiometry are responsible for the species specificity of fertilization [6]. A heteropolysaccharide of sulfated fucose—fucoidan—inhibits binding between the sperm and the ZP in several mammals [10]. Sulfated fucose has therefore been proposed as part of the recognition signal of the ZPGPs with regard to acrosin in human spermatozoa [11, 12].
Recently, participation of boar ß-acrosin in the binding to ZPGPs was confirmed [13–15]. Using synthetic peptides and recombinant proteins, Jansen et al. [13] found that a fragment that included residues 3 to 275 was able to bind 125I-ZPGPs in the same way as native proacrosin. Later, Crosby et al. [14] confirmed these results by showing that the whole molecule of boar ß-acrosin was necessary to achieve a binding capacity similar to that of proacrosin. Furthermore, they demonstrated that the catalytic activity of acrosin was not necessary for the maintenance of binding to ZPGPs. On the other hand, Jansen et al. [15] showed, using site-directed mutagenesis, that two groups of basic residues on the boar ß-acrosin surface—His47, Arg50, and Arg51, together with Arg250, Lys252, and Arg253—were crucial for the maintenance of binding to ZPGPs. Deletion or change of these amino acids severely reduced the affinity of the binding.
The binding site of rabbit acrosin to the ZP has also been determined [16]. The authors of that study produced a recombinant rabbit ß-acrosin protein of only 48 amino acids that was able to bind ZPGPs with 50% of the maximal binding capacity. Using site-directed mutagenesis, they identified three arginines (Arg47, Arg50, and Arg51) as the critical residues permitting ZP binding. Moreover, they used three-dimensional protein modeling of rabbit proacrosin to suggest that these basic residues were localized on the surface of the molecule, very near to the catalytic site.
All these results support the hypothesis that the polysulfate binding sites of the proacrosin/acrosin system are formed by a restricted number of basic amino acids, localized on the surface of the molecule, with specific orientation complementary to the sulfate groups of the ZPGPs. Therefore, the aim of the present investigation was to study the contribution of boar ß-acrosin to the secondary binding to the intact ZP. We used two boar recombinant proteins: ß-acrosin and a mutant of the catalytic site, ß-acrosin Ser/Ala222 [14]. We demonstrated the binding capacity of iodinated recombinant protein to intact ZPs and determined that this binding is distributed on the entire surface of the ZP. We also showed that the recombinant proteins did not affect gamete interaction.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recombinant boar ß-acrosin proteins were produced and purified as described elsewhere [14]. We used two recombinant boar ß-acrosin truncated proteins: one normal, and one a mutant of the catalytic site, Ser/Ala222. Analysis of proteolytic activity of both recombinant protein stocks revealed that they were inactive.
Proacrosin was extracted from boar spermatozoa at pH 3.0, as described previously [14]. For the binding assays, approximately 10 ng of recombinant ß-acrosin protein or native proacrosin was iodinated with 125I-Na and Iodogen (Pierce Chemical Co., Rockford, IL) as described elsewhere [13].
Isolation of the ZP
Pig ovaries were obtained from a local slaughterhouse and brought to the laboratory within 2 h of slaughter. ZPs were isolated through nylon mesh screens as described elsewhere [17]; purified on a 10%, 20%, and 30% Percoll gradient; washed several times in PBS (NaCl, 136.89 mM; Na2HPO4, 8.45 mM; NaH2PO4, 1.66 mM; KCl, 2 mM; pH 7.4); and used immediately.
Protein Binding to the Intact ZP and Competition Assay (Experiment 1)
To quantify specific binding of iodinated recombinant ß-acrosin or native proacrosin to intact ZPs, we incubated increasing concentrations of iodinated proteins for 3 h at 39°C with 100 ZPs in BSA-blocked Eppendorf tubes. In the competition assays, 100 ZPs were incubated for 1 h with 10 µg/ml "cold" proacrosin or 100 µM fucoidan; then the samples were incubated for another hour with 100 000 cpm of recombinant ß-acrosin protein (10.8 cpm/pmol) or with iodinated native proacrosin (33.5 cpm/pmol) in 100 µl of sterile PBS and subsequently washed three times. In each experiment, ZPs were changed from one solution to another by hand using fine-bore pipettes under a dissecting microscope. Radioactivity binding was detected in a gamma counter (LKB Wallac 1261 Multigamma Counter; Rockville, MD). Nonspecific binding was defined as the amount of probe remaining on the tube in the absence of ZPs, and specific binding as the amount of probe bound to ZPs minus the nonspecific background binding. Maximum binding was defined as the amount of probe bound to ZPs in the presence of the target protein but in the absence of the competing agent.
Autoradiographic Assay (Experiment 2)
To determine the localization of iodinated ß-acrosin recombinant bound to intact ZP, we designed a micro-autoradiographic system using the samples processed as above. ZPs incubated with 100 000 cpm of the iodinated recombinant protein (with or without competitor) were fixed for 15 min with 2.5% glutaraldehyde in PBS. After washing, ZPs were immobilized over glass slides previously treated with 0.05% poly-L-lysine (Sigma Chemical Co., St. Louis, MO). Finally, the slides were covered with Hypercoat LM-1 (Amersham Life Science, Buckinghamshire, UK) autoradiographic emulsion, exposed, and developed as described by the manufacturer. ZPs were analyzed under an Olympus (Burlingame, CA) BH-2A brightfield microscope attached to a CCD IRIS/FGB Sony (Park Ridge, NJ) video camera. The images were digitized with Xclaim Video Player 1.0 software (ATI Technologies, Thornhill, ON, Canada). The three-dimensional densitometry utilized Scion Image software (Scion Corporation, Frederick, MD; http://www.scioncorp.com).
Sperm Collection and Preparation
Sperm-rich semen fractions, from the ejaculates of three Large White boars of proven fertility, were obtained from the breeders, Sociedad Agrícola El Monte (El Monte, Chile). Precautions were taken during collection to avoid cold-shock damage to the spermatozoa. The semen was filtered through gauze; the sperm concentration was adjusted to 1.5 x 108 spermatozoa/ml with Beltsville thawing solution extender [18], and the suspension was maintained at 15°C in the dark for up to 48 h.
For the fertilization assay, 3 ml of the diluted semen was washed by centrifugation through a two-step Percoll gradient (35% and 70%) [19]. The sperm pellet was resuspended in 0.3 ml of the lower-percentage Percoll medium, and the final concentration was calculated. Finally, spermatozoa were resuspended in a capacitation medium (113 mM NaCl, 2.95 mM KCl, 20 mM Tris, 10.98 mM D-glucose, 10 mM sodium pyruvate, 7.7 mM CaCl2) supplemented with 4 mg/ml of BSA (A-4378; Sigma) at a concentration of 50 x 106 spermatozoa/ml and incubated for 1.5 h at 39°C in an atmosphere of 5% CO2 in air, for use in in vitro fertilization assays.
Maturation of Pig Oocytes In Vitro
Ovaries were obtained from a local slaughterhouse and washed three times in prewarmed PBS, and cumulus-oocyte complexes were aspirated from 3- to 5-mm-diameter follicles using a 38-gauge syringe. Pig oocyte in vitro maturation followed published protocols [20–23]. Briefly, groups of 20 oocyte complexes, each with a compact cumulus, were cultured in maturation medium with hormonal supplement for 24 h at 39°C in an atmosphere of 5% CO2 in air. The complexes were cultured for a further 24 h in maturation medium without hormonal supplement, after which expanded cumulus cells were removed by a 4-min vortex treatment. Finally, the oocytes that presented evenly granulated cytoplasm and normal appearance [22, 23] were selected manually under a dissecting microscope, washed once in fertilization medium, and placed in groups of 10 oocytes in 50-µl drops under mineral oil in 35-mm Petri dishes (Falcon 3001; Becton Dickinson, Lincoln Park, NJ).
The basic maturation medium consisted of tissue culture medium 199, pH 7.4, supplemented with 10% fetal calf serum (Life Technologies, Gaithersburg, MD), 3.05 mM D-glucose, 2.92 mM calcium lactate, 0.91 mM sodium pyruvate, 0.57 mM L-cysteine, 12 mM taurine, and 1 µg/ml of gentamycin. The hormonal supplement consisted of 10 U/ml of hCG, 10 U/ml eCG, and 1 µg/ml of estradiol-17ß (all from Sigma). Fertilization medium consisted of basic maturation media without hormonal supplement, supplemented with 2 mM caffeine and 4.5 mM CaCl2.
Effect of Boar Recombinant ß-Acrosin Proteins on In Vitro Fertilization (Experiment 3)
To determine whether the recombinant proteins had any effect on sperm penetration of the ZP in a fertilization event, we incubated a group of the denuded matured oocytes with 100 pmol/zona of each recombinant ß-acrosin protein, for 3 h at 39°C, in 50 µl of fertilization medium. Capacitated spermatozoa were added to the drops containing oocytes, to reach a final concentration of 1–1.5 x 106 cells/ml, and coincubated for an additional period of at least 3 h at 39°C in 5% CO2 in air. Finally, the inseminated eggs were washed using a fine-bore pipette of diameter slightly greater than that of the oocyte (approximately 150 µm), fixed with 2.5% glutaraldehyde, and stained with 200 µg/ml of propidium iodide; the fertilization state was analyzed using a confocal microscope (Zeiss LSM-410 Axiovert; Carl Zeiss, Thornwood, NY).
To analyze the acrosomal state of the spermatozoa attached to the ZP, some of the fixed eggs were processed for scanning electron microscopy. Briefly, the inseminated eggs were fixed, dehydrated in a series of acetone of increasing concentration, critical point dried in CO2 (Sorvall, Newtown, CT; model 49300), sputter-coated with palladium-gold (SEM Coating Unit 9000; PELCO, Redding, CA), and analyzed using a scanning electron microscope (Jeol-JSM-25 SII; Peabody, MA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recombinant ß-acrosin protein bound to intact ZP (Fig. 1), with the binding increasing in proportion to the concentration of the probe. Binding was maximal at 3 h of incubation (Fig. 2) and did not differ from the binding of native proacrosin. Moreover, the binding was stable over time; more than 90% of the radioactive probe remained with the zonae after they were washed, stored for a further 12 h at 39°C, and washed again (data not shown).
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Two different competitors, "cold" proacrosin and fucoidan, were able to displace each of the probes, iodinated ß-acrosin and iodinated proacrosin (Fig. 3). Maximal binding of the control was 2063 cpm for ß-acrosin recombinant protein and 3030 cpm for proacrosin. Cold proacrosin (10 µg/ml) inhibited 55% of the binding of the recombinant protein and 76% of the native proacrosin binding capacity. Fucoidan (100 µM) inhibited approximately 44% of the binding capacity of each probe.
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Localization of Binding to the Intact ZP (Experiment 2)
The micro-autoradiographic assay, described earlier, enabled us to visually localize the binding of 125I-ß-acrosin to intact ZPs. After 1.5 h of incubation, the distribution of binding was heterogeneous over the surface of the zona (Fig. 4A), while after 3 h of incubation the radioactive label had covered the whole ZP (Fig. 4B). Competition with 10 µg/ml of cold proacrosin resulted in almost no label on the ZP (Fig. 4C). Densitometric representations of the radioactive probe bound to the ZP clarified the amount of binding. These are shown at the bottom of each digitized image (Fig. 4, D–F).
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Effect of Recombinant Boar ß-Acrosin Proteins on In Vitro Fertilization (Experiment 3)
When pig oocytes were treated with recombinant boar ß-acrosin proteins and then inseminated in vitro with capacitated boar sperm, the motility of the sperm was not affected, and the spermatozoa were able to bind in great numbers (more than 60 sperm per ZP; data not shown). The numbers of bound spermatozoa per ZP of the treated oocytes did not differ from the numbers bound to the control oocytes. Scanning electron microscopy of similarly treated eggs revealed that this treatment did not block ZP induction of the acrosome reaction (Fig. 5). Treated inseminated oocytes were also evaluated for sperm penetration using confocal microscopy. We defined as penetrated eggs all inseminated oocytes that showed sperm heads in the thickness of the ZP, in the perivitelline space, or in the oocyte cytoplasm or those that contained a sperm pronucleus. Based on these criteria, fertilization rates were 63 ± 26% in controls, 51 ± 11% in competition with recombinant ß-acrosin protein, and 56 ± 15% in competition with ß-acrosin Ser/Ala222 (Fig. 6 and Table 1). These values were not significantly different (chi-square test, P < 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this work, we have proved that recombinant ß-acrosin and native proacrosin are able to bind to intact ZPs with equal binding capacity. This binding occurs in a manner dependent on the dose of the iodinated recombinant protein and reaches saturation after 3 h of incubation. This confirms our previous work using solubilized ZPGPs [14]. We have also shown that the binding is specific; it was inhibited by 10 µg/ml of cold native proacrosin and by 100 µM of fucoidan, thus confirming our dot-blot results [14]. However, the inhibition was not 100%, as indicated by the dot-blot assays, possibly because it would be more difficult for the competitor to dissociate the interaction between an intact zona and a purified protein in solution than between two purified proteins in solution.
We showed that the binding between recombinant ß-acrosin protein and intact ZPs was maintained for up to 12 h after coincubation. This indicates that under the present experimental conditions, in which recombinant ß-acrosin was not enzymatically active, ß-acrosin was unable to break its interaction with ZPGPs. If the recombinant protein had been enzymatically active, it would have digested its ligand and detached itself from the ZP. The present results do not, therefore, invalidate the binding-releasing model [2, 16], which explains the process of sperm penetration through the ZP. The model considers that the process of enzymatic activation, induced by the ZPGPs, occurs over the zymogen, proacrosin, which is normally anchored to the acrosomal matrix.
Using the technique of micro-autoradiography that we developed for this work, we observed recombinant ß-acrosin protein binding over the entire surface of the ZP. We demonstrated that this binding was initially distributed heterogeneously and finally covered the entire ZP. This evidence suggests that there could be two types of binding sites for ß-acrosin in the ZP: sites of high binding affinity, distributed heterogeneously over the ZP and occupied first, and other sites of lower affinity that permit ß-acrosin to cover the entire surface of the ZP. More work needs to be done to confirm this hypothesis, but it could have paramount implications for the definition of selected sites of sperm penetration.
From these results, we conclude that recombinant ß-acrosin specifically and selectively binds over the surface of the intact ZP. This validates the assays, discussed below, that were used to determine the role of ß-acrosin in sperm penetration through the ZP.
Role of ß-Acrosin in Sperm Penetration
The rates of in vitro fertilization by boar sperm in the presence of recombinant ß-acrosin proteins (normal or the catalytic site mutant Ser/Ala222) did not differ significantly from rates for controls. They correlated well with published data, according to which fertilization rates average around 70%, with high incidence of polyspermy [21, 23–26]. We did not use native proacrosin as a competitor because we know that when incubated with the ZP, it influences fertilization rates by causing proteolytic modification of the ZP rather than by blocking acrosin binding sites (unpublished results).
From these results, we conclude that under the present experimental conditions, blocking acrosin binding sites does not affect the process of sperm penetration through the ZP. This may be explained by one of the following mechanisms: 1) when all the secondary binding sites are blocked by the recombinant protein, the native proacrosin, present in the acrosomal matrix of the reacted spermatozoa, may have higher affinity for the ligands and therefore displace the recombinant protein already at the site; 2) native proacrosin, present in the acrosomal matrix of the penetrating spermatozoa, may become enzymatically active, break through the blocked acrosin binding sites, and bind to inner binding sites; 3) other molecules present in the acrosomal matrix of the reacted spermatozoa may be responsible for the secondary binding, by binding to sites different from those used by acrosin [27–29]. This last idea is consistent with recent results using proacrosin gene knockout mice [3, 4]. These mice carry a targeted mutation of the proacrosin gene and are fertile in vivo. However, while their sperm were able to fertilize homologous oocytes in vitro, this took 30 min longer than in the controls [3]. Moreover, Adham et al. [4] showed that in in vitro fertilization assays in which wild-type spermatozoa competed with those lacking a functional acrosin gene, only wild-type spermatozoa were able to fertilize the oocytes. They concluded that spermatozoa lacking the acrosin gene had a selective disadvantage compared with normal sperm. On the other hand, new acrosomal serine proteases, which are exposed after the acrosome reaction, have recently been identified [27–29]. Their deduced amino acid sequences indicate that they are synthesized as zymogen forms, and therefore they must be processed into a mature enzyme to exhibit enzymatic activity. Because of the characteristics of their cleavage site, its activation might depend on a trypsin-like protease, as yet unknown. However, one of these proteases is most likely to be an inactive form due to the replacement of one of the amino acids of the catalytic triad. Nevertheless, these proteases may be other candidates, besides acrosin, for sperm-egg secondary binding.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: J.A. Crosby, Laboratorio de Embriología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Casilla 114-D, Alameda 340, Santiago, Chile. FAX: 56 2 222 5515; jcrosby{at}latinmail.com
Accepted: July 20, 1999.
Received: May 21, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This article has been cited by other articles:
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  K. J. Mengerink and V. D. Vacquier Glycobiology of sperm-egg interactions in deuterostomes Glycobiology, April 1, 2001; 11(4): 37R - 43R. [Abstract] [Full Text] [PDF]
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