Proteolysis of the zona pellucida by acrosin: the nature of the hydrolysis products

Boar sperm acrosin was previously shown to hydrolyze the porcine zona pellucida in a specific and limited fashion. The action of acrosin on its presumed physiological substrate was investigated further in terms of the hydrolysis products formed. Peptide mapping experiments of zona pellucida glycoprotein families using acrosin demonstrated the formation of several products 2-4K smaller than the original susceptible families. When zona pellucida hydrolysates were examined with gel filtration, the hydrolysis products were associated in large macromolecular aggregates. These observations suggest that zona pellucida solubilization by acrosin may not be a relevant criterion for assessing acrosin's role in sperm penetration of the zona pellucida.     

Contraceptive potential of porcine zona pellucida in cats

The contraceptive potential of solubilized porcine zona pellucida (spZP) was studied in 2 groups of cats after active immunization using slightly different protocols. Cats from Group 1 (n = 3) were immunized with a total of 300 8g spZP divided in 4 s.c. multisite injections (each of 37.5 8g) given at 10 day intervals followed by a booster 150 days after the initial immunization. Cats from Group 2 (n = 5) were immunized with a total of 400 8g spZP divided in 4 i.m. injections (each of 50 8g) given at 2 wk intervals followed by a booster 92 days after initial immunization. Immunogen was emulsified in Complete Freund Adjuvant for the first dose and in Incomplete Freund Adjuvant for the following 3 doses. The respective controls were immunized in the same manner using only adjuvant and PBS. Immunofluorescence studies showed intense fluorescence on the zona pellucida (ZP) of the oocytes isolated from immunized cats, as well as on the ZP of porcine and cat oocytes preincubated in immune sera. Sera from cats immunized with spZP inhibited in vitro binding was demonstrated in oocytes isolated from immunized group 1 cats. In vivo fertility data in Group 2 cats revealed that only 1 of 5 cats became pregnant, the one with the lowest anti-spZP titer. The results from the experiments reported above, suggest that in this preliminary study spZP can elicit antibodies with contraceptive potential in actively immunized female cats.     

Differences in the macromolecular composition of the zona pellucida isolated from pig oocytes, eggs, and zygotes

The macromolecular differences in the zona pellucida (ZP) isolated from pig oocytes, eggs, and zygotes were investigated using two-dimensional polyacrylamide gel electrophoresis. The ZP was isolated from individual cells or zygotes using micropipettes, radiolabeled with 125I and analyzed using disulfide bond reducing and nonreducing conditions. The reduced ZP isolated from oocytes was composed of four glycoprotein components. The gel pattern of the ZP isolated from a single oocyte was indistinguishable from that isolated en masse. The ovulated egg ZP contained the four oocyte components plus three additional macromolecules. Relative to the egg ZP, the zygote ZP lacked one component but had three additional smaller macromolecules. We concluded that: the macromolecular differences between the oocyte and egg ZPs are caused by the addition of macromolecules to the ZP as the egg transits the oviduct, the macromolecular differences between the egg and the zygote ZPs reflect hydrolytic processing of ZP glycoproteins probably by enzymes derived from the egg cortical granules, and the microheterogeneity of the pig ZP glycoproteins is due to posttranslational modification and is not due to population genetic variation.     

Analysis of N-linked glycans of porcine zona pellucida glycoprotein ZPA by MALDI-TOF MS: a contribution to understanding zona pellucida structure

The mammalian oocyte is encased by a transparent extracellular matrix, the zona pellucida (ZP), which consists of three glycoproteins, ZPA, ZPB, and ZPC. The glycan structures of the porcine ZP and the complete N-glycosylation pattern of the ZPB/ZPC oligomer has been recently described. Here we report the N-glycan pattern and N-glycosylation sites of the porcine ZP glycoprotein ZPA of an immature oocyte population as determined by a mass spectrometric approach. In-gel deglycosylation of the electrophoretically separated ZPA protein and comparison of the pattern obtained from the native, the desialylated and the endo-beta-galactosidase-treated glycoprotein allowed the assignment of the glycan structures by MALDI-TOF MS by considering the reported oligosaccharide structures. The major N-glycans are neutral biantennary complex structures containing one or two terminal galactose residues. Complex N-glycans carrying N-acetyllactosamine repeats are minor components and are mostly sialylated. A significant signal corresponding to a high-mannose type chain appeared in the three glycan maps. MS/MS analysis confirmed its identity as a pentamannosyl N-glycan. By the combination of tryptic digestion of the endo-beta-galactosidase-treated ZP glycoprotein mixture and in-gel digestion of ZPA with lectin affinity chromatography and reverse-phase HPLC, five of six N-glycosylation sites at Asn(84/93), Asn268, Asn316, Asn323, and Asn530 were identified by MS. Only one site was found to be glycosylated in the N-terminal tryptic glycopeptide with Asn(84/93.) N-glycosidase F treatment of the isolated glycopeptides and MS analysis resulted in the identification of the corresponding deglycosylated peptides.     

Diversity of antigenic determinants of porcine zona pellucida revealed by monoclonal antibodies

Monoclonal antibodies derived from ten hybrid cell clones, generated against porcine zona pellucida gave strong immunofluorescence with zona but the pattern varied from patchy, thin rim to heavy precipitation type of rim. Five of the 6 monoclonals studied prevented the binding of the porcine epididymal sperm to homologous oocytesin vitro, whereas the sixth one was partially effective. All of the 6 monoclonale of this batch inhibited the lysis of zonae by proteolytic enzymes even at dilutions up to 1 × 10⁻³. Three of the four monoclonals prepared in a subsequent batch gave strong immunofluorescent reactions and had high titres as determined by enzyme immunoassay. These monoclonals did not, however, protect the zonae against lysis by proteolytic enzymes. These properties are suggestive of the heterogeneity of the antigenic determinants in zona and emphasize the employment of appropriate bioassays for screening and selection of bioeffective antibodies.     

Biochemical characterization of five glycoprotein molecules of the porcine zona pellucida

Five glycoproteinmolecules with the molecular masses of 17 000; 38 000; 42 000; 50 000 and 67 000 were purified by high performance liquid chromatography following solubilization of isolated porcine zonae pellucidae by treatment with lithium-3,5-diiodosalicylate. The N-terminal amino acid residues were identified as arginine for 67 000, alanine for 50 000, arginine for 42 000, alanine for 38 000 and histidine for 17 000. The glycopeptides 42 000 and 17 000 were found to be rich on carbohydrates and 67 000 contained 7, 16% sialic acids. The latter moieties were tentatively identified as 5-N-acetylneuraminic acid, 5-N-glycolylneuraminic acid and 5-N-acetyl-7,8,9 tri-O-acetylneuraminic acid. The five components of the zona were resolved by thin layer chromatography in a solvent system of propanol/butanol/HCl (2:1:1) and showed Rf-values of 0.17, 0.42, 0.46, 0.50 and 0.55 respectively. The glycoprotein with the molecular mass of 38 000 possesses spermatozoal receptor properties. This receptor molecule showed a pI of 5.9 upon isoelectric focusing.     

Predominance of beta-structure in solubilized zona pellucida from porcine ova

The isolated zona pellucida from porcine ova was effectively solubilized in water at 60 degrees C within one hour. The circular dichroic spectra of zona in water with and without dithiothreitol showed the beta-form. Although sodium dodecyl sulfate partially induced helical structure, the beta-form was considerably retained. These results indicate that the zona glycoproteins have a structure-forming potential for the beta-structure and the hydrogen bonds of the beta-structure stabilize the supramolecular complex of the zona pellucida. The beta-form was also detected in zona solubilized by tryptic digestion. Porcine acrosin, however, did not solubilize the zona.     

Ultrastructural studies of the porcine zona pellucida during the solubilization process by Li-3,5-diiodosalicylate

Isolated porcine zonae pellucidae were investigated using transmission electron microscopy. It was found that the fine structure of the zona is not homogenous. The region near the oocyte consists of a more tightly packed micellar structure than the structure near the external surface. However, no clear boundary between the two structural features could be detected. The assumption that the molecular structure of the external and internal surface of the zona is not the same is substantiated by the finding that antibodies against the whole zona structure react apparently only with the more loosely bound structure on the external surface. This effect is attributed to differences in the spacial arrangements of the micellar structures rather than to differences in chemical composition. Solubilization of the zona results in a reorientation of the otherwise randomly interconnected structure. At lower Li-3,5-diiodosalicylate concentration (0.05 M) the fibrils seems to expand so that the individual fibers lie almost parallel to each other. At a higher Li-3,5-diiodosalicylate concentration (0.2 M) the individual micelles begin to break up from the zona surface, while at a still higher concentration (0.3 M) the rigid structure of the zona is completely solubilized. In the latter case no residual material could be detected in the sediment following high speed centrifugation of the mixture. These electron microscopic results correlated with the protein concentrations in the supernatant indicating that the maximal protein content (35 ng/zona) is obtained at 0.3 M or higher Li-3,5-diiodosalicylate concentrations.     

Neutral oligosaccharide structures linked to asparagines of porcine zona pellucida glycoproteins

N-Linked sugar chains were liberated by hydrazinolysis from porcine zona pellucida glycoproteins obtained from ovarian follicular oocytes. Neutral sugar chains were separated from acidic ones by paper electrophoresis and fractionated with a serial lectin column chromatography and Bio-Gel P-4 column chromatography. Their structural analysis by sequential glycosidase digestion in combination with methylation analysis revealed that the neutral sugar chains are of bi-, tri-, and tetraantennary complex type with a fucosylated trimannosyl core. Twenty-six percent of the sugar chains contain N-acetyllactosamine repeating structures in their outer chain moieties. Only linear N-acetyllactosamine repeats, the maximum size of which is hexasaccharide, are detected. A characteristic feature is that 39% of the sugar chains contain N-acetylglucosamine residues at their nonreducing termini in spite of the absence of bisected sugar chains. This study provided, for the first time, the substantial information about the sugar chain structures of mammalian zona pellucida glycoproteins.     

Sperm binding to the pig zona pellucida and inhibition of binding by solubilized components of the zona pellucida

An assay to determine the binding of pig spermatozoa to the zona pellucida (ZP) of pig oocytes was developed using conditions compatible with in-vitro fertilization of pig eggs and with pig sperm penetration of zona-free hamster ova. These conditions were used to define which of the pig oocyte ZP components were involved in sperm binding by a competitive inhibition approach. Assay variables that were optimized included: the method of sperm preparation; sperm preincubation time; sperm-oocyte coincubation time; sperm concentration and temperature; and methods for the separation of free from oocyte-bound spermatozoa. Inclusion of solubilized ZP in the sperm preincubation medium inhibited sperm binding approximately 50%. Both the 55K and 90K components inhibited sperm binding although the 55K component was more effective. The two polypeptides derived from chemical deglycosylation of the 55K families did not inhibit sperm binding. Of several monoclonal antibodies to the ZP components tested, only one directed against the 55K alpha glycoprotein family inhibited sperm binding. Sperm binding to pig oocyte ZP is therefore dependent on the carbohydrate moiety of the glycoproteins and appears to involve more than a single ZP glycoprotein.     

Isolation, composition, and biological activity of sugar chains of porcine oocyte zona pellucida 55K glycoproteins

ZP3, a preparation of the 55K families of porcine oocyte zona pellucida, possesses carbohydrate-dependent ligand activity for boar sperm. The aim of the present study was to analyze ZP3 N- and O-linked oligosaccharides with respect to size distribution, composition, and role in sperm-zona recognition events. Digestion of denatured ZP3 with peptide N-glycosidase F (PNGaseF) released the majority of N-glycans which fractionated on Sephadex G-75 resin as a polydisperse population with apparent molecular masses ranging from 1,900-8,200 Da. The higher molecular weight N-glycans were characterized by the presence of strongly anionic sulfated/sialylated polylactosamine structures. Alkaline-borohydride treatment of the PNGaseF-digested core proteins liberated O-glycans as a heterogeneous population of oligosaccharide alcohols, which were fractionated on a Sephadex G-50 column. Compositional analyses indicated sulfated polylactosamine units associated with the higher molecular weight O-glycans. Preincubation of boar sperm with ZP3 or purified O-glycans, but not N-glycans, inhibited subsequent attachment to zona-encased oocytes. Purified O-glycans were, however, 2 to 3 orders of magnitude less effective than ZP3 as competitive ligands. The results document the extreme heterogeneity of the ZP3 carbohydrate moiety, in large part attributable to a broad spectrum of variably sized N- and O-linked sulfated polylactosamines. Ligand competition bioassays suggest that O-glycans mediate, at least in part, the sperm adhesive properties of ZP3 and strongly imply that high-affinity interaction of ZP3 sugar chains with complementary sperm receptors is dependent upon their covalent association with core proteins.     

Boar acrosin digestion of the porcine egg coat, zona pellucida, and rearrangement of the zona proteins

Mechanically isolated, structurally intact porcine zonae pellucidae composed of three families of glycoproteins (PZP1-3) were digested with purified boar acrosin, and then the solublized and unsolubilized fractions were separately analyzed by HPLC and/or SDS-PAGE. In isotonic solution, PZP1 was first degraded and then PZP2, whereas PZP3 was quite resistant to acrosin, reflecting the organization of these families in the zona structure. As the proteolytic hydrolysis proceeded, high-molecular-weight products appeared in the unsolubilized fraction. These products disintegrated on treatment with beta-mercapto-ethanol. The zona materials solubilized by acrosin were separated into seven fractions by reverse phase HPLC. The total yield of the latter was only about 5% by weight. Thus, limited sites of the porcine zona were cleaved by the homologous sperm acrosin. Since five fractions contained peptides that were more hydrophilic than the original proteins, these peptides seemed likely to be present on the outer surface of the zona structure. SDS-PAGE of the unsolubilized fraction showed that acrosin cleaved the zona at many more sites in hypotonic solution than in isotonic solution. Thus, structural relaxation of the inner region of the zona was indicated to be induced under hypotonic conditions. However, no high-molecular-weight products were formed in hypotonic solution, suggesting that the native architecture of the zona is a prerequisite for their formation.     

Evaluation of somatic and reproductive immunotoxic effects of the porcine zona pellucida vaccination

Immunological, immunocytochemical and fertility analyses were performed to determine the potential toxic side effects of porcine zona pellucida (pZP) vaccinations on target animals, including horses and dogs. The study was designed to determine the effect of antibodies, raised against highly purified pZP, on somatic tissues. Immunocytochemical studies performed with fixed tissues showed that rabbit anti-pZP antiserum did not crossreact with brain, heart, lung, kidney, liver, bladder, stomach, small intestine, large intestine, muscle, skin, spleen, pancreas, or lymph node of either the dog or horse. To determine the effect or oral intake on nontarget animals, female rabbits were fed a contraceptive vaccine containing pZP glycoproteins and the synthetic trehalose dicorynomycolate in drakeol (S-TDCM) adjuvant. Enzyme-linked immunoadsorbent assay (LISA) analyses showed that rabbits fed with the adjuvanted pZP proteins did not develop circulating anti-pZP IgG antibodies that crossreacted with pZP. Furthermore, fertility studies performed on rabbits fed with adjuvanted pZP revealed no significant differences in the number of embryos or stage of the embryos produced between the treated and control animals. Results of these studies suggest that the pZP vaccine delivered to dogs or horses in field studies have no recognizable somatic tissue effects. Moreover, there were no side effects on nontarget animals should they eat the vaccine. This substantiates field trials results about the safety of the pZP immunocontraceptive vaccine.     

Identification of the carboxyl termini of porcine zona pellucida glycoproteins ZPB and ZPC

The extracellular matrix surrounding mammalian oocytes plays important roles in fertilization and is known as the zona pellucida (ZP). The ZP consists of three glycoproteins, ZPA, ZPB, and ZPC, which contain homologous regions known as ZP domains. The ZP domain is also found in many other secretory glycoproteins. Putative transmembrane domains present at the C-termini of ZP glycoprotein precursors are removed as the proteins proceed through the secretory pathway. However, the details of this processing have been unclear. In particular, the precise locations of the C-termini of mammalian zona proteins have not yet been determined. In this study, the C-terminal residues of porcine ZPB and ZPC were identified as Ala-462 and Ser-332, respectively, by mass spectrometry of C-terminal polypeptide fragments of these proteins. These results suggest that ZPB is processed at its furin consensus site, whereas ZPC is processed N-terminal to the furin consensus site. In addition, the analyses of porcine ZPB and ZPC fragments revealed that disulfide bonds within the ZP domains are divided into two groups, suggesting that the ZP domain consists of two subdomains.     

Immunogold study on lectin binding in the porcine zona pellucida and granulosa cells

An ultrastructural localization of lectin receptors on the zona pellucida (ZP) of porcine antral oocytes and on the granulosa cells was performed using a panel of horseradish peroxidase-labelled lectins in conjunction with antiperoxidase antibody and protein A-gold. In some cases, lectin incubation was preceded by sialidase digestion. WGA-, Con-A-, UEA-I-, RCA-I-, PNA- and SBA-reactive sites were distributed differently in the porcine ZP. Sialidase digestion increased the positivity obtained with RCA-I and it was necessary to promote PNA and SBA reactivity. These results indicated that the ZP contained N-acetylglucosamine, a-mannose, a-fucose, b-Gal-(1-4)GlcNAc, b-Gal- (1-3)GalNAc, b-GalNAc and sialic acid residues. We also observed the presence of vesicles in both the ooplasm and granulosa cells, showing a similar lectin binding pattern to that of the ZP, thus suggesting that the oocyte and granulosa cells are the site of synthesis of ZP glucidic determinants.