Immunocontraception of captive exotic species. III. Contraception and population management of fallow deer (Cervus dama)

Immunocontraception has become an increasingly valuable tool in the population management of captive exotic ungulates. Although porcine zona pellucida vaccine (PZP) was used successfully in other cervids, a previous study with fallow deer (Cervus dama) suggested that the vaccine did not work in this species. In the current study, PZP was tested in two captive herds of fallow deer. Antibody titers were monitored over a 3‐year period to evaluate three different adjuvant protocols, and the vaccine was applied to an entire herd to determine the impact on fawning rates. In a semi‐free‐ranging herd, antibody titers rose from preimmunization levels of 2.6% of positive control serum to 56.5% 4 weeks after initial inoculations, to 65.1% at 1 year, and to 81.3% at 2 years, after a single annual booster was applied. Fawn production in this herd was reduced significantly over 3 years. The adjuvant protocol of Freund's Modified Adjuvant® (FMA) for the initial inoculation followed by a booster with Freund's Incomplete Adjuvant® (FIA), and the protocol of FMA for the initial inoculation followed in 3 weeks by a booster with FMA both produced significantly higher antibody titers than the 3× FIA (3 weeks apart) protocol after year 1. The FMA+FMA protocol produced significantly higher titers than the 3× FIA protocol at year 2, but was not different from the titers produced by the FMA+FIA protocol at year 2. Zoo Biol 22:261–268, 2003. © 2003 Wiley‐Liss, Inc.     

Solubilization of porcine zonae pellucidae by trypsin and pronase

The porcine zona pellucida was dissolved with difficulty by trypsin in isotonic solution, whereas it was efficiently dissolved by pronase. A structural change of the zona was induced in hypotonic solution, resulting in acceleration of dissolution by these proteases. The solubilization rate of three families (PZP1-3) of zona protein by both enzymes was analyzed by HPLC. In hypotonic solution, PZP1 was solubilized first, followed by PZP2; and PZP3 was then finally released. In isotonic solution, PZP1 and PZP2 were also solubilized faster than PZP3, which was almost completely resistant to trypsin, showing that the solubilization of the zona depended on that of PZP3. Noticeably, high molecular weight products were produced as the proteolytic hydrolysis proceeded in PBS. Circular dichroic spectra and electrophoretograms of the tryptic hydrolysates showed that the zona may have a regular supramolecular structure.     

Immunocontraception in Wild Horses: One Inoculation Provides Two Years of Infertility

ABSTRACT ABSTRACT Previous studies reported one year of contraception associated with a 1-injection porcine zona pellucida (PZP) vaccine. We have subsequently determined contraceptive effectiveness of a presumptive 1-injection, 2-year-duration PZP vaccine in free-roaming wild horses (Equus caballus) in Nevada, USA. In January 2000, we captured, freeze-branded, treated, and subsequently released 96 adult females that received 1) a primary dose of vaccine emulsion consisting of aqueous PZP and Freund's Complete Adjuvant, and 2) booster doses of PZP and adjuvant in controlled-release polymer pellets. We determined PZP release characteristics of pellets in vitro, prior to field use. We determined reproductive success in treated and untreated females through October 2004 via measurement of estrone sulfate and progesterone metabolites in fresh feces collected from the ground and by twice-annual foal counts. Among treated females, annual reproductive success from 2001 though 2004 sequentially was 5.9%, 14.0%, 32.0%, and 47.5%. Untreated females showed average reproductive success of 53.8 ± 1.3% across this period. This study revealed that: 1) PZP acted as an effective contraceptive for 2 years posttreatment; 2) some residual contraceptive effect remained in year 3; and 3) fertility returned to control levels by year 4 posttreatment. It appears that controlled-release technology can replace both the second(1-month) and annual booster injection of PZP vaccine, thereby decreasing cost and increasing efficiency of use of this vaccine in wild horse management.     

Use of mucosal immunization with porcine zona pellucida (PZP) in mice and rabbits

Rabbits (Oryctolagus cuniculus) and two strains of mice (Mus musculus, one inbred and one outbred) were immunized against porcine zona pellucida (PZP) antigen. Alginate microspheres or cholera toxin B were used alone or in combination when mucosal immunization routes were used. Serum antibody responses and fertility were assessed. Neither rabbit or mouse groups immunized by mucosal routes generated significant antibody responses to PZP as compared to parenteral immunization (ANOVA, P > 0.05). The study shows that porcine zona pellucida is not an effective mucosal antigen in small mammals.     

Immunocontraception of captive exotic species. I. Przewalski's horses (Equus przewalskii) and banteng (Bos javanicus)

A contraceptive vaccine made of porcine zonae pellucidae (PZP) was tested in three Przewalski's mares and five banteng cows. The vaccine antigen consisted of the complete family of glycoproteins of the porcine zona pellucida, including the sperm receptor ZP3. All mares and three of five banteng were inoculated with 2 or 3 i.m. injections of approximately 65 μg of antigen given over a 6 week period. Two other banteng received inoculations of only 35 μg of antigen on the same schedule. Two of the three mares and three of five banteng cows were pregnant at the time of inoculation. No new pregnancies, as a result of postinoculation breedings, occurred among either the mares 36 months after 65 μg antigen inoculations or among the banteng for 24 months after 65 μg inoculations. One postinoculation pregnancy resulted among the two banteng receiving only 35 μg of antigen. Differences in fertility between treated and control mares and between preinoculation and postinoculation reproductive performance of the banteng were significant (P < 0.05). Urinary ovarian steroid metabolites and behavioral observations indicated follicular development and ovulations were occurring among treated mares during the year following PZP inoculations. PZP immunization produced progressively elevated anti-PZP antibodies in both species, which provided contraceptive protection. PZP immunization appears to be an effective form of contraception in both species. © 1995 Wiley-Liss, Inc.     

Molecular analysis of the antigenicity and immunogenicity of recombinant zona pellucida antigens in a primate model.

The studies reported here are the first to demonstrate that recombinant zona pellucida (ZP) proteins will elicit a humoral immune response that recognizes native ZP proteins. Three cDNAs encoding rabbit ZP protein antigens expressed in bacteria were used to immunize cynomolgus monkeys. Four groups of six monkeys each were immunized with bacterially expressed cro-beta-galactosidase recombinant proteins encoded by a full-length cDNA (rc55) encoding the 55-kDa rabbit ZP recombinant protein (rec55), two partial cDNAs (rc75a and rc75b) encoding two recombinant peptides (rec75a and rec75b) of the 75-kDa rabbit ZP protein, and the plasmid-encoded cro-beta-galactosidase control protein. Initial immunizations with these fusion proteins using the muramyl dipeptide adjuvant did not elicit significant levels of antibodies to native or recombinant ZP proteins. Further immunizations were therefore carried out using recombinant ZP proteins conjugated to either protein A or keyhole limpet hemocyanin. Antibodies were detected in the groups immunized with the rec55 and rec75a; however, no antibodies were generated against the rec75b protein. These antibodies have been characterized by two-dimensional PAGE immunoblotting and shown to recognize antigenic domains associated with two of the native rabbit ZP proteins. Reprobes of these immunoblots with sheep anti-total native rabbit ZP proteins, affinity-purified on pig ZP, further demonstrate that a fourth distinct rabbit ZP antigen may be present. The characterization of species-conserved antigenic domains of mammalian ZP proteins is important for studies of the functional regions of ZP proteins and is critical for the design of safe and effective contraceptive vaccines.     

Immunocontraception of captive exotic species

The biological efficacy of porcine zona pellucida (PZP) immunocontraception and the timing of booster inoculations were determined in 24 species of ungulates and five species of non‐ungulates across 10 years of treatment and for which no technical problems were identified. The collective contraceptive efficacy for 265 ungulates, 25 bears, and 11 sea lions, across 517 contraceptive intervals was 93.3%, and contraceptive efficacy ranged from a low of 60% in nyala (Taurotragus angasi) to 100% in 16 other species. The timing of annual booster inoculations was adjusted, between 6 months and 12 months, depending on the length of the breeding season and the species‐specific immune responses. The high degree of efficacy in bears and sea lions suggests that the PZP vaccine has some application in certain non‐ungulate species. Technical problems in the timing and delivery of the vaccine can cause non‐biological failures and were identified in an additional 20 animals, not included above, and included inoculating pregnant animals, the use of needles too short for effective intramuscular injections, failure to complete the initial inoculation series, and failure to give booster inoculations at the prescribed time. Zoo Biol 0:1–10, 2005. © 2005 Wiley‐Liss, Inc.     

Estimating Abundance and Simulating Fertility Control in a Feral Burro Population

Overabundant populations of feral equids are negatively affecting rangelands in the western United States. To better manage these populations, robust estimates of abundance and demographic rates and cost-effective methods of reducing abundance are necessary. From August 2015 to April 2017, we estimated the abundance of feral burros (Equus asinus) at the Fort Irwin National Training Center (NTC; California, USA) using a double-observer-sightability aerial survey method; captured, radio-collared, and inoculated female burros with porcine zona pellucida (PZP), an immunocontraceptive control agent; estimated female demographic rates; and used matrix population models to simulate how changes in demographic and PZP delivery rates would influence burro abundance. We estimated there were 690 (95% CI = 618–752) feral burros within the surveyed area, but these are part of a much larger population that is not geographically isolated from those in the survey area. Sighting probabilities ranged from 0.19–0.98 and were most strongly influenced by distance from observer and group size. We estimated age-specific demographic rates at the NTC and compiled mean rates across burro populations in arid environments from the literature. Mean fecundity varied from 0.17 to 0.58 foals/adult female with younger females having lower fecundity. Mean survival was 0.90 for foals, 0.98 for yearlings, and 0.96 for adults. The PZP vaccine treatment strategies that suppressed fertility for up to 10 years predicted that burro abundance would be reduced by 67–88% after 15 years (compared with no treatment), but none of these strategies resulted in population extirpation. Population growth rates shifted from increasing to decreasing at adult survival rates below 0.84 and the population was predicted to become extirpated when adult survival declined to <0.60. In the absence of other methods to reduce burro numbers, our findings indicate that current formulations of PZP immunocontraception, which require multiple doses, would be inadequate for controlling population growth rates at the NTC. Our fieldwork also highlighted the difficulty of administering PZP vaccination to large, free-ranging animals. Development of longer-term fertility reduction agents or more efficient vaccine delivery techniques would likely improve the efficacy of fertility control for overabundant ungulate populations. Lack of geographic closure (physical barriers to migration) further complicated efforts to reduce burro numbers. © 2021 The Wildlife Society.     

Inhibition of pig oocyte in vitro fertilization by the action of components of the zona pellucida

The aim of the present study was to determine whether the previous addition of porcine zona pellucida (ZP) components to spermatozoa of the same species has an inhibitory effect on in vitro fertilization (IVF). Boar spermatozoa were exposed to whole porcine solubilized zona pellucida (SZP), ZP glycoproteins (55 kDa and 90 kDa) and peptides (37 kDa, 40 kDa and 68kDa). Doses tested were 40, 70 and 100 mug/ml. In vitro fertilization was clearly inhibited by each component when the oocytes were compared with those fertilized with untreated spermatozoa. All the components had an effect in a dose dependent manner.     

Expression of the rabbit sperm protein Sp17 in cos cells and interaction of recombinant Sp17 with the rabbit zona pellucida

This study extends our analysis of rabbit recombinant Sp17 (rSp17) by examining whether rSp17 synthesized in transfected COS cells will show a particular localization within the cell and whether the COS cell will bind with zona pellucida. We show, using the cross-linking, reagent DSS that rSp17 can bind to rabbit zona glycoprotein R45 or R55. © 1995 Wiley-Liss, Inc.     

Preliminary evaluation of porcine zona pellucida (PZP) immunocontraception for behavioral effects in feral horses (Equus caballus)

Successful management of captive populations of wild animals requires effective control of reproduction. Contraception is one tool for controlling reproduction of animals in zoos; however, the options available to the animal manager are limited. Contraceptives vary in efficacy, reversibility, and side effects, and thus may not be suitable for widespread use. One consideration when selecting a contraceptive is its potential for side effects on behavior, especially given the fact that reproduction plays such a prominent role in the biology of any species. To date, there have been few evaluations of contraceptives for behavioral effects, and those that have been conducted have focused on hormone-based contraceptives. This study sought to evaluate a novel method of population control, immunocontraception, for behavioral effects in a population of feral horses. Porcine zona pellucida (PZP) immunocontraception prevents fertilization of ova and does not alter normal hormone secretion patterns. It therefore should leave the animal behaviorally intact in terms of reproductive behavior. The study examined the behavior of 43 sexually mature mares on Assateague Island during the 1997 breeding season and, with help from Earthwatch volunteers, collected observations over a 3-month period. The study found no significant differences between treated and untreated mares in general activity budget, aggression given or received, and spatial relationships relative to the stallion. These preliminary findings indicate that PZP contraception seems to have no acute behavioral effects on the behavior of individuals. The study findings also suggest that PZP could be a desirable and effective management tool for captive species in which social behavior plays an integral role in group dynamics. Analyses of group level effects and population level effects are continuing.     

Proteolytic processing of human zona pellucida proteins.

Formation of the egg's extracellular matrix, the zona pellucida, is critical for fertilization and development of growing embryos. Zona pellucida glycoproteins, ZP1, ZP2, and ZP3, are secreted to form an insoluble extracellular matrix surrounding mammalian eggs. All cloned mammalian zona pellucida sequences contain a furin consensus cleavage site, RX(K)/(R)R, upstream of a putative transmembrane domain, which suggests processing by an endoprotease of the furin-proprotein-convertase family. Recombinant expression of human (h) ZP1, ZP2, and ZP3 produces glycoproteins that are secreted and have migration patterns in SDS-PAGE identical to those of native human zona pellucida proteins. Because a C-terminal epitope tag that is present in the cell-associated zona proteins is, however, absent from the secreted zona proteins, secreted recombinant zona pellucida proteins lack their C-terminal regions. Three different strategies were used to explore processing events in the C-terminal region: site-directed mutagenesis of the furin cleavage site, treatment with a competitive inhibitor of all furin family members, and interference with Golgi modifications by Brefeldin A. All treatments altered the SDS-PAGE migration of recombinant hZP3, concordant with cleavage by a furin family member and Golgi glycosylation of secreted hZP3. Furthermore, cleavage of cell-associated hZP3 by exogenous furin converts the migration of cell-associated hZP3 to that of secreted hZP3. To determine whether a similar cleavage pattern exists in zona pellucida proteins that are assembled in the zona matrix, "hZP3 rescue" mouse zonae pellucidae were employed. Immunoblotting experiments revealed that hZP3, assembled and functional in the "hZP3 rescue" mouse zona pellucida, lacks the furin cleavage site, supporting the hypothesis that formation of the zona pellucida matrix involves regulated proteolysis by a member of the furin convertase family.     

Molecular identities of human sperm proteins that bind human zona pellucida: Nature of sperm–zona interaction,tyrosine kinase activity,and involvement of FA-1

The present study was conducted to investigate the molecular identities, nature of interaction, and tyrosine phosphorylation activity of the spermzona pellucida binding proteins in humans. Sperm proteins belcnging to four major molecular regions, namely 95, 63, 51, and 14–18 kDa, reacted with zona pellucida proteins in the Western blot and immunoprecipitation procedures. In these procedures, zona pellucida protein that reacted strongest with the sperm proteins belonged to the molecular region of 55 kDa (ZP3), besides weakly reacting proteins in the 110-kDa (ZP1/ZP2) and 14–18-kDa molecular regions. The major forces involved in the sperm-zona protein interactions were of hydrophobic and ionic in nature. Three (95, 51, and 14–18 kDa) of the four molecular regions of sperm proteins that bound to the zona pellucida proteins also seem to involve o-phospho-L-tyrosine residues in their interaction, and these proteins demonstrated the presence of phosphotyrosine residues, and the 51-kDa protein also showed autophosphorylating activity in the in vitro kinase assay. The sperm binding zona protein of 55 kDa also demonstrated autophosphorylating activity. Using specific monoclonal antibody to the well characterized sperm-specific glycoprotein, designated FA-1, and the competitive inhibition in the immunoprecipitation procedure, it was found that the 51 kDa protein is indeed FA-1 antigen. Besides elucidating the molecular nature of the spermzona interaction, these antigens will find application in the development of a multivalent contraceptive vaccine, and may also help in specific diagnosis and treatment of infertility mediated through defective gamete (sperm or oocyte) function. © 1994 Wiley-Liss, Inc.     

Structure of the acidic N-linked carbohydrate chains of the 55-kDa glycoprotein family (PZP3) from porcine zona pellucida.

N-linked carbohydrate chains of the major 55-kDa family, PZP3, of porcine zona pellucida glycoproteins are composed of neutral (28%) and acidic (72%) complex-type chains. The structures of the main components of the neutral chain have been established [Noguchi, S., Hatanaka, Y., Tobita, T. & Nakano, M. (1992) Eur. J. Biochem. 204, 1089-1100]. Here we report the structures of the acidic chains. Only two kinds of acidic fragments were released from PZP3 by endo-beta-galactosidase digestion following beta-elimination of O-linked chains. 500-MHz one-dimensional and two-dimensional 1H-NMR spectroscopy revealed their structures to be Sia alpha(2-3)Gal beta(1-4) [HSO3-6]GlcNAc beta(1-3)Gal and HSO3-6GlcNAc beta(1-3)Gal, showing that the sulfate-containing acidic chains are constructed with non-branched N-acetyllactosamine repeats which have sialic acid(s) at the non-reducing end(s) and sulfate at the C-6 position of GlcNAc residues. The acidic N-linked chains obtained from PZP3 by hydrazinolysis were separated into diantennary chains (34%) and tri- and tetra-antennary chains (66%) by concanavalin-A--agarose gel chromatography. The diantennary chains and their sialidase digests were fractionated by DEAE-HPLC. From the analyses of the endo-beta-galactosidase digests of each fraction, structures of the diantennary acidic chains were determined. They are classified into four groups. The first group is the sialylated chains without the sulfated N-acetyllactosamine repeating unit. The other three groups have the chains of various lengths differing in the number of monosulfated N-acetyllactosamine unit. These chains are extended from the Man alpha(1-3) branch of the trimannosyl core in the second group, from the Man alpha(1-6) branch in the third group, and from both branches in the fourth group. The structural features of the tri- and tetra-antennary acidic chains are also presented.     

Improving protective immunity induced by DNA-based immunization: priming with antigen and GM-CSF-encoding plasmid DNA and boosting with antigen-expressing recombinant poxvirus

Intramuscular immunization with a naked DNA plasmid expressing the Plasmodium yoelii circumsporozoite protein (pPyCSP) protects mice against challenge with P. yoelii sporozoites. This protection can be improved either by coadministration of a plasmid expressing murine GM-CSF (pGMCSF) or by boosting with recombinant poxvirus expressing the PyCSP. We now report that combining these two strategies, by first mixing the priming dose of pPyCSP with pGMCSF and then boosting with recombinant virus, can substantially increase vaccine effectiveness. Not only were immune responses and protection improved but the pPyCSP dose could be lowered from 100 microg to 1 microg with little loss of immunogenicity after boost with recombinant poxvirus. Comparing mice primed by the 1-microg doses of pPyCSP plus 1 microg pGMCSF with mice primed by 1-microg doses of pPyCSP alone, the former were better protected (60% vs 0) and had higher concentrations of Abs (titers of 163, 840 vs 5, 120 by indirect fluorescent Ab test against sporozoites), more ex vivo CTL activity (25% vs 7% specific lysis), and more IFN-gamma-secreting cells by enzyme-linked immunospot assay (1460 vs 280 IFN-gamma spot-forming cells/106 cells). Priming with plasmid vaccine plus pGMCSF and boosting with recombinant poxviruses strongly improves the immunogenicity and protective efficacy of DNA vaccination and allows for significant reduction of dose.     

Role of adjuvants in inhibitory influence of immunization with porcine zona pellucida antigen (ZP-3) on ovarian folliculogenesis in bonnet monkeys: a morphological study

Female bonnet monkeys were immunized with 55 kDa porcine zona antigen (ZP-3), with either complete Freund's adjuvant (CFA) or sodium phthalylated lipopolysaccharide (SPLPS) as adjuvant. Anti-ZP-3 antibody titers were monitored and the effect of immunization on the ovarian morphology was assessed by high-resolution light microscopy. The study demonstrated that both adjuvants used were equally potent in eliciting antibody response against ZP-3. Although no morphological damage to ovarian components was observed in animals immunized with SPLPS as adjuvant, immunization using CFA resulted in profound ovarian follicular atrophy, sparing only the primordial follicles. The atrophic phenomenon involved those follicles that either already had or were in the process of forming zona pellucida. The results of this study indicate that choice of adjuvant may be an important consideration for immunization against zona antigens. These findings encourage further investigations for developing better immunization regimen aimed at using zona antigens for immunocontraception.     

Influence of zona pellucida thickness on fertilization, embryo implantation and birth

Defective sperm-zona pellucida binding and penetration are the main causes of IVF failure. The purpose of this study was to evaluate the effect of zona pellucida thickness in fertilization failure and test the influence of zona pellucida thickness on implantation and birth in rabbits. Embryos and oocytes were collected from 72 females on Day 2 post-insemination. A total of 559 normal embryos were recovered; 402 embryos were transferred by laparoscopy and 157 embryos were used to measure the zona pellucida thickness using the ImageJ program. Laparoscopies were also performed on all does at Day 12 of gestation to record the number of implanted embryos. Litter size at birth was recorded. The mean zona pellucida thickness of the 157 embryos and of the 64 control group oocytes (18.3 ± 0.2 and 18.5 ± 0.3 μm, respectively) was significantly less than the zona pellucida thickness of the 74 failed fertilization oocytes (19.2 ± 0.3 μm). The probabilities of the regression coefficient being positive were 0.72 and 0.74 for implantation and birth, respectively, and the subsequent means of the coefficient were 2.92 and 0.03 for implantation and birth, respectively. In conclusion, the zona pellucida thickness has an important influence on in vivo fertilization and implantation processes, but not on birth.     

重组人卵透明带蛋白(rhZP3)的生物活性研究

为了研究毕赤酵母表达的重组人卵透明带蛋白(rhZP3)的生物活性,分别用空白培养液,含孕酮或rhZP3的培养液对人精子进行顶体诱发实验,用考马斯亮蓝染色法对顶体状态进行评价;用不同浓度的rhZP3以及空白培养液分别处理精子,然后再与卵子进行结合实验,观察经过不同处理的精子在精卵结合中的情况;用抗rhZP3抗血清与阴性血清分别处理卵子,再与精子进行结合实验,观察经过不同处理后的卵子在精卵结合中的情况。rhZP3诱发顶体反应实验结果显示,rhZP3处理组与空白对照组之间差异显著(P<0.01);精卵结合实验结果显示,各实验组和对照组之间存在显著性差异(P<0.01),rhZP3、抗rhZP3抗体均能抑制精卵结合。实验结果表明,rhZP3具有天然人卵透明带蛋白相似的活性。     

Differences between antigenic determinants of pig and cat zona pellucida proteins

Despite many efforts, the control of reproduction in feral cat populations is still a problem in urban regions around the world. Immunocontraception is a promising approach; thus the present study examined the suitability of the widely used pig zona pellucida proteins (pZP) for contraception in feral domestic cats. Purified zona pellucida proteins obtained from pig and cat ovaries were used to produce highly specific antisera in rabbits. Antibodies against pZP raised in rabbits or lions were not effective inhibitors of either in vitro sperm binding (cat spermatozoa to cat oocytes) or in vitro fertilization in cats, whereas antibodies against feline zona pellucida proteins (fZP) raised in rabbits showed a dose-dependent inhibition of in vitro fertilization. Immunoelectrophoresis, ELISA and immunohistology of ovaries confirmed these results, showing crossreactivity of anti-fZP sera to fZP and to a lesser extent to pZP, but no interaction of anti-pZP sera with fZP. It is concluded that cat and pig zonae pellucidae express a very small number of shared antigenic determinants, making the use of pZP vaccine in cats questionable. A contraceptive vaccine based on feline zona pellucida determinants will be a better choice for the control of reproduction in feral cats if immunogenity can be achieved.     

Protease activity involvement in the passage of mammalian sperm through the zona pellucida

The interaction between acrosome-reacted sperm and zona pellucida proteins is not yet fully understood. Serine protease acrosin and its zymogen proacrosin have been proposed to fulfill this function due to their capacity to bind zona pellucida glycoproteins. However, the molecular mechanism underlying this interaction has been merely speculative. Here we show that fucoidan (a sulfated polysaccharide) and solubilized zona pellucida glycoproteins, but not soybean trypsin inhibitor, are able to detach bound spermatozoa, which suggests that live sperm binds to the zona pellucida in a non-enzymatical way. Interestingly, mild proteolytic digestion with acrosin or trypsin does not modify the structure of the zona pellucida, but rather results in fewer spermatozoa binding to the zona. These results agree with a model where the active site of acrosin digests the zona pellucida and binds through the polysulfate-binding domain through a three-dimensional zona structure rather than a single ligand.