Uptake of an oviductal antigen by the hamster zona pellucida

Using an antiserum raised against hamster oviductal zona pellucida, we observed specific immunogenic components of the reproductive tract on the zonae of oviductal eggs and in oviductal fluid. Results of immunohistochemical studies suggested that these oviductal components may originate from epithelial cells of the isthmus and, to a lesser extent, of the ampulla and fimbria. The oviductal immunogenic components have also been observed within the bursal cavity, which contains the ovary. These observations suggest that these oviductal components may play an important role in the first steps of the hamster reproductive process.     

Ultrastructural evidence for an association between an oviductal glycoprotein and the zona pellucida of the golden hamster egg

The ultrastructural localization of an oviductal glycoprotein, designated ZP-0 in golden hamster oviductal eggs, was investigated by immunolabeling methods using a monoclonal antibody (C11E8). Immunofluorescence staining showed that C11E8 specifically reacted with the zona pellucida of the oviductal egg but not the ovarian egg. In an immunoelectron microscopic study applying the protein-A gold technique, gold particles were distributed throughout the zona pellucida of the oviductal eggs and were also associated with the perivitelline matrix. Structures within the eggs and cumulus cells did not react with C11E8. Quantitative evaluations of the degree of labeling demonstrated that a large number of gold particles was bound to the zone pellucida, especially in the middle layer. Moreover, in bovine testicular hyaluronidase-treated eggs the density of labeling decreased only in the outer third of the zona pellucida. These results show that ZP-0 to the was associated with the zona pellucida and perivitelline matrix of the golden hamster egg after ovulation and suggest that there are topographical differences in the binding activity of ZP-0 to the zona pellucida. In addition, the decrease in labeling density of ZP-O induced by hyaluronidase appears to be related to changes in the properties of the outer layer of the zona pellucida.     

A glycoprotein of oviductal origin alters biochemical properties of the zona pellucida of hamster egg

Eggs were isolated from ovaries and oviducts of the golden hamster and the components of zonae pellucidae were examined using density gradient SDS-polyacrylamide electrophoresis. Zonae of ovarian eggs (ZP-OVA) had three major components corresponding to the so-called ZP-1, ZP-2, and ZP-3. Zonae of recently ovulated eggs collected from oviducts (ZP-OVI) had a 200–240 K component (ZP-O) in addition to the three components present in ZP-OVA. When ovarian and oviductal eggs were stained with FITC-conjugated B. simplicifolia-1 lectin (BS-1), which specifically binds to alpha-D-galactose- or alpha-N-acetyl-D-galactosamine-like terminal saccharide residues, ZP-OVI was intensely stained, while ZP-OVA was not. ZP-OVA gained the ability to bind to BS-1 after a brief treatment with oviduct extracts. These results suggest that biochemical properties of hamster zonae change after transport of eggs from ovary to the oviduct. The addition of the 200–240 K component of oviductal origin to preexisting zona components seems to be responsible for this change.     

In vivo versus in vitro oviductal glycoprotein (OGP) association with the zona pellucida (ZP) in the hamster and baboon

The goal of this study was to determine if differences exist between in vivo vs. in vitro OGP association with the ZP and to quantitate those differences. Ovarian oocytes were harvested 12.5 or 27 hr post-hCG from hyperstimulated hamsters or baboons, respectively. Hamster and baboon ovarian oocytes were incubated in vitro in media +/- homologous OGP (100 or 200 microg/100 microl) or in some studies with 100 microl oviductal fluid for 3, 6, or 24 hr at 37 degrees C. Some of the baboon ovarian oocytes were transferred immediately after harvesting to the ampulla of both oviducts using a tom cat catheter and retrieved after a 3 hr in situ incubation. Hamster oviductal oocytes were collected 3, 6, and 24 hr following ovulation. After incubation or oocyte retrieval from the oviduct, cumulus cells were removed, oocytes were washed extensively and binding of OGP to the ZP was examined by immunofluorescence. Fluorescence intensity was quantified using densitometric scanning of photographic negatives with the background of each negative as an internal control. In all studies, OGP association with the ZP was significantly greater in vivo than in vitro (P < 0.05). In vitro OGP association with the ZP did not significantly increase with incubation time or OGP concentration; however, a small nonsignificant increase in OGP association with the ZP in the oviduct was detected over time. Differences did not appear to be due to depletion of OGP from the in vitro incubation media, since Western blot analysis of the media showed that OGP was still present. Although OGP concentration in vivo is unknown, Western blots showed similar intensity comparing 100 microg of OGP media and oviductal fluid. Immunolocalization of OGP using laser confocal microscopy showed regional differences in OGP binding. The outer half of the zona pellucida had significantly more OGP bound than the inner half on oviductal oocytes. No regional differences were detected for in vitro incubated oocytes. In conclusion, OGP association with the ZP is greater in vivo vs. in vitro, suggesting that one must be cautious in designing and evaluating in vitro studies of OGP function.     

Immunocytochemical evidence for the transfer of an oviductal antigen to the zona pellucida of hamster ova after ovulation

The zona pellucida (ZP), a glycoprotein layer that encloses the mammalian oocyte, is formed during follicular development in the ovary, persists at the time of fertilization within the oviduct, and then surrounds the embryo until implantation in the uterus. Although the structure and chemical properties of the ZP have been extensively studied, the precise site of origin of the ZP remains a matter of controversy. Moreover, the mechanism of synthesis and secretion of the ZP constituents is not fully elucidated. We have recently developed monoclonal antibodies (MAbs) against oviductal ZP of the golden hamster. We have used one of these MAbs (an immunoglobulin G) and the protein A-gold technique to study the localization of the corresponding antigenic sites, and we report here their distribution in the oviduct and within the cumulus oophorus complex of the superovulated hamster. In the oviductal epithelium, immunolabeling was observed in non-ciliated secretory cells in structures involved in protein secretion. In the cumulus masses collected from the oviduct, the sites of immunoreactivity were localized exclusively in the ZP encompassing the oocyte. Gold particles were evenly distributed throughout the entire thickness of the ZP. Treatment of the cumulus masses with hyaluronidase prior to preparation of isolated oocytes for immunocytochemistry did not affect this uniformity. The ZP of the preovulatory oocytes in ovarian follicles was not labeled. Our study provides immunocytochemical evidence for the secretion of an oviductal antigen that becomes intimately associated with the ZP of the oocytes during their passage through the oviduct.     

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.     

Immunoelectron microscopic localization of an oviductal antigen in hamster zona pellucida by use of a monoclonal antibody

The zona pellucida is an extracellular matrix of glycoproteins which surrounds the mammalian oocyte and preimplantation embryo. We have recently developed monoclonal antibodies against oviductal zona pellucida of the golden hamster. We applied the post-embedding immunocytochemical method using a monoclonal antibody (IgGl,k) to determine the precise location of antigenic sites in the cumulus oophorus complex of the superovulated hamster. By applying the high-resolution protein A-gold technique, we demonstrated that the sites of immunoreactivity were exclusively in the zona pellucida encompassing the oocyte. Other structures within the oocyte and neighboring cumulus cells were not labeled by gold particles. Moreover, gold particles were evenly distributed throughout the entire thickness of the zona pellucida, indicating that this extracellular layer is at least in part made up of an antigen recognized by the monoclonal antibody that is uniformly distributed in the zona matrix.     

Texture of the zona pellucida of the mature pig oocyte. The mammalian egg envelope revisited

The zona pellucida (ZP) of mature pig oocytes is believed to consist of a dense filamentous meshwork, less compact on the inner and outer faces. The uneven surface of the ZP is made of unordered and stretched fibrils surrounding deep funnels which are the openings of the radial canaliculi. The topography of the ZP surface may contribute to the initial interplay between male and female gametes. Using cytochemical techniques for transmission electron microscopy (TEM), such as tannic acid and ruthenium red treatments, we found that the ZP of pig oocytes was essentially made of bundles of fibrils distributed in concentric layers (except in the innermost and outer parts). A correlation appears between the dense structure of the core layer of the ZP and its texture: it is constituted of superposed layers of fibril bundles, whereas only a random meshwork is found in a very thin innermost and in the outer layer. The fascicular configuration may control the permeability of the ZP, giving its semi-rigidity and elasticity, and may facilitate sperm penetration. The liquid crystal-like design of the core layer of the ZP is similar to textures found in the the vitelline envelope (zona radiata) of other vertebrates and possibly of all the deuterostomes. Such texture is probably related to the unique ZP protein composition and to a coordinated synthesis.     

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.     

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.     

A contraceptive peptide vaccine targeting sulfated glycoprotein ZP2 of the mouse zona pellucida

In this study, we have mapped and characterized a B cell epitope of sulfated glycoprotein ZP2 (ZP2) as a step toward the development of a multi-epitope zona pellucida (ZP) vaccine. Recombinant polypeptides expressed by random deoxyribonuclease-digested fragments of ZP2 cDNA were screened for binding to IE-3, a monoclonal antibody to murine ZP2. Positive clones contained cDNA inserts encoding polypeptide corresponding to ZP2(103-134). When normal or ovariectomized female mice were immunized with three overlapping peptides that span this region of ZP2 (101-120, 111-130, 121-140), only ZP2(121-140) elicited IgG antibodies that reacted with mouse ovarian ZP, indicative of the presence of native B epitope and helper T cell epitope in ZP2(121-140). To more finely map the ZP2 B cell epitope, a random peptide display library was screened with the IE-3 antibody, and a consensus tetramer sequence VxYK that matched the ZP2(123-126) sequence VRYK was located. Competitive immunofluorescence analysis with single alanine-substituted VxYK peptides ranked the relative contribution of the three critical B cell epitope residues as Y > V > K. A chimeric peptide was constructed that contained the YRYK motif of ZP2 and a bovine RNase T cell epitope. Although (C57BL/6xA/J) F1 (B6AF1) female mice immunized with the chimeric peptide developed ZP antibody response, this peptide elicited antibody only in mice of the histocompatibility complex (MHC) H-2(k or b) haplotype. In contrast, ZP2(121-140) peptide elicited antibody in inbred mice with three additional mouse MHC haplotypes. Moreover, although ZP2(121-140) contained a T cell epitope, no oophoritis was observed after immunization of B6AF1 mice with ZP2(121-140) in complete Freund's adjuvant (CFA). In a preliminary trial, female B6AF1 mice immunized with ZP2(121-140) in CFA had reduced litter sizes as compared with mice injected with CFA alone.     

Localization of neutral N-linked carbohydrate chains in pig zona pellucida glycoprotein ZPC.

Zona pellucida, a transparent envelope surrounding the mammalian oocyte, plays important roles in fertilization and consists of three glycoproteins; ZPA, ZPB and ZPC. In pig, neutral complex-type N-linked chains obtained from a ZPB/ZPC mixture possess sperm-binding activity. We have recently reported that among neutral N-linked chains triantennary and tetraantennary chains have a sperm-binding activity stronger than that of diantennary chains. Triantennary and tetraantennary chains are localized at the second of the three N-glycosylation sites of ZPB. In this study, we focused on the localization of neutral N-linked chains in ZPC. ZPB and ZPC can not be separated from each other unless the acidic N-acetyllactosamine regions of their carbohydrate chains are removed by endo-beta-galactosidase digestion. A large part of the acidic N-linked chains becomes neutral by the digestion, but the main neutral N-linked chains are not susceptible to the enzyme. N-glycanase digestion indicated that ZPC has three N-glycosylation sites. Three glycopeptides each containing one of the N-glycosylation sites were obtained by tryptic digestion of ZPC and the N-glycosylation sites were revealed as Asn124, Asn146 and Asn271. The carbohydrate structures of the neutral N-linked chains from each glycopeptide were characterized by two-dimensional sugar mapping analysis taking into consideration the structures of the main, intact neutral N-linked chains of ZPB/ZPC mixture reported previously. Triantennary and tetraantennary chains were found mainly at Asn271 of ZPC, whereas diantennary chains were present at all three N-glycosylation sites. Thus, ZPC has tri-antennary and tetra-antennary chains as well as ZPB, but the localization of the chains is different from that in ZPB.     

Localization of N-linked carbohydrate chains in glycoprotein ZPA of the bovine egg zona pellucida.

The zona pellucida, a transparent envelope surrounding the mammalian oocyte, consists of three glycoproteins, ZPA, ZPB and ZPC, and plays a role in sperm-egg interactions. In bovines, these glycoproteins cannot be separated unless the acidic N-acetyllactosamine regions of the carbohydrate chains are removed by endo-beta-Galactosidase digestion. Endo-beta-Galactosidase-digested ZPB retains stronger sperm-binding activity than ZPC. It is still unclear whether ZPA possesses significant activity. Recently, we reported that bovine sperm binds to Man5GlcNAc2, the neutral N-linked chain in the cow zona proteins. In this study, we investigated the localization of the sperm-ligand active high-mannose-type chain and the acidic complex-type chains in bovine ZPA. Three N-glycopeptides of ZPA, containing an N-glycosylation site at Asn83, Asn191 and Asn527, respectively, were obtained from endo-beta-Galactosidase-digested ZPA. Of these glycosylation sites, only Asn527 is present in the ZP domain common to all the zona proteins. The carbohydrate structures of the N-linked chains obtained from each N-glycopeptide were characterized by two-dimensional sugar mapping analysis, while considering the structures of the N-linked chains of the zona protein mixture reported previously. Acidic complex-type chains were found at all three N-glycosylation sites, while Man5GlcNAc2 was found at Asn83 and Asn191, but there was very little of this sperm-ligand active chain at Asn527 in the ZP domain of ZPA.     

Changes to the meiotic spindle and zona pellucida of mature mouse oocytes following different cryopreservation methods

This study is to investigate the change of morphology of the meiotic spindle and the extent of zona hardening relating to the morphological survival and developmental competence of thawed oocytes. Four- to 8-week-old female mice (C57BL/6) primed with an intraperitoneal injection of pregnant mare's serum gonadotropin and human chorionic gonadotropin. Cryopreserved oocytes using two protocols: vitrificaton using ethylene glycol (EG) and slow freezing using propanediol (PROH). The freezing oocytes were thawed and were fertilized and subsequently cultured in vitro. Spindle/chromosome imagery, dissolution of zona pellucida, and post-thawing survival and development were comparable between two groups. The vitrification cryopreservation method proved to be better than the slow-freezing protocol when comparing the frequency of normal-shaped spindle development post-thawing. The difference in the time required for the dissolution of the zona pellucida under treatment of pronase that was determined to exist between the two cryopreservation methods was statistically significant (P<0.005). The survival rate of post-thawed mature oocytes was significantly greater for the vitrification group than it was for the slow-freezing cryopreservation group (P=0.005). The vitrification cryopreservation of mature murine oocytes would appear to be more satisfactory than the slow controlled-rate freezing method as regards the post-thawing oocyte survival and also the incidence of the normal spindle apparatus in the ooplasm.     

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.     

Evidence for a role of the major glycoprotein in the structural maintenance of the pig zona pellucida

The functional domains of the glycoproteins of the pig zona pellucida have been analysed using lectin binding, peptide mapping, and immunoblotting in conjunction with analysis by high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and protein detection with the silver-based colour stain. Two of the pig zona pellucida glycoproteins identified in 2D-PAGE were differentially proteolysed within the intact matrix by a variety of enzymes. This proteolysis of specific proteins, however, did not affect the suprastructure of the matrix, or inhibit spermatozoa from adhering to the surface of the zona pellucida. The major glycoprotein appears to be involved in the structural maintenance of the zona pellucida because dissolution of the matrix correlated with proteolysis of this glycoprotein by proteinase K. These glycoproteins were further evaluated by lectin blotting with Ricinus communis agglutinin (RCA) and wheat germ agglutinin (WGA) before and after proteolysis of zona pellucida with trypsin. The lectins bound to all charge species of the three major zona pellucida glycoproteins. Only the most acidic components of the major glycoprotein family, which are not extensively digested, were recognized by these lectins after proteolysis. These studies provide evidence that the major glycoprotein family I of the pig zona pellucida is primarily responsible for maintaining the integrity of the matrix.