On the macromolecular composition of the zona pellucida from porcine oocytes

The chemical and immunological relation between the glycoprotein components of the pig oocyte zona pellucida resolved by two-dimensional polyacrylamide gel electrophoresis was investigated. After disulfide bond reduction, four microheterogeneous glycoprotein components with apparent molecular weights of 25K, 55K, 65K, and 90K were resolved. When disulfide bonds were left unreduced, two microheterogeneous glycoprotein components were resolved with apparent molecular weights of 55K and 90K. Actin was present, but as a contaminant of the zona pellucida rather than as a true component. The structural relation of these components was investigated using deglycosylation with trifluoromethane-sulfonic acid, limited proteolysis with Staphylococcus aureus V8 protease, amino acid and carbohydrate composition analyses, sequence analysis, and monoclonal antibodies. The 25K and 65K components comigrated with the 90K component when disulfide bonds were not reduced. When the intermolecular disulfide bonds crosslinking the two components were reduced, the 25K and 65K components behaved independently. The 25K and 65K components were derived from the 90K glycoprotein family by proteolysis. The 25K component originated from the C-terminal end, and the 65K component from the N-terminal end of the 90K glycoprotein. The 55K component was composed of two chemically and antigenically distinct glycoproteins, termed 55K alpha and 55K beta, that electrophoretically comigrated. The N-terminal amino acid of the 55K alpha family was blocked. The 55K beta family had an N-terminal amino acid sequence of Asp-Val-Pro-Thr-Ile-Gly-Leu-Ser-X-Ala-Pro-Thr. Thus, the two to four electrophoretic components of the zona pellucida observed on gel electrophoresis are derived from three glycoprotein families.     

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.     

Isolation of the zona pellucida and purification of its glycoprotein families from pig oocytes

The isolation of the porcine zona pellucida, the glycoprotein envelope surrounding the mammalian oocyte, and the purification of its glycoprotein families is described. The zona pellucida was prepared from oocytes isolated from pig ovaries using a razor blade device and sieving procedures with Teflon or nylon screens. In 6-7 man h, the zona pellucida from 5 X 10(5) oocytes was obtained yielding 12 mg protein and 2.2 mg carbohydrate. The absorptivity of heat solubilized and filtered zona pellucida was A1%280 nm, 1 cm = 10.8. The four glycoprotein families of the zona pellucida were purified by two-dimensional polyacrylamide gel electrophoresis and electrophoretic elution. The electrophoretic purity of these families was greater than 90%. The protein and carbohydrate content and the amino acid and monosaccharide compositions of each of the glycoprotein families were determined.     

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.     

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.     

N-glycosylation of zona glycoproteins during meiotic maturation is involved in sperm-zona pellucida interactions of porcine oocytes

The objective was to determine whether N-glycosylation of zona pellucida (ZP) glycoproteins occurred during meiotic maturation of porcine oocytes, and whether this had a role in fertilization. In the first of three experiments, carbohydrate residues in the ZP of in vitro matured porcine oocytes were blocked with various lectins and the influence of such blocking on sperm-ZP interactions was studied. The second experiment used a lectin-binding assay to determine whether the number of GlcNAc residues in ZP was changed by N-glycosylation during in vitro maturation (IVM) of porcine oocytes. The last experiment determined the effects of tunicamycin, a specific N-glycosylation inhibitor, for various intervals during IVM, on sperm-ZP interactions in porcine oocytes. The primary findings were that: 1) N-glycosylation of GlcNAc residues in porcine ZP occurred during the first 24 h of IVM; and 2) such glycosylation was indispensible for sperm-ZP interactions, e.g., number of sperm bound to ZP, acrosome-reacted sperm, sperm penetration rate, and level of polyspermy (P < 0.05). However, blocking N-glycosylation by tunicamycin treatment during IVM did not adversely influence the progression of oocytes to meiotic metaphase II and male pronucleus formation, indicating that this glycosylation was involved only in the initial stages of fertilization. We inferred that the increase in terminal GlcNAc residues in ZP glycoprotein through new N-glycosylation during the first 24 h of meiotic maturation played a critical role in porcine ZP acquiring the capacity to accept sperm.     

Differences in proteinase digestibility of the zona pellucida of in vivo and in vitro derived porcine oocytes and embryos

Embryo transfer practitioners know very well that, in a variety of species, there are differences between in vitro- and in vivo-derived embryos. It is assumed that these differences are results of suboptimal in vitro conditions leading to cytoplasmic and nuclear imperfections that will result in decreased embryo viability. In the present investigation the resistance of the zona pellucida of in vivo- and ex vivo-derived porcine embryos to a proteolytic enzyme is addressed. Ovulated but unfertilized oocytes, in vitro and in vivo-derived embryos of various developmental stages were exposed to a 0.5% pronase solution. The zonae of ovulated oocytes and in vivo-derived embryos at various stages of development took much longer to be digested than zonae of comparable in vitro-stages. Residence of in vitro derived embryos in an oviduct, no matter whether excised or in situ, significantly increased the zona resistance to pronase digestion. Embryonal stages normally residing in the uterus exhibited a distinctly decreased zona resistance to pronase. The culture of IVF embryos in an excised oviduct brought about a 6- to 14-fold increase in time required for zona digestion. A 24 h residence of IVF-derived embryos in the oviduct of a live recipient resulted in a digestion time for the zona of, on average, 48 h versus 1.4 h in the controls. Individual differences were substantial. The observations suggest that there must be structural changes in the zona pellucida or some sort of protective coating deposited while ova or embryos reside in the oviduct supplying protection against the action of proteolytic enzymes.     

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 antigenie 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. © 1995 wiley-Liss, Inc.     

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.     

Analysis of zona pellucida modifications due to cortical granule exocytosis in single porcine oocytes, using enhanced chemiluminescence

The present study was carried out to determine whether modification of zona pellucida (ZP) of a single oocyte following the cortical granule (CG) exocytosis induced by electrical stimulation could be analyzed using enhanced chemiluminescent (ECL) detection of the biotinylated ZP in a porcine oocyte. When a biotinylated ZP derived from a single oocyte matured in vitro was subjected to SDS-PAGE, 3 major bands (ZP1, ZP2 and ZP3) were observed following ECL detection. In these oocytes, CGs staining with fluorescein isothiocyanate (FITC)-labeled peanut agglutinin (FITC-PNA) had formed a monolayer underlying the plasma membrane. Electrical stimulation to induce artificial activation caused a decline in the fluorescent intensity of the CGs with a concomitant decrease in the amounts of ZP1 and ZP2 bands. However, the mobility changes of ZP1 and ZP2 on SDS-PAGE were not found under the inhibitory condition of the CG exocytosis in which oocytes were treated with ethylene glycol-bis(beta-aminoethyl ether) N, N, N',N'-tetraacetic acid (EGTA) or 1, 2-bis(2-aminophenoxy)ethane-N, N, N', N'-tetraacetic acid tetrakis(acetoxymethyl) ester (BAPTA/AM). In addition, when a time-dependent decrease in amounts of ZP1 and ZP2 bands on SDS-PAGE was observed in a single oocyte during activation, a maximum decrease in these bands was detected in oocytes incubated for at least 3.5 h after electrical stimulation. These results show that the method employed, ECL detection of the biotinylated ZP of a single oocyte, is a valuable tool for the analysis of ZP modification resulting from a decrease in amounts of ZP1 and ZP2 glycoproteins in combination with exocytosis of CGs, and that the prolonged period after activation is required for complete ZP modification in porcine oocytes.     

Reversible changes in dissolution of the zona pellucida of immature bovine oocytes

The influence of holding immature bovine oocytes in in vitro bovine oviducts on the dissolution of zona pellucida in 0.1% pronase, the role of cumulus cells in this process and the possibility of reversing the process were examined. For the study, 1,045 oocytes were obtained from 2 to 6 mm ovarian follicles. Cumulus-free oocytes were placed in isolated bovine oviducts at 37 degrees C. The average dissolution time of the zona pellucida increased in proportion to the holding time of oocytes in oviducts: 9.9, 13.8, 48.3, 239.3 and 788.3 min after 5, 20, 40, 80 and 120 min in the oviduct, respectively. For the control group, only 4.6 min were required for dissolution of the zona. When cumulus-free and cumulus enclosed oocytes were held for 120 min, no differences were seen in the average lytic time of the zona pellucida of cumulus enclosed oocytes compared with the control group. When cumulus-free oocytes were held in vitro for 120 min and then immersed in follicular fluid from 30 min to 18 h, there was a significant reversal in the sensitivity of the zona pellucida to proteolysis.     

Sperm binding activity of the zona pellucida of immature mouse oocytes

Immature oocytes taken from ovarian follicles are sometimes used in studies of sperm-zona interaction in species for which it is difficult to obtain ovulated eggs. As yet, however, there has been no quantitative comparison of the sperm binding capacities of immature and ovulated oocytes. We report here that in mice there is no significant difference in the numbers of sperm which bind to the zonae pellucidae of immature and ovulated oocytes in vitro. These results support the use of immature oocytes in studies of sperm-zona interaction. We have also analyzed the sources of variability in sperm binding assays, and we make suggestions for the most efficient design of experiments.     

Proteolysis of specific porcine zona pellucida glycoproteins by boar acrosin

The morphologic and biochemical effects on the structure and constituent glycoproteins of the zona pellucida (ZP) by a specific sperm enzyme, acrosin, and a nonsperm enzyme, trypsin, have been evaluated. Intact porcine ZP matricies, exposed to either acrosin or trypsin, were analyzed microscopically. Changes in specific glycoproteins were monitored by high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and the silver-based color stain, GELCODE. Although these enzymes did not alter the macroscopic properties of the ZP matrix, the 2D-PAGE ZP protein patterns were markedly altered. The high molecular weight glycoprotein families (II and III) were sensitive to proteolytic digestion, whereas the major glycoprotein family (I) of the porcine zona was only partially proteolyzed by acrosin and trypsin. Furthermore, it was demonstrated that acrosin had unique substrate specificity compared to that of trypsin, since the ZP peptide patterns were found to be different. These studies are the first to demonstrate which integral glycoproteins of the native porcine ZP matrix are specifically proteolyzed by acrosin from the homologous species and that this proteolysis occurs without the dissolution of the native porcine matrix.     

Effect of oviductal and cumulus cells on zona pellucida and cortical granules of porcine oocytes fertilized in vitro with epididymal spermatozoa

The objective of this study was to evaluate the effects of porcine oviductal epithelial cell (POEC) monolayers and cumulus cells on the zona pellucida (ZP) and cortical granules (CG) of in vitro matured porcine oocytes. Denuded and cumulus-enclosed oocytes were exposed to POEC before or during in vitro fertilization (IVF). The functional effects of the co-culture system were the tested on the ZP resistance, measured by the time necessary to dissolve the ZP with 0.1% pronase, and the distribution and density of the cortical granules. CG density in the equator and cortex of each oocyte was evaluated by confocal microscopy after staining with fluorescein isothiocyanate-labelled peanut agglutinin (FITC-PNA). Both variables were assessed immediately after an in vitro maturation period (IVM group), 3 and 6h after culture with or without (Control) oviductal cells (Experiment 1) and 3h after insemination with frozen-thawed epididymal spermatozoa in the presence or absence (Control) of oviductal cells (Experiment 2). The time to dissolve the ZP of oocytes from IVM group was 440.4 +/- 61.7 s and no difference was observed among groups in Experiment 1. In contrast, the density of CG was affected; oocytes pre-incubated for 6h had a higher density than those pre-incubated for 3 h (P <0.001). Oocytes fertilized in vitro in the presence of POEC (Experiment 2) had a similar ZP digestion time as control oocytes 3 h after insemination. The presence of POEC during IVF as well as the presence of cumulus cells had no effect on the density and distribution of CG. However, a significant decrease in the density of CG was observed in the fertilized oocytes compared to in vitro matured oocytes (P <0.001). It is concluded that under the conditions employed the oviductal and cumulus cells in the perifertilization period had no effect on ZP hardening and CG density. However, an increase in CG density was observed when oocytes were maintained in culture. In addition, no hardening of ZP was observed after IVF, and denuded and cumulus-enclosed oocytes showed similar cortical reactions after insemination with epididymal spermatozoa regardless of the presence of POEC.     

Further fractionation of the glycoprotein families of porcine zona pellucida by anion-exchange HPLC and some characterization of the separated fractions

The glycoproteins of porcine zonae pellucidae have been fractionated into three families (PZP1-3) by gel filtration HPLC [Nakano et al. (1987) Biochem. Int. 14, 417-423]. However, they still comprise heterogeneous molecular species differing in electric charge. We found that sulfate, but not phosphate, is contained in PZP1-3 by a simple and rapid method for microanalysis of the anionic groups. These families were efficiently separated into many fractions by anion-exchange HPLC. When elution was performed by stepwise increase in NaCl concentration in 8 M urea/20 mM Tris-HCl, pH 8.0, a single distinctive peak emerged for each step. The analyses of amino acids, monosaccharides, and anions of the eight separated fractions of the major family, PZP3, showed that larger amounts of sulfated lactosamine linked to the constituent proteins are present in the fractions that are eluted later: the chain length and/or the chain number of these polylactosamines and the sulfate content increased with stepwise increase in NaCl concentration. Composition analyses also revealed that twice as much N-glycolylneuraminic acid is present as N-acetylneuraminic acid in all fractions. The contents of these sialic acids in the fractions slightly increased in the order of elution. These results together with those of the analyses of endo-beta-galactosidase digests showed that the charge heterogeneity of the porcine zona proteins is due mainly to differences in the amount of sulfated lactosamine, which is predominantly distributed in the non-reducing regions of the sugar chains.     

Sperm binding capacity and ultrastructure of the zona pellucida of stored canine oocytes

Sperm binding to the zona pellucida is a prerequisite for fertilization, and tests that evaluate this function have been described for several species. When carrying out such tests in the canine species, ovaries or oocytes have to be stored to obtain a sufficient number of oocytes at the time of testing. In the present study, the sperm binding capacities of salt-stored oocytes and oocytes from deep frozen ovaries were measured and compared with that of fresh oocytes. Two different procedures for washing the sperm-oocyte complexes (gentle and tough) were used before evaluating the number of bound spermatozoa. The total number of oocytes that bound spermatozoa was significantly lower for both salt-stored and deep frozen oocytes compared with fresh oocytes. Significantly fewer spermatozoa bound to stored oocytes than to fresh oocytes (P 相似文献   

Involvement of carbohydrates in the hardening of the zona pellucida of mouse oocytes

The effect of lectins with different saccharide specificity (ConA, LCA, DBA, WGA and PNA) on enzymatic digestion of the zona pellucida (ZP) of mouse oocytes was studied. All lectins tested, except PNA, induced ZP hardening with different degrees of efficiency. Moreover, extensive ZP digestion with mixed exoglycosidase prevented "spontaneous" ZP hardening. These observations suggest that changes of the carbohydrate moieties can be involved in the hardening of the zona pellucida of mouse oocytes.