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PubMedDate not supplied

The hemizona assay (HZA) as an experimental model to evaluate the inhibition of sperm binding to the murine zona pellucida by isolated zona pellucida protein.

Compelling evidence has demonstrated that zona binding represents gamete recognition by sperm binding with high affinity and specificity to complex glycoproteins of the zona pellucida. In the present study we evaluated the hemizona assay (HZA) in the investigation of the interaction of mouse spermatozoa with unfertilized murine oocytes and hemizonae after exposure to solubilized murine zonae pellucidae proteins. The zonae pellucidae were isolated from ovarian tissue following described mincing techniques. The sperm binding characteristics of murine spermatozoa were studied by using SDS-PAGE isolated ZP2 (+/- 120 Kd) and ZP3 (+/- 83 Kd) components of the zona pellucida. Sperm receptor activity was examined in a competitive gamete binding fashion using the HZA as an indicator of sperm/zona interaction. The results illustrated that isolated, solubilized ZP2 and ZP3 glycoprotein moieties of the zona pellucida inhibited sperm binding to hemizonae and oocytes when compared to controls, and that the HZA can be utilized as an internally controlled homologous bioassay to evaluate the effects of zona pellucida proteins on tight binding of spermatozoa to the zona pellucida.

Animals↗
PubMed2000

Structural analysis of the asparagine-linked glycan units of the ZP2 and ZP3 glycoproteins from mouse zona pellucida.

Zona pellucida (ZP), the extracellular glycocalyx that surrounds the mammalian egg plasma membrane, is a relatively simple structure consisting of three to four glycoproteins. In the mouse, the ZP is composed of three glycoproteins, namely ZP1 (200 kDa), ZP2 (120 kDa), and ZP3 (83 kDa). Extensive studies in this species have resulted in the identification of primary (mZP3) and secondary (mZP2) binding sites for spermatozoa. The two zona components are highly glycosylated containing N-linked and O-linked glycan units. In an attempt to characterize N-linked glycan units, mZP2 and mZP3 were purified and the N-linked carbohydrate chains were released by exhaustive digestion with N-glycanase. The released oligosaccharides (OSs) were radiolabeled by reduction with NaB3H4 and resolved by gel filtration on a column of Bio-Gel P-4. The OSs separated into several peaks indicating the presence of a variety of N-linked glycans. Interestingly, the radioactive peaks resolved from mZP2 and mZP3 were quite different, a result suggesting qualitative and quantitative differences in the glycans. The [SH]-labeled glycans present in mZP2 and mZP3 were pooled separately and fractionated by serial lectin chromatography. Experimental evidence included in this report strongly suggests that mZP3 (but not mZP2) contains polylactosaminyl glycan with terminal, nonreducing alpha-galactosyl residues. The mZP3 glycans eluted from the immobilized lectin columns were further characterized by lectin and sizing column chromatography before or after digestion with endo-/ exo-glycohydrolases. Data revealed the presence of a variety of OSs, including poly-N-acetyllactosaminyl, bi-, tri-, and tetraantennary complex-type, and high-mannose-type glycans. Taken together, these results provide additional evidence on the complex nature of the glycan chains present on mZP glycoconjugates.

Animals↗
PubMed1987

Analysis of the biological properties of antibodies raised against intact and deglycosylated porcine zonae pellucidae.

Zonae pellucidae (ZP) were isolated from 1,500 porcine ovaries and heat solubilized to generate approximately 15 mg ZP glycoprotein. Analysis of this material by isoelectric focusing, one-dimensional electrophoresis, and gas chromatography indicated the presence of a major glycoprotein species that exhibited considerable microheterogeneity with respect to its charge (pI 7.5-3.5) and molecular mass (45-85 kDa) and that contained 39.6% carbohydrate, predominantly N-acetylglucosamine. Chemical deglycosylation of porcine ZP using trifluoromethanesulphonic acid (TFMS) resulted in the production of five discrete protein bands on one-dimensional sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/PAGE) with molecular masses of 66, 52, 36, 32, and 16 kDa. Antisera raised in rabbits and marmosets to ZP and/or deglycosylated ZP (DGZP) were used in immunoblotting experiments to demonstrate the retention of immunogenicity by DGZP and the cross-reactivity of the antisera with their heterologous antigen. These studies indicated that antisera that were capable of inhibiting the fertility of primates in vivo and the penetration of the human ZP in vitro reacted preferentially with 3 of the 5 products of deglycosylation, with molecular masses of 66, 52, and 36 kDa. Anti-DGZP antibodies were also shown to interact with intact porcine and human ZP and, with the latter, to block the ability of human spermatozoa to both bind to and penetrate this structure.

Animals↗
PubMed2005

Efficacy of antibodies against a chimeric synthetic peptide encompassing epitopes of bonnet monkey (Macaca radiata) zona pellucida-1 and zona pellucida-3 glycoproteins to inhibit in vitro human sperm-egg binding.

Immunocontraception achieved by immunization with zona pellucida (ZP) glycoproteins is invariably associated with ovarian dysfunction. Use of ZP glycoprotein-based synthetic peptides as immunogens has been proposed to overcome adverse side effects on ovaries. In the present study, a chimeric peptide encompassing the epitopes of bonnet monkey (Macaca radiata) ZP glycoprotein-1 (bmZP1; amino acid residues 251-273) and ZP glycoprotein-3 (bmZP3; amino acid residues 324-347), separated by a tri-glycine spacer, was synthesized and conjugated to diphtheria toxoid (DT). Immunization of female BALB/cJ mice and bonnet monkeys with the chimeric peptide led to generation of antibodies that reacted with the chimeric peptide, individual bmZP1 & bmZP3 peptides, and also recombinant bmZP1 and bmZP3 proteins expressed by E. coli in an ELISA. Indirect immunofluorescence studies revealed that the immune serum also recognized human as well as bonnet monkey ZP. A significant inhibition of human sperm binding to ZP was observed with antibodies generated against the chimeric peptide in mice (P = 0.0001) as well as monkeys (P = 0.0002) in a hemizona assay (HZA). The inhibition efficacy was significantly higher than that observed by using antibodies against the individual bmZP1 and bmZP3 peptides. Interestingly, no ovarian pathology was observed in female bonnet monkeys immunized with the chimeric peptide. These studies have demonstrated that the chimeric peptide encompassing peptides of multiple ZP glycoproteins may be a promising candidate antigen for designing immunocontraceptive vaccines.

Amino Acid Sequence↗
PubMed1995

In vivo fertility of rams in relation to sperm-zona pellucida binding and sperm-zona pellucida penetration of ovine oocytes.

Sperm-zona pellucida (zona) binding and sperm-zona penetration have been suggested for use as in vitro bioassays of fertility since both are essential steps in the fertilization process. The correlations of sperm-zona binding and sperm-zona penetration with the in vivo fertility of sheep were investigated in this study. In vivo fertility was estimated from a heterospermic insemination trial using cryopreserved ram semen. Neither zona binding, zona penetration nor the ability to undergo an acrosome reaction was significantly correlated with the in vivo fertility of the rams (P = 0.78, P = 0.66, and P = 0.85, respectively). These results suggest that the zona binding and zona penetration bioassays may not be useful estimators for assessing cryopreserved ram sperm fertility.

Journal Article↗
PubMed1997

Polarized light microscopy and digital image processing identify a multilaminar structure of the hamster zona pellucida.

The zona pellucida (zona) is a glycoprotein coat that envelopes the oocyte and embryo, binds sperm during fertilization and facilitates transfer of the embryo through the Fallopian tube. Before implantation can occur, the blastocyst must hatch from the zona. Several lines of evidence suggest that the zona is multilaminar. We hypothesized that the multilaminar structure of the zona filaments could be imaged non-destructively with the polarized light microscope. A recent modification of the polarized light microscope (pol-scope), which combines innovations in polarization optics with novel image processing software, allows measurement of birefringence at all points of the image. Hamster metaphase II oocytes were placed on glass coverslips which replaced the bottom of culture dishes, imaged under differential interference contrast (DIC) and pol-scope optics, then digitized and processed to measure birefringence magnitude and orientation. The pol-scope revealed the zona to be divided into outer and inner layers separated by a zone of low retardance. This finding is consistent with filaments in the outer layer oriented tangentially and in the inner layer oriented radially. The multilaminar structure of the mammalian zona suggested by differential lectin binding and by scanning electron microscopy could be imaged non-destructively with the pol-scope. Because the pol-scope provides a non-destructive method to identify macro-molecular organization of the zona, it may prove useful in developmental studies of hatching and to direct resection of the zona.

Animals↗
PubMed1986

In vitro studies of the golden hamster sperm acrosome reaction: completion on the zona pellucida and induction by homologous soluble zonae pellucidae.

We have studied the occurrence of the golden hamster sperm acrosome reaction (AR) in vitro during interaction with the oocyte investments: the cumulus cell matrix and the zona pellucida. Hamster sperm were capacitated in a defined medium that does not induce the AR. These spermatozoa were allowed to interact with the ovum vestments, the events of which were recorded using high-speed videomicrography. Frame-by-frame analysis revealed that sperm did not complete the AR in the cumulus cell matrix, but did so on the zona pellucida. Furthermore, a higher percentage of sperm completed the AR on the zona pellucida of cumulus-invested than on cumulus-free eggs. We also investigated the effect of solubilized hamster and mouse zonae pellucidae on the hamster sperm AR. Addition of solubilized hamster zonae to capacitated sperm elicited the AR within 15 min. Solubilized mouse zonae were significantly less effective, indicating that the zona-induced AR in hamster sperm may be species specific. These results suggest that the hamster zona pellucida is an inducer of the AR in the intact or soluble form, and that the majority of spermatozoa traverse the cumulus cell matrix without completing the AR in our in vitro system.

Acrosome↗
PubMed2006

Pig zona pellucida 2 (pZP2) protein does not participate in zona pellucida formation in transgenic mice.

The zona pellucida, an extracellular matrix surrounding mammalian oocytes, is composed of three or four glycoproteins. It is well known that the zona pellucida plays several critical roles during fertilization, but there is little knowledge about its formation. The purpose of this study is to examine whether a pig zona pellucida glycoprotein 2 (pZP2) would assemble with mouse zona pellucida. A transgene construct was prepared by placing a minigene encoding pZP2 downstream from the promoter of mouse ZP2. The result showed that the transgenic protein was synthesized in growing oocytes but not incorporated into the zona pellucida. Furthermore, the pZP2 transgene did not rescue the phenotype in ZP2-knockout zona-deficient mice. These results indicate that pZP2 does not participate in mouse zona pellucida formation and the zona pellucida is constituted from its component proteins in a molecular species-specific manner between mice and pigs.

Animals↗
PubMed2005

Structural conservation of mouse and rat zona pellucida glycoproteins. Probing the native rat zona pellucida proteome by mass spectrometry.

The mammalian zona pellucida is an egg extracellular matrix to which sperm bind. Mouse zonae are composed of three glycoproteins (ZP1, ZP2, and ZP3), while rat zonae contain four (ZP1, ZP2, ZP3, and ZP4/ZPB). Mouse sperm bind to zonae comprised solely of mouse ZP2 and ZP3. In this report, we show that rat sperm also bind to these zonae, indicating that ZP2 and ZP3 contain a "minimum structure(s)" to which rodent sperm can bind, and ZP1 and ZP4/ZPB are dispensable in these two rodents. These data are consistent with our mass spectrometric analysis of the native rat zona pellucida proteome (defined as the fraction of the total rat proteome to which the zonae glycoproteins contribute) demonstrating that the rat zonae glycoproteins share a high degree of conservation of structural features with respect to their mouse counterparts. The primary sequences of the rat zonae proteins have been deduced from cDNA. Each zona protein undergoes extensive co- and post-translational modification prior to its secretion and incorporation into an extracellular zona matrix. Each has a predicted N-terminal signal peptide that is cleaved off once protein translation begins and an anchoring C-terminal transmembrane domain from which the mature protein is released. Mass spectrometric analysis with a limited amount of native material allowed determination of the mature N-termini of rat ZP1 and ZP3, both of which are characterized by cyclization of glutamine to pyroglutamate; the N-terminus of ZP2 was identified by Edman degradation. The mature C-termini of ZP1 and ZP3 end two amino acids upstream of a conserved dibasic residue that is part of, but distinct from, the consensus furin cleavage sequence, while the C-terminus of ZP2 was not determined. Each zona protein contains a "zona domain" with eight conserved cysteine residues that is thought to play a role in the polymerization of the zona proteins into matrix filaments. Partial disulfide bond assignment indicates that the intramolecular disulfide patterns in rat ZP1, ZP2, and ZP3 are identical to those of their corresponding mouse counterparts. Last, nearly all potential N-glycosylation sites are occupied in the rat zonae glycoproteins (three of three for ZP1, six or seven of seven for ZP2, and four or five of six for ZP3). In comparison, potential O-glycosylation sites are numerous (59-83 Ser/Thr residues), but only two regions were observed to carry O-glycans in rat ZP3.

Amino Acid Sequence↗
PubMed1997

Three-dimensional structure of the zona pellucida.

The zona pellucida is the extracellular coat that surrounds the mammalian oocyte. It forms a spherical shell of remarkably uniform thickness (5-10 microns in eutherian mammals). The mouse is currently the largest source of data on the zona pellucida and this review is built largely on these data. The zona pellucida is composed of three proteins in both mice and humans: ZP1, ZP2 and ZP3. These proteins are glycosylated and, in mice, have mature relative molecular masses of 200,000, 120,000 and 83,000, respectively. ZP1 is a dimer of two apparently identical subunits. All three mouse proteins have been sequenced and possess transmembrane domains at their C-terminal ends coupled with furin cleavage sites immediately upstream. Sequence data have been used to provide an accurate assessment of the mole ratios of the three proteins. The ratio of ZP2:ZP3 is close to 1:1, whereas ZP1 is approximately 9% of the combined mole amounts of ZP2 and ZP3. Ultrastructural evidence suggests that the mouse zona pellucida is composed of filaments constructed by head-to-tail association of globular proteins. The coordinate synthesis of the three zona pellucida proteins coupled with the near 1:1 stoichiometry of ZP2 and ZP3 is consistent with a model in which ZP2-ZP3 heterodimers are the basic repeating units of the filament, with cross-linking of filaments by dimeric ZP1. This model is also consistent with data from ZP2 and ZP3 gene knockout and antisense experiments. However, the structure remains unproven. The small amount of ZP1 relative to ZP2 and ZP3 may have important implications for the distribution of ZP1 cross-links, since the number of cross-linking sites potentially exceeds the number of ZP1 dimer molecules by a considerable margin. The evidence that ZP1, ZP2 and ZP3 are all synthesized via a membrane-bound step is discussed and two models are proposed for the assembly of the zona pellucida. The cortical reaction and its effect on the zona pellucida are examined in detail. It is shown that the amount of material released by cortical granules could be of the order of 30% by mass of ZP1, and that if this material was distributed predominantly on the inner face of the zona pellucida, its local concentration could approach that of ZP1. A model in which the zona block to polyspermy is caused by direct titration of zona pellucida binding sites is suggested as an alternative to the explanation that relies on enzyme cleavage of ZP2 to ZP2f. Finally, some of the major experimental and structural issues that remain to be addressed are identified.

Animals↗
PubMed1999

Structure and function of the mammalian egg zona pellucida.

The zona pellucida is a thick extracellular coat that surrounds all mammalian eggs and preimplantation embryos. The zona pellucida supports communication between oocytes and follicle cells during oogenesis; protects oocytes, eggs, and embryos during development, and regulates interactions between ovulated eggs and free-swimming sperm during and following fertilization. Mutant females that produce eggs that lack a zona pellucida are infertile. The functions of the zona pellucida during fertilization now can be ascribed to certain of its glycoproteins. Here we describe some aspects of zona pellucida structure and function as they relate to mammalian fertilization. J. Exp. Zool. (Mol. Dev. Evol.) 285:251-258, 1999.

Animals↗
PubMed2006

Fourier transform infrared spectroscopic analysis of the intact zona pellucida of the mammalian egg: changes in the secondary structure of bovine zona pellucida proteins during fertilization.

The zona pellucida is the acellular transparent envelope surrounding the mammalian oocyte. An analysis of the changes in the structures of zona pellucida proteins is essential for understanding the molecular mechanisms underlying the important physiological roles of the zona during fertilization and preimplantation. The hardening of the zona caused by the structural changes during fertilization is generally accepted to be responsible for blocking polyspermy. In this study, we analyzed changes in the secondary structure of the zona during fertilization by Fourier transform infrared (FTIR) spectroscopy and transmission electron microscopy. The predominance of beta-sheet structure in porcine ovarian egg zona proteins in water was ascertained using FTIR spectra. Alpha-helix structure was also present. The attenuated total reflection (ATR)-FTIR spectrum of intact, unsolubilized porcine zonae pellucidae from ovarian eggs indicated that the zona proteins in the native zona pellucida also have beta-structure as the main constituent. Attenuated total reflection-FTIR spectroscopy of intact bovine zona pellucida obtained from ovarian and fertilized eggs at the blastocyst stage revealed that the beta-structure content increased during fertilization. Furthermore, a reduction of the thickness of the zona during fertilization was observed using transmission electron microscopy. Therefore, the change in the zona architecture that causes hardening of the zona during fertilization is accompanied by changes in the secondary structure of the zona proteins.

Animals↗
PubMed1999

Porcine sperm surface beta1,4galactosyltransferase binds to the zona pellucida but is not necessary or sufficient to mediate sperm-zona pellucida binding.

The binding of sperm to the zona pellucida is an integral part of the mammalian fertilization process, investigated most extensively in the mouse. Several sperm receptors for the murine zona pellucida have been studied (Snell WJ, White JM. 1996. Cell 85:629-637; Wassarman PM. 1999. Cell 96:175-183), but the most compelling evidence exists for beta-1,4-galactosyltransferase (GalTase). Considering that GalTase is present on the surface of porcine sperm (Larson JL, Miller DJ. 1997. Biol Reprod 57:442-453), we investigated the role of GalTase in porcine sperm-zona binding. Sperm surface GalTase catalyzed the addition of uridine diphosphate-[(3)H]galactose to the 55 kDa group of the porcine zona pellucida proteins implicated in sperm binding, demonstrating that GalTase binds the porcine zona. The functional importance of GalTase-zona pellucida binding was tested. Addition of uridine diphosphate galactose, a substrate that completes the GalTase enzymatic reaction and disrupts GalTase mediated adhesion, had no effect on binding of sperm to porcine oocytes. Furthermore, removal of the GalTase zona ligand by incubation of oocytes with N-acetylglucosaminidase had no effect on binding of sperm to oocytes. These results suggest that GalTase is not necessary for sperm to bind to the zona pellucida. Digestion of isolated porcine zona proteins with N-acetylglucosaminidase did not affect the biological activity of soluble porcine zona proteins in competitive sperm-zona binding assays, suggesting that GalTase alone is not sufficient to mediate sperm-zona attachment. From these results, it appears that, although GalTase is able to bind porcine zona proteins, its function in porcine sperm-zona binding is not necessary or sufficient for sperm-zona binding. This supports the contention that porcine sperm-zona binding requires redundant gamete receptors.

Acetylglucosaminidase↗
PubMed1994

Disordered acrosome reaction of spermatozoa bound to the zona pellucida: a newly discovered sperm defect causing infertility with reduced sperm-zona pellucida penetration and reduced fertilization in vitro.

It is believed that during the process of human fertilization, acrosome-intact spermatozoa bind to the surface of the zona pellucida which triggers the acrosome reaction and the enzymes released facilitate sperm penetration through the zona pellucida. We describe here reduced frequency of the acrosome reaction on the zona pellucida as a cause of infertility in 10 couples with long durations of infertility (average 6 years) and low (< 15%, n = 3) or zero (n = 7) fertilization rates in vitro. Sperm concentration, motility, velocity (Hamilton-Thorn), morphology and DNA normality were within the normal range in all the patients. Electron microscopy of spermatozoa did not reveal any specific ultrastructural defects. All couples were negative for antisperm antibodies by immunobead tests. Oocytes from other patients which failed to fertilize in in-vitro fertilization and normal donor spermatozoa were used as controls for sperm-zona pellucida binding and penetration experiments. Acrosome status of spermatozoa bound to the zona pellucida was assessed with a fluorescent lectin and electron microscopy. The mean number of spermatozoa bound to the zona pellucida was not significantly different between patients and controls. However, the acrosome reaction of spermatozoa bound to the zona pellucida after 2 h incubation was significantly lower (P < 0.001) in the patients (mean 5%, range 0-16) than in the controls (mean 68%, range 44-96). No zona pellucida (out of 40) was penetrated by patient spermatozoa whereas most (39/40) zonae were penetrated by control spermatozoa (average 27 spermatozoa/four zonae pellucidae).(ABSTRACT TRUNCATED AT 250 WORDS)

Acrosin↗
PubMedDate not supplied

Zona pellucida piercing enhances zona penetration by spermatozoa from normospermic and teratospermic domestic cats.

Poor quality ejaculates appear associated with reduced zona penetration and poor fertilization in vitro in some felid species or populations. Even morphologically normal sperm from these teratospermic ejaculates are compromised in ability to penetrate the zona pellucida and fertilize oocytes. A domestic cat model and zona piercing were used to study the function of normal and malformed sperm. Male cats naturally producing different proportions of morphologically normal sperm (group I, > 60%; group II, 45-55%; group III, < 40%) were identified. Electroejaculates were subjected to sperm washing (WS) and half of these to swim-up (SU) processing following WS, and all samples were assessed for a sperm motility index (SMI) over time. Zonae of salt-stored (SS), domestic cat oocytes were mechanically pierced (ZnPd) three times each. Control (nonmanipulated) SS oocytes (n = 430) and ZnPd-SS oocytes (n = 430) were coincubated with sperm in vitro. The proportion of morphologically normal sperm/ejaculate differed (P < 0.05) among groups averaging 68.8% for group I, 53.5% for group II, and 34.7% for group III. After SU processing, the percent normal sperm among groups was similar (P > 0.05; range, 70.8-80.2%). Average SMI for all groups was high (> 70) at 0 hour and decreased over time, but the descent of slope was greater (P < 0.05) for group III than groups I and II. The proportion of oocytes penetrated was not influenced (P > 0.05) by sperm-processing treatment. For all groups, inner zona penetration was greater (P < 0.05) in ZnPd-SS than SS oocytes (group I, 52.3% vs. 34.9%; group II, 37.9% vs. 20.3%; group III, 32.0% vs. 17.2%).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Fertilization antigen (FA-1) completely blocks human sperm binding to human zona pellucida: FA-1 antigen may be a sperm receptor for zona pellucida in humans.

The effects of purified human sperm fertilization antigen-1 (FA-1), affinity-purified monoclonal Fab' antibody to FA-1, and monoclonal Fab' antibody to phosphotyrosine residues on human sperm-zona interaction were investigated. The purified FA-1 antigen completely blocked sperm binding to zona pellucida (P < 0.0001). Also, the monoclonal Fab' antibodies to FA-1 antigen and phosphotyrosine residues significantly (P < 0.05) reduced sperm-zona pellucidae and the antibodies were preincubated with sperm before insemination and not vice versa. These results suggest that the tyrosine phosphorylation especially of FA-1 antigen has an important role in zona pellucida receptor recognition and binding. These findings also suggest that FA-1 antigen may be the sperm receptor involved in zona pellucida binding in humans.

Animals↗
PubMed1989

Genomic organization of a sex specific gene: the primary sperm receptor of the mouse zona pellucida.

The zona pellucida is an extracellular matrix which surrounds mammalian oocytes and plays an important role in the species-specific fertilization of mammals. The mouse zona is composed of three sulfated glycoproteins one of which, ZP3, serves as the primary sperm receptor in the initial interaction between egg and sperm. We have previously cloned a ZP3 cDNA and shown that the gene for ZP3 is expressed in a precise temporal and spatial fashion only in growing oocytes. We now describe the genomic organization of this gene whose expression is germ-line and female sex-specific. The gene contains 8 exons spanning approximately 8.6 kilobases in the mouse genome where it appears to be present as a single copy gene. S1 analyses of the 5' and 3' ends of the gene define the transcription start and stop sites and show that ZP3 mRNA has very short untranslated regions. Sequence determination has revealed a novel tandem repeat which is reiterated six times in the 5' flanking region and five times in the seventh intron.

Amino Acid Sequence↗
PubMed1999

Disulfide formation in bovine zona pellucida glycoproteins during fertilization: evidence for the involvement of cystine cross-linkages in hardening of the zona pellucida.

The time for solubilization of the bovine zona pellucida in a hypotonic buffer containing 5% (v/v) beta-mercaptoethanol and 7 mol urea l-1 increased by 10% after fertilization. Coupling with a specific fluorescent thiol probe, monobromobimane (mBBr), was markedly greater in the zona pellucida of ovarian eggs compared with fertilized eggs, indicating that the cysteine residues in the zona pellucida of unfertilized eggs are oxidized to cystines during fertilization. After endo-beta-galactosidase digestion to remove N-acetyllactosamine repeats of the carbohydrate chains, three zona pellucida glycoproteins (ZPA, ZPB and ZPC) coupled with the fluorescent bimane groups were fractionated efficiently by reverse-phase HPLC. Estimation of bimane groups in the three components and SDS-PAGE revealed that intramolecular disulfide bonds in ZPA and intra- and intermolecular disulfide bonds in ZPB were formed during fertilization, but oxidation of cysteine residues in ZPC was low. Specific proteolysis of ZPA during fertilization was also observed. These results indicate that the formation of disulfide linkages together with specific proteolysis result in the construction of a rigid zona pellucida structure, which is responsible for hardening of the zona pellucida.

Animals↗