Culture media and their components differ in their ability to scavenge reactive oxygen species in the plasmid relaxation assay.

OBJECTIVE: To investigate the modulation of DNA-damaging effects of reactive oxygen species by media composition. DESIGN: In vitro study. SETTING: Academic medical center. PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Plasmid relaxation. RESULT(S): Ham's F-10 medium, 1% Percoll, superoxide dismutase (1, 10, or 100 IU), and synthetic serum substitute did not affect DNA damage by reactive oxygen species and did not have any effect on plasmid DNA damage. Plasmid DNA damage was partially inhibited in the presence of P-1 and human tubal fluid media. Human serum albumin, phenol red, glucose, polyvinyl alcohol, polyvinylpyrrolidone, sucrose, and HEPES also were found to protect DNA from damage. CONCLUSION(S): In vitro fertilization media and their components vary widely in the way they affect DNA damage by reactive oxygen species.

Human zona pellucida glycoproteins: characterization using antibodies against recombinant non-human primate ZP1, ZP2 and ZP3.

Characterization and classification of human zona pellucida glycoproteins is essential to understand the functions of these components during fertilization. To achieve this, antibodies were raised in rabbits against recombinant non-human primate [Bonnet Monkey (Macaca radiata)] zona pellucida proteins, bmZP1, bmZP2 and bmZP3 expressed in Escherichia coli. Antibodies against the three recombinant zona proteins reacted with human zonae as revealed by indirect immunofluorescence. Such antibodies were used as specific probes to further characterize human zona pellucida glycoproteins in Western blot of heat solubilized human zonae pellucidae (hSIZP) resolved by one dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Under non-reduced conditions human (h) hZP1, hZP2 and hZP3 resolved as 60, 100 and 53 kDa bands respectively. Under reduced conditions, dominant reactivity of hZP1, hZP2 and hZP3 was localized to 63, 65 and 58 kDa and faint reactivity to 53, 96 and 138 kDa bands respectively. In two-dimensional SDS-PAGE, hZP1 was shown to comprise two chains at 63-58 and 55-45 kDa, each consisting of multiple isomers. hZP2 was less acidic when compared with hZP1 and hZP3 and comprised a major component of 65 kDa and a minor component of approximately 96 kDa. The 65 kDa component displayed a higher degree of charged isomers in comparison with the 96 kDa component. hZP3 comprised a broad band in the range 68-58 kDa. These studies show conclusively that the hZP1 heavy train overlaps with hZP3 and that in previous studies, hZP2 was likely to have been misinterpreted as being hZP1. Our studies failed to distinguish two distinct species of hZP3, unlike previous reports. These studies will further help in our understanding of the nature of human zona pellucida glycoproteins.

Hetero-oligomerization-dependent binding of pig oocyte zona pellucida glycoproteins ZPB and ZPC to boar sperm membrane vesicles.

The zona pellucida surrounding the pig oocyte contains two Mr 55,000 glycoproteins, pZPB and pZPC, which are orthologues of mouse zona proteins ZP1 and ZP3, respectively. We previously reported that isolated boar sperm membrane vesicles possess high affinity binding sites for partially purified pZPB, but not pZPC. Interestingly, co-incubation experiments also implicated pZPB-pZPC complexes as potential ligands. We now report that when depleted of a minor pZPC contaminant by size exclusion chromatography, pZPB lacks independent binding activity. In solid phase binding assays employing immobilized boar sperm membranes, pZPB failed to compete with biotin-(pZPB+pZPC) probe, and biotin-labeled pZPB yielded negligible binding. However, when co-incubated with pZPC prior to the binding assays, pZPB acted as a potent competitor, and biotin-labeled pZPB exhibited high affinity, saturable binding. Binding activity was attributed to pZPB-pZPC heterocomplexes, which were detected in co-incubation mixtures by size exclusion chromatography and Western blot analysis. In the pig, therefore, sperm membranes possess a zona-binding protein with high affinity sites for pZPB-pZPC heterocomplexes, but not free glycoprotein subunits. Consequently, associative interactions between zona molecules can contribute toward both the assembly of the zona matrix and generation of ligands important for sperm-zona interactions.

Induction of native protein reactive antibodies by immunization with peptides containing linear B-cell epitopes defined by anti-porcine ZP3 beta monoclonal antibodies.

To identify pertinent target epitopes for contraceptive vaccine development, rabbit polyclonal antibodies were raised against four peptides synthesized from the deduced amino acid (aa) sequence of porcine zona pellucida macromolecule ZP3 beta and coupled to diphtheria toxoid (DT). Synthetic peptides consisted of: P1, 23-37 aa; P2, 164-179 aa with an additional C-terminal cysteine; P3, 246-263 aa with an extra C-terminal cysteine; and P4, 310-321 aa residues corresponding to pZP3 beta precursor protein. Selected sequences were based upon B cell epitopes identified previously by monoclonal antibodies. Immune sera reacted with their respective peptides and DT in an ELISA, and also recognized porcine SIZP and pZP3 beta both in ELISA and Western blot and zona pellucida of porcine oocytes in an indirect immunofluorescence assay. None of the four anti-peptide sera recognized pZP3 alpha in Western blot, emphasizing the specificity of these antibodies to pZP3 beta. The anti-peptide sera, individually, failed to inhibit in vitro attachment of boar sperm to antibody treated zona encased porcine oocytes. However, combinations of immune sera against peptides such as P1 + P4, P2 + P4 and P1 + P2 + P4, did significantly inhibit porcine sperm-oocyte interaction. These results identify combinations of peptides that could potentially be used in the design of an immunocontraceptive vaccine based upon synthetic peptides corresponding to pZP3 beta or its homologues in other species.

Mapping of epitopes on porcine zona pellucida-3 alpha by monoclonal antibodies inhibiting oocyte-sperm interaction.

Zona pellucida glycoprotein 3 alpha (ZP3 alpha) has been designated as the primary sperm receptor ligand in porcine gamete interaction. In this study, epitopes were mapped on porcine ZP3 alpha (pZP3 alpha) by using monoclonal antibodies (mAbs) possessing in vitro contraceptive efficacy. Using Western blots, we tested recombinant pZP3 alpha fragments expressed as fusion proteins in Escherichia coli and corresponding to pZP3 alpha precursor protein amino acid residues 18-142 (F1), 140-243 (F2), 239-363 (F3), and 359-462 (F4), for reactivities with mAbs. MAb-403 reacted with F3, and mAbs-412 and -421 with F2. MAb-420 showed weak reactivity with F1. Synthesis of overlapping 12-mer peptides further resolved the epitope for mAb-420 to amino acid residues 133-144, mAb-421 to 157-168, mAb-412 to 205-216, and mAb-403 to 301-312. MAbs-412 and -420 inhibited the binding of boar sperm to zona-encased porcine oocytes. These results, the first to define peptide epitopes of pZP3 alpha, should assist in the design of a synthetic peptide-based immunocontraceptive vaccine.

Localization of epitopes for monoclonal antibodies at the N-terminus of the porcine zona pellucida glycoprotein pZPC.

Zona pellucida glycoproteins play an important role in fertilization. In this study, attempts have been made to identify and define epitopes of monoclonal antibodies (mAbs) possessing contraceptive efficacy in vitro. The porcine zona glycoprotein pZPC, a homologue of mouse/human ZP3, was reduced and alkylated and subsequently digested with trypsin. Reverse-phase HPLC of the tryptic digest yielded twenty two peaks (T1-T22). When tested against mAbs reactive against sequential determinants on pZPC, T11 was immunoreactive with two mAbs, mAb-455 and mAb-467, as shown by antigen inhibition ELISA. IC50 values of 3.1 nM and 8.6 nM were recorded versus mAb-455 and mAb-467 respectively, and approximated the IC50 values obtained with intact pZPC. Amino acid analysis, Edman degradation, and FAB-MS identified T11 as the N-blocked decapeptide pyro-Gln-Pro-Val-Trp-Gln-Asp-Glu-Gly-Gln-Arg derived from the N-terminus of pZPC. Synthesis of overlapping octapeptides further identified VWQDE and WQDE as the minimum motifs with antigenic activity for mAb-455 and mAb-467, respectively. Glycine replacement peptides confirmed residues W,Q,E as critical for binding mAb-455 and W,Q,D,E as critical for binding mAb-467. Both mAbs inhibited binding of boar sperm to zona-encased porcine oocytes. These results, the first to define peptide epitopes of porcine zona glycoprotein, will assist in the design of an immunocontraceptive vaccine based on synthetic peptides corresponding to pZPC or its homologues in other species.

Generation and characterization of antibodies against synthetic peptides of porcine zona pellucida ZP3 alpha.

OBJECTIVE: Use of synthetic zona peptides as target immunogens is a promising approach to an anti-fertility vaccine. To elucidate contraceptive epitopes, we raised polyclonal antibodies against synthetic peptides of pig ZP3 alpha and evaluated for immunogenicity, ability to inhibit in vitro boar sperm-pig zona attachment, and cross-reactivity with human zonae. METHODS: Five synthetic peptides were chosen based on hydropathicity analysis of ZP3 alpha, synthesized and coupled to keyhold limpet haemocyanin (KLH). Pairs of male rabbits were immunized with each peptide-KLH conjugate using a multi-site intradermal injection protocol. Resulting antisera were evaluated for immunoreactivity with cognate peptide and ZP3 alpha/EBGD (purified endo-beta-galactosidase-digested ZP3 alpha), contraceptive potential using an in vitro pig gamete bioassay, and cross-reactivity with human zonae using indirect immunofluorescence. RESULTS: All anti-peptide-KLH sera demonstrated immunoreactivity with cognate peptide. Anti-peptide sera, except for anti-alpha-3-KLH serum, were less immunoreactive with ZP3 alpha/ EBGD. Antisera directed against alpha-1-KLH, alpha-2-KLH, and alpha-3-KLH demonstrated significant (P < .00001) inhibition of boar sperm-pig zona attachment. Only anti-alpha-2-KLH and anti-alpha-3-KLH sera cross-reacted with human zonae. CONCLUSION: Synthetic peptides of pig ZP3 alpha are immunogenic, elicit antibodies cross-reactive with human zonae, and have in vitro contraceptive activity. These data support continued investigation of synthetic ZP3 alpha peptides as potential target immunogens for anti-fertility vaccine development.

Immunoreactivity with native zona pellucida of antibodies against a 19 amino acid synthetic peptide corresponding to human ZP3.

Immunization of female Syrian hamsters with a 19mer-synthetic peptide corresponding to amino acid sequence 323-341 of human ZP3 and coupled to diphtheria toxoid (DT) led to generation of antibodies against both the peptide as well as DT. Antisera showed positive reaction in ELISA with solubilized isolated zona pellucida (SIZP) from human oocytes. In an indirect immunofluorescence assay, the anti-peptide antibodies bound to zona pellucida of human and bonnet monkey but failed to recognize that of mouse, rabbit, hamster and dog. These studies will help in designing a synthetic peptide based ZP immunocontraceptive vaccine for human application.

The effects of in vitro cocaine exposure on human sperm motility, intracellular calcium, and oocyte penetration.

OBJECTIVE: To determine if cocaine exposure affects human sperm motility, intracellular calcium level, and fertilizing capability. DESIGN AND METHODS: Human semen samples were treated with 1 to 1,000 microM cocaine hydrochloride for up to 2 hours in vitro. Sperm motion kinematics were measured by computer-assisted semen analysis (CASA). Spermatozoan intracellular calcium was determined by laser cytometry. The sperm fertilizing capability was assessed using the zona-free hamster oocyte penetration test. RESULTS: After a short exposure (15 minutes) to cocaine, the sperm motion kinematic parameters, straight line velocity and linearity, were decreased in the high concentration groups. However, after a longer exposure (2 hours) to cocaine, the differences were no longer significant. Cocaine treatment did not alter spermatozoa intracellular calcium levels. Most importantly, human sperm treated with cocaine at a high concentration were fully capable of penetrating zona-free hamster oocytes. CONCLUSION: Human spermatozoa acutely exposed to high concentrations of cocaine initially demonstrate a decrease in two motion kinematics, straight line velocity and linearity. However, overall, cocaine exposure had no significant effects on sperm motility and fertilizing capability.

Cloning and characterization of zona pellucida genes and cDNAs from a variety of mammalian species: the ZPA, ZPB and ZPC gene families.

Full length zona pellucida cDNAs from cat, dog and pig that are homologous to the ZP2/rc75 genes from mouse, human and rabbit, a full length zona pellucida cDNA from cat and a gene and full length cDNA from human that are homologous to the rc55/ZP3 alpha genes from rabbit and pig, and full length zona pellucida cDNAs from cat, cow, dog, pig and rabbit that are homologous to the ZP3 genes from mouse, hamster, human and marmoset have been cloned and characterized. The members of these gene families are herein referred to as ZPA, ZPB and ZPC genes to avoid the confusion that currently exists in the zona pellucida of nomenclature. This report is the first to describe the presence all three major zona pellucida genes within individual mammalian species. Within the ZPA, ZPB and ZPC gene families, the DNA and deduced amino acid sequences are highly homologous to each other, and are most homologous between members of the same order within the class mammalia. These results imply that all or most mammalian species express the ZPA, ZPB and ZPC proteins, which form the zona pellucida layer surrounding the oocyte.

Block in porcine gamete interaction by polyclonal antibodies to a pig ZP3 beta fragment having partial sequence homology to human ZP3.

A murine monoclonal antibody (MA-30) recognizing a sequential epitope on porcine zona pellucida-3 beta glycoprotein (ZP3 beta) and capable of inhibiting sperm-egg interaction was previously described. Polyclonal antibodies against a approximately 6 kDa fragment from the tryptic digest of ZP3 beta, reacting with MA-30, can also inhibit porcine gamete interaction in vitro. Partial N-terminal sequencing of the smallest fragment from the ZP3 beta tryptic digest having reactivity with MA-30 shows sequence homology with human ZP3.

Porcine zona pellucida ZP3 alpha glycoprotein mediates binding of the biotin-labeled M(r) 55,000 family (ZP3) to boar sperm membrane vesicles.

The two M(r) 55,000 glycoproteins, ZP3 alpha and ZP3 beta, of porcine zona pellucida copurify as a preparation designated ZP3. Gamete binding assays have implicated ZP3 alpha, but not ZP3 beta, as participating in sperm-zona recognition events. We now report that boar sperm contain membrane-associated binding sites with specificity for ZP3 alpha. Biotin-labeled (b-) preparations of ZP3 bind to intact boar sperm in a saturable manner, with localization on the anterior head region. Membrane vesicles obtained from capacitated sperm by nitrogen cavitation retain b-ZP3 binding sites as determined by an enzyme-linked method employing alkaline phosphatase-conjugated strepavidin. In competitive binding assays using b-ZP3 (0.1 microgram/ml) as probe, heat-solubilized zonae and ZP3 were effective competitors, whereas the nonzona molecules fetuin and fucoidin were not. Digestion of ZP3 with endo-beta-galactosidase, an enzyme that trims polylactosamines, enhanced its affinity for membrane receptors. In contrast treatments such as chemical deglycosylation, pronase digestion, or disruption of disulfide bonds abolished the ligand activity of ZP3. Finally, purified ZP3 alpha was an at least 100-fold better antagonist than purified ZP3 beta. The results demonstrate that binding of b-ZP3 to isolated boar sperm membranes is mediated by sperm receptors with specificity for the ZP3 alpha macromolecular component and reveal a complex contribution of both carbohydrate and protein moieties toward the ligand activity of this sperm adhesive zona molecule.

Mapping of immunogenic domains on porcine zona pellucida 3 alpha and beta glycoproteins by murine monoclonal antibodies.

PROBLEM: Immunization with zona pellucida (ZP) leads to block in fertility with variable degree of ovarian dysfunctions. To design an immunocontraceptive vaccine based on synthetic peptides of zona pellucida, it is imperative to identify and define epitopes involved in sperm binding. METHOD: Epitopic domains recognized by monoclonal antibodies (MAbs) specific to either porcine ZP3 alpha or ZP3 beta glycoproteins were delineated in an enzyme-linked immunosorbent assay (ELISA) based on the ability of a MAb in solution to inhibit the binding of biotinylated ZP3 to another MAb coated on a microtitration plate. Immunoblot studies were carried out to determine the nature of reactive determinants. Porcine oocytes preincubated with MAbs were tested for sperm binding in vitro. RESULTS: Out of 23 MAbs generated, 10 had specificity for ZP3 alpha and 13 for ZP3 beta. By using these antibodies, eight epitopic domains on both ZP3 alpha and ZP3 beta were discernible. On ZP3 beta, epitopic domain DI partially overlaps with DII and DV with DVI, whereas on ZP3 alpha domains DI to DV were in close proximity with a partial overlap, suggesting the dominance of this region. All 10 MAbs against ZP3 alpha, and 10 out of 13 against ZP3 beta recognized deglycosylated forms of antigens. Seven antibodies having specificities for ZP3 alpha and ZP3 beta respectively recognized linear epitopes. MA-30, having specificity for ZP3 beta and MA-420 for ZP3 alpha and recognizing linear epitopes significantly inhibit the binding of boar sperm to porcine oocytes in vitro. CONCLUSIONS: Collectively, these studies indicate the value of utilizing MAbs for identifying and characterizing functionally significant ZP determinants. MAbs recognizing sequential epitopes will help in the elucidation of the amino acid sequence of the epitopes, which will subsequently help in design of synthetic immunocontraceptive vaccines.

Nucleotide sequence of cDNA encoding ZP3 alpha, a sperm-binding glycoprotein from zona pellucida of pig oocyte.

We isolated a cDNA encoding the pig oocyte zona pellucida protein ZP3 alpha from a pig ovary lambda gt11 cDNA library. The 1699 bp cDNA contains a short 3' untranslated region characteristic of cDNAs encoding zona proteins. The deduced amino acid sequence for ZP3 alpha consists of 536 amino acid residues and shares 66% overall identity with a 55 kDa rabbit zona protein. Important features of the ZP3 alpha polypeptide include a predicted N-terminal signal sequence, twenty-two cysteine residues, an O-glycosylated domain and potential attachment sites for five N-linked sugar chains. A multibasic tetrapeptide occurs upstream of a predicted C-terminal transmembrane sequence; this suggests proteolytic processing of an integral membrane precursor within the constitutive secretory pathway.

Generation and characterization of site-directed antisera against an amino-terminal segment of a 55 kDa sperm adhesive glycoprotein from zona pellucida of pig oocytes.

ZP3 alpha, a 55 kDa zona pellucida glycoprotein, mediates attachment of boar spermatozoa to zona-encased oocytes. In the study reported here, site-directed antipeptide antibodies were used to probe the topography and biological activity of the amino-terminal domain of this sperm adhesive zona molecule. A synthetic peptide (alpha 8-18) corresponding to an amino-terminal sequence of ZP3 alpha (residues 8-18) was coupled to keyhole limpet haemocyanin (KLH) using either m-maleimidobenzoic acid N-hydroxysuccinimide ester or glutaraldehyde. Male rabbits were immunized with peptide-KLH conjugates using Freund's adjuvant, and antibody production was monitored by ELISA. High-titred antipeptide sera were obtained and further characterized. Western blotting experiments using deglycosylated ZP3 alpha and ZP3 beta core proteins demonstrated reactivity of antipeptide sera with homologous protein. Preincubation of antisera with cognate peptide blocked binding to ZP3 alpha thus confirming site-specific binding of antibodies to the targeted sequence in the intact protein. Anti-KLH-alpha 8-18 sera reacted poorly with fully glycosylated ZP3 alpha, but ZP3 alpha immunoreactivity was markedly enhanced by digestion of polylactosamines with endo-beta-galactosidase, chemical deglycosylation or protein unfolding. Finally, zona-encased pig oocytes were preincubated with the antipeptide sera, washed and exposed to boar spermatozoa. Pretreatment with anti-KLH-alpha 8-18 sera significantly diminished attachment of boar spermatozoa to zonae. These studies demonstrate that the amino-terminal domain of pig oocyte zona protein, ZP3 alpha, is partially occluded, in part by carbohydrate, and that site-directed antipeptide sera targeted to this portion of the molecule can interfere with sperm-zona attachment in vitro.

Delineation of epitopes on porcine zona pellucida relevant for binding of sperm to oocyte using monoclonal antibodies.

Seven monoclonal antibodies (MAs) generated against porcine zona pellucida glycoprotein, ZP3 (comprising both ZP3 alpha and ZP3 beta) were characterized for their specificities to ZP3 alpha (MA 7 and MA 28) or ZP3 beta (MA 1, MA 2, MA 10, MA 27 and MA 30) and their relative affinities in competitive ELISA. Among the seven MAs tested, MA 28 showed the highest affinity for ZP3 and ZP3 alpha and MA 30 for ZP3 beta. All the antibodies bound to the zona pellucida in an indirect immunofluorescence assay, but only four (MA 7, MA 28, MA 10 and MA 30) were able to inhibit the binding of boar sperm to the porcine oocyte. Reduction followed by carboxyamidomethylation of the antigen or its chemical deglycosylation reduces reactivity to MA 7 and MA 10, suggesting that these antibodies read conformational or discontinuous determinants. The epitope recognized by MA 28 is sequential or conformational, stabilized by disulfide bonds while MA 30 reads a sequential determinant. ZP3 alpha digested with alpha-chymotrypsin, trypsin and V8 protease, respectively, revealed fragments in the range of 27-20 kDa with MA 28 in immunoblots. Proteolytic digests of ZP3 beta show that MA 30 recognizes approximately 14 kDa fragment of an alpha-chymotrypsin digest and a approximately 6 kDa fragment of a tryptic digest. These studies will help in delineation of smaller determinants of ZP involved in sperm binding.

Characteristics of monoclonal antibodies against porcine zona pellucida-3 and their functional relevance.

Seven monoclonal antibodies (MAs) against 55 kDa glycoprotein family of porcine zona pellucida (ZP3) reacting with either ZP3 alpha (MA-7, MA-27, MA-28) or ZP3 beta (MA-1, MA-2, MA-11, MA-30) have been described. MA-1, -2, -27, -28 and -30 do not recognize carbohydrate determinants as shown by their reactivity to the deglycosylated (DG) ZP3 alpha and ZP3 beta. Indirect immunoperoxidase studies showed that all MAs reacted with zona pellucida from porcine and monkey ovaries. Only MA-1 and -27 reacted with ZP from rabbit ovary as well, while none of the MAs recognised mouse ZP, MA-7, -11, -27, -28 and -30 inhibited in vitro, the zona lysis by trypsin as well as the binding of ZP3 to sperm membrane vesicle as investigated by ELISA.

Porcine oocyte zona pellucida M(r) 55,000 glycoproteins: identification of O-glycosylated domains.

The distribution of O-linked oligosaccharides on the M(r) 55,000 glycoproteins, ZP3 alpha and ZP3 beta, of the porcine oocyte zona pellucida was examined. Purified preparations of endo-beta-galactosidase digested ZP3 alpha and ZP3 beta were reduced and carboxamidomethylated and digested with trypsin. When the trypsin digests were mapped by HPLC, each glycoprotein yielded only one N-acetylgalactosamine containing glycopeptide. Purification of the O-glycopeptides was achieved by a two-step protocol. Tryptic digests were applied to jacalin-agarose and specifically-bound O-glycopeptides (alpha OGP and beta OGP) were eluted with buffer containing 50 mM alpha-methylgalactoside as the haptenic sugar. Further purification of each O-glycopeptide was accomplished by reverse phase HPLC. Purified O-glycopeptides were characterized with respect to amino acid and carbohydrate compositions and sequenced by automated Edman degradation; alpha OGP was a 41-residue glycopeptide with three O-linked sugar chains. Sequence comparisons revealed a 75% identity between alpha OGP and a corresponding segment of rabbit rec55 zona protein; beta OGP was a 25-residue glycopeptide characterized by the presence of one N-linked and five O-linked sugar chains and a trypsin-resistant internal arginine residue. Sequence alignments revealed an 80% or greater identity between beta OGP and internal peptides of mouse, hamster and human ZP3 zona proteins. These studies demonstrate that in the case of ZP3 alpha and ZP3 beta, the pig homologues of rabbit rec55 and mouse ZP3, respectively, O-linked oligosaccharides are confined within delimited domains rather than widely dispersed on the polypeptide backbone. Such clustering of O-linked oligosaccharides may represent an essential determinant of the structure and biological activity of zona proteins.

Histology of ovaries of female rabbits immunized with deglycosylated zona pellucida macromolecules of pigs.

Female rabbits (n = 36, 6 per group) were immunized with: (i) solubilized isolated porcine zona pellucida (SIZP), which contains ZP1, 82 kDa; ZP3 alpha, 55 kDa; and ZP3 beta, 55 kDa; (ii) a purified preparation of ZP3 alpha and ZP3 beta (ZP3); (iii) purified endo-beta-galactosidase digested glycoproteins ZP3 alpha-(EBGD) and (iv) ZP3 beta-(EBGD) (each about 30% deglycosylated); (v) chemically deglycosylated core proteins ZP3 alpha-(DG) and (vi) ZP3 beta-DG (each greater than 92% deglycosylated). Rabbits injected with saline (n = 6) or Freund's adjuvant (n = 6) served as controls. Rabbits were bled weekly to monitor titres. Every six weeks two animals from each group (n = 16) were selected for unilateral oophorectomy followed by histological examination. Sections were scored for numbers of primary, secondary and tertiary follicles. Anti-ZP3 titres developed in all treatment groups and correlated with carbohydrate content (peak per cent [125I]-labelled ZP3 binding by radioimmunoassay: SIZP 71.9 +/- 1.2, ZP3 70.0 +/- 2.5, ZP3 alpha-EBGD 60.9 +/- 5.3, ZP3 beta-EBGD 56.4 +/- 5.0, ZP3 alpha-DG 56.4 +/- 4.0, ZP3 beta-DG 53.5 +/- 4.3) (means +/- SEM). Animals immunized with SIZP, ZP3 and ZP3 beta-EBGD showed a statistically significant reduction in the number of primary, secondary and tertiary follicles compared with controls (P less than 0.01, MANOVA), whereas animals immunized with ZP3 alpha-EBGD, ZP3 alpha-DG and ZP3 beta-DG did not (P greater than 0.05, MANOVA). These results demonstrate that immunization with purified ZP3 alpha macromolecules (ZP3 alpha-EBGD, ZP3 alpha-DG) or ZP3 beta-DG does not produce histopathological changes in ovaries. Such deglycosylated ZP macromolecules represent potential target antigens for immunocontraceptive development.