Differences between antigenic determinants of pig and cat zona pellucida proteins

Despite many efforts, the control of reproduction in feral cat populations is still a problem in urban regions around the world. Immunocontraception is a promising approach; thus the present study examined the suitability of the widely used pig zona pellucida proteins (pZP) for contraception in feral domestic cats. Purified zona pellucida proteins obtained from pig and cat ovaries were used to produce highly specific antisera in rabbits. Antibodies against pZP raised in rabbits or lions were not effective inhibitors of either in vitro sperm binding (cat spermatozoa to cat oocytes) or in vitro fertilization in cats, whereas antibodies against feline zona pellucida proteins (fZP) raised in rabbits showed a dose-dependent inhibition of in vitro fertilization. Immunoelectrophoresis, ELISA and immunohistology of ovaries confirmed these results, showing crossreactivity of anti-fZP sera to fZP and to a lesser extent to pZP, but no interaction of anti-pZP sera with fZP. It is concluded that cat and pig zonae pellucidae express a very small number of shared antigenic determinants, making the use of pZP vaccine in cats questionable. A contraceptive vaccine based on feline zona pellucida determinants will be a better choice for the control of reproduction in feral cats if immunogenity can be achieved.     

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.     

Modifications of the lectin binding pattern in the rat zona pellucida after in vivo fertilization

The zona pellucida (ZP) is an extracellular matrix surrounding the mammalian oocyte. It is involved in the sperm-egg adhesion phenomenon, induces the acrosome reaction, and participates in the late blockage to polyspermy. Thus, during the process of fertilization the cortical reaction is induced and the biochemical and biological properties of the ZP are modified. Some of these changes have been suggested to prevent the polyspermy. However, the mechanisms behind most of these changes are not well understood. Carbohydrate residues of the ZP glycoproteins have been shown to play a key role in the early step of fertilization. In the present study, the changes produced in the terminal oligosaccharide sequences of the rat ZP glycoproteins after in vivo fertilization were investigated by means of lectin-gold cytochemistry. A comparative quantitative analysis of the density of labeling in the ZP before and after fertilization was carried out by automatic counting of gold particles. The ZP of fertilized and unfertilized eggs were labeled by a battery of lectins including PNA, LFA, MAA, AAA, DSA, RCA I, and WGA. For all lectin studied in both fertilized and unfertilized eggs the labeling was preferentially located in the inner region of the ZP. After fertilization, binding of PNA, LFA, MAA, AAA, and DSA decreased in both inner and outer regions of the ZP. Labeling of RCA l-binding sites only decreased in the inner ZP, whereas reactivity to WGA was increased in the inner area of the ZP. Digestion of the thin-sections with neuraminidase prior to labeling with WGA resulted in a decrease of labeling for WGA binding sites. However, the labeling density of WGA binding sites was similar in both unfertilized and fertilized eggs upon treatment with neuraminidase. The present results demonstrate that the oligosaccharide chains contained in the rat ZP are modified after fertilization of the oocyte. Cortical granules of the oocytes might be involved in these modifications by two mechanisms: 1) by hydrolysis of terminal carbohydrate residues of ZP glycoproteins by specific glycosidases contained in the granules; and 2) by addition of new glycoproteins to the ZP after the exocytosis of the cortical granules (cortical reaction). © 1996 Wiley-Liss, Inc.     

Lysophosphatidylcholine induces changes in physicochemical, morphological, and functional properties of mouse zona pellucida: a possible role of phospholipase A2 in sperm-zona pellucida interaction

Previous evidence has suggested the implication of phospholipase A2 (PLA2) in the interaction of the sperm with the egg's zona pellucida (ZP). We have tested this hypothesis by observing the effect of one of the products of the enzyme reaction, namely lysophosphatidylcholine (LPC), on the functional, physicochemical and morphological properties of ZP. In vitro treatment of oocytes with LPC produced a dramatical increase in the ability of ZP to bind sperm. This increased binding was completely abolished if sperm were pre-treated with pertussis toxin to block the acrosome reaction. ZP was the primary target of LPC since the same effect occurred on isolated ZP. LPC treatment of isolated ZP also produced alterations in its physicochemical properties as deduced from both a decreased binding of fluorescent lectins and an increased solubility of ZP at low pH. As assessed by scanning electron microscopy, LPC also produced dramatic changes in the structure of the ZP surface which lost its typical rough aspect and became smooth. These morphological changes were observed upon LPC treatment of both the intact oocyte and the previously isolated ZP. The chemically related (and precursor molecule of LPC in PLA2 reaction) phosphatidylcholine (PC) had no effect on any of the above studied parameters. Most importantly, LPC had no effect on ZP from two-cell embryos, a structure that has physiologically become refractory to further sperm binding. Analysis of radioiodinated ZP proteins by SDS-PAGE did not indicate an important effect of LPC on these proteins, although the ZP3 protein seemed to be slightly more iodinated.     

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.     

“Spontaneous” hardening of the zona pellucida of mouse oocytes during in vitro culture

Culture in vitro causes a slow, progressive hardening of the zona pellucida (ZP) of fully grown dictyate oocytes isolated from the mouse ovary. Hardening cannot be prevented by inhibitors of peroxidase or by a tyrosine analogue. Culture in anaerobic conditions is very effective in preventing ZP hardening. If the oocyte is cultured surrounded by its own follicle cells or in contact with cumuli oophori obtained from superovulated females, hardening is much reduced. The results suggest that the “spontaneous” hardening in cultured ovarian oocytes is not due to a cortical reaction, and that a diffusible factor is produced by follicle cells that protect the ZP from hardening.     

Coculturing cumulus oocyte complexes with denuded oocytes alters zona pellucida ultrastructure in in vitro matured bovine oocytes

Oocyte quality is a key factor affecting success of in vitro embryo production in cattle. Improving the microenvironment of oocytes during in vitro maturation (IVM) can increase developmental rate and embryo quality. Therefore, the objective was to determine whether denuded oocytes (DO) affect embryo development and ultrastructure of the zona pellucida (ZP) in in vitro matured bovine oocytes. Intact immature cumulus-oocytes complexes (COC) obtained from a local abattoir or by ovum pick-up (OPU) were cocultured with and without abattoir-obtained DO at a COC:DO ratio of 1:5. After IVM, DO were removed and intact DO were either fertilized or observed by scanning electron microscopy. Blastocyst quality was evaluated using a TUNEL assay. The ZP pore size decreased after IVM in COC + DO coculture, regardless of their origin (OPU, 310.5 ± 92.5 vs. 428.9 ± 148.5 nm; abattoir, 317.5 ± 68.5 vs. 358.9 ± 128.5 nm; P < 0.05; mean values ± standard deviation). Moreover, the number of ZP pores in OPU COC + DO and COC + DO was greater than those in OPU COC and COC (control) groups (56 ± 4 and 55 ± 7 vs. 50 ± 6 and 42 ± 4; P < 0.05). The rate of blastocyst development in COC + DO and OPU COC + DO groups was greater those in control and OPU COC groups (36.6% and 55.5% vs. 28.1% and 40.0%; P < 0.05). Moreover, the total cell numbers of blastocysts in COC + DO group exceeded that of control (132.91 ± 30.90 vs. 115.44 ± 24.95; P < 0.05), with no significant between OPU COC + DO and OPU COC groups (139.31 ± 42.51 vs. 137.00 ± 61.34). In conclusion, in vitro embryo development competence and quality improved when oocytes were cocultured with DO. Furthermore, there more, but smaller, ZP pores.     

Structure and function of the zona pellucida: Identification and characterization of the proteins of the mouse oocyte's zona pellucida

The zona pellucida is an acellular coat which surrounds the plasma membrane of fully grown mammalian oocytes and which performs a variety of important functions during oogenesis, fertilization, and preimplantation development. In this investigation the proteins of the mouse oocyte's zona pellucida have been identified and characterized by using zonae pellucidae isolated individually from fully grown oocytes with mouth-operated micropipets. Various morphological and biochemical criteria were employed to assess the purity of the isolated zonae pellucidae and, in most cases, they were found to be virtually free of contamination by other oocyte proteins. It was determined that each zona pellucida contains 4.8 ng of protein, which represents 80% or more of the dry weight of the zona pellucida and about 17% of the oocyte's total protein. Electrophoretic analyses of as few as five isolated zonae pellucidae treated with diazotized [¹²⁵I]iodosulfanilic acid revealed the presence of only three radiolabeled proteins, designated ZP1, ZP2, and ZP3. The same three proteins were identified by Coomassie blue staining when large numbers of isolated zonae pellucidae (approximately 750) were subjected to SDS-polyacrylamide gel electrophoresis. These three proteins migrate as broad bands on SDS-polyacrylamide gels, consistent with their being glycoproteins, with apparent molecular weights of 200,000 (ZP1), 120,000 (ZP2), and 83,000 (ZP3). The same proteins were radiolabeled when intact oocytes were treated with diazotized [¹²⁵I]iodosulfanilic acid, a reagent which does not penetrate the oocyte's plasma membrane, or when isolated zonae pellucidae were treated with ³H-labeled 1-dimethylaminonaphthalene-5-sulfonyl chloride. Results of amino acid analysis and high-resolution two-dimensional electrophoresis of the individual proteins suggest that each protein represents a unique polypeptide chain. The proteins ZP1, ZP2, and ZP3 represent about 36, 47, and 17%, respectively, of the total protein of the zona pellucida. In the presence of reducing agents which cause dissolution of the zona pellucida, ZP1 is converted into a species which migrates with an apparent molecular weight of 130,000, suggesting that it exists as an oligomer, stabilized by disulfide bonds, in the unreduced state. The results of these experiments are discussed in terms of the properties of the zona pellucida before and after fertilization and are compared with results obtained using vitelline envelopes of eggs from nonmammalian animal species.     

Mammalian zona pellucida glycoproteins: structure and function during fertilization

Zona pellucida (ZP) is a glycoproteinaceous translucent matrix that surrounds the mammalian oocyte and plays a critical role in the accomplishment of fertilization. In humans, it is composed of 4 glycoproteins designated as ZP1, ZP2, ZP3 and ZP4, whereas mouse ZP is composed of ZP1, ZP2 and ZP3 (Zp4 being a pseudogene). In addition to a variable sequence identity of a given zona protein among various species, human ZP1 and ZP4 are paralogs and mature polypeptide chains share an identity of 47%. Employing either affinity purified native or recombinant human zona proteins, it has been demonstrated that ZP1, ZP3 and ZP4 bind to the capacitated human spermatozoa and induce an acrosome reaction, whereas in mice, ZP3 acts as the putative primary sperm receptor. Human ZP2 only binds to acrosome-reacted spermatozoa and thus may be acting as a secondary sperm receptor. In contrast to O-linked glycans of ZP3 in mice, N-linked glycans of human ZP3 and ZP4 are more relevant for induction of the acrosome reaction. Recent studies suggest that Sialyl-Lewis^x sequence present on both N- and O-glycans of human ZP play an important role in human sperm?Cegg binding. There are subtle differences in the downstream signaling events associated with ZP3 versus ZP1/ZP4-mediated induction of the acrosome reaction. For example, ZP3 but not ZP1/ZP4-mediated induction of the acrosome reaction is dependent on the activation of the G_i protein-coupled receptor. Thus, various studies suggest that, in contrast to mice, in humans more than one zona protein binds to spermatozoa and induces an acrosome reaction.     

Effect of vitrification on the zona pellucida hardening and follistatin and cathepsin B genes expression and developmental competence of in vitro matured bovine oocytes

The purpose of our study was to assess the effect of vitrification with or without the presence of calcium in the vitrification solution on the: 1) diameter of oocytes and thickness of the zona pellucida, 2) zona pellucida hardening, 3) expression of mRNA follistatin (FST) and cathepsin B (CTSB) in oocytes and 4) developmental competence of embryos derived from in vitro matured and vitrified oocytes.The results of our study demonstrate, that vitrification did not alter thickness of the zona pellucida and diameter of the oocytes, however it triggered hardening of the zona pellucida. The presence of calcium in the vitrification solutions intensified hardening of zona in immature and mature oocytes (P < 0.04, P < 0.001, respectively) and provoked increased mRNA FST expression in oocytes matured in vitro compared to immature oocytes (P < 0.01) and those vitrified without calcium (P < 0.004). CTSB mRNA expression was increased in immature oocytes and oocytes vitrified with calcium compare to mature oocytes (P < 0.02). The developmental potential of vitrified oocytes was impaired compared to non-vitrified oocytes, being more evident in oocytes vitrified with calcium.In summary, vitrification did not change the oocyte diameter and thickness of the zona pellucida and expression of FST and CTSB mRNA. It diminished developmental potential of the vitrified oocytes. The presence of calcium in the vitrification solutions increased hardening of zona pellucida as well as affected the level of FST and CTSB mRNA in oocytes and developmental potential of these oocytes.     

Zonadhesin: characterization, localization, and zona pellucida binding.

Zonadhesin is a multiple-domain transmembrane protein that is believed to function as a sperm-zona pellucida binding protein. In this study we sequenced zonadhesin from rabbit testis and analyzed its processing, expression, localization, and zona pellucida binding. We show that the precursor protein occurs exclusively in the testis and that proteolytic processing results in the formation of three fragments: p43 (D1 domain), p97 (D2-D4 domains), and p58 (D4 domain-C-terminal). In mature spermatozoa the p43 and p97 fragments exist as disulfide-bonded dimers. During spermatogenesis, synthesis of zonadhesin mRNA chiefly occurs in primary spermatocytes, whereas the protein is abundant in both Sertoli cells and spermatids. In spermatozoa the protein is localized exclusively to the anterior acrosome but is not available for binding antibody on live spermatozoa. Once the acrosome reaction is induced, zonadhesin is lost from the spermatozoon, but remains with the acrosomal shroud. We show that recombinant D4 domain can bind zona pellucida, and we propose that zonadhesin functions after the acrosome reaction has been initiated to bind the acrosomal shroud to the zona pellucida.     

Cryopreservation of mammalian oocytes and embryos: current problems and future perspectives

Cryopreservation techniques for mammalian oocytes and embryos have rapidly progressed during the past two decades,emphasizing their importance in various assisted reproductive technologies.Pregnancies and live births resulting from cryopreserved oocytes and embryos of several species including humans have provided proof of principle and led to the adoption of cryopreservation as an integral part of clinical in vitro fertilization.Considerable progress has been achieved in the development and application of the cryopreservation of mammalian oocytes and embryos,including preservation of the reproductive potential of patients who may become infertile,establishment of cryopreserved oocyte banks,and transport of oocytes and embryos internationally.However,the success rates are still far lower than those obtained with fresh oocytes and embryos,and there are still obstacles that need to be overcome.In this review,we address the major obstacles in the development of effective cryopreservation techniques.Such knowledge may help to eliminate these hurdles by revealing which aspects need improvement.Furthermore,this information may encourage further research by cryobiologists and increase the practical use of cryopreservation as a major part of assisted reproductive technologies for both humans and animal species.     

Lectin-binding properties of hamster egg zona pellucida and plasma membrane during maturation and preimplantation development

The structure and organization of the zona pellucida and plasma membrane of the hamster egg at various stages of maturation and development were examined using lectin-mediated agglutination and the binding of fluorescent-labeled lectins. Ricinus communis I and Dolichos biflorus lectins specifically agglutinated the zona pellucida of both unfertilized and fertilized eggs, while wheat germ agglutinin (WGA) only agglutinated eggs which had been pretreated with protease. Six other lectins failed to agglutinate even eggs pretreated with protease. A comparison of the lectin-binding sites on the zona pellucida of eggs in various stages of maturation and development revealed that the intensity of binding and distribution of fluorescent-labeled lectins remain unchanged. Zona-free eggs were agglutinated by every lectin tested except those recognizing -fucose-like residues. Fertilized zona-free eggs were slightly more agglutinable by concanavalin A (ConA), Lens culinaris and WGA than unfertilized eggs. When the surfaces of zona-free eggs were examined with fluorescent ConA, Ricinus communis I and WGA, maximal binding was seen when eggs reached full maturity and binding decreased during the later stages of preimplantation development.     

Equine marker genes: Polymorphism for plasminogen

Polymorphism for two autosomal alleles of equine plasminogen, PLG ¹ and PLG ², was demonstrated in plasma by isoelectric focusing and immunofixation, with a goat anti-human plasminogen antibody. The frequency of PLC ² was 0.16 in 150 Standardbreds, 0.20 in 96 Thoroughbreds, and 0.39 in 32 Shetland ponies. No evidence for linkage of PLG with any of 13 marker loci was found.     

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.     

Changes in the properties and composition of zona pellucida of pigs during fertilization in vitro.

Several hundred fertilized pig eggs were prepared by an in vitro fertilization (IVF) technique in which follicular phase ovarian eggs were matured in vitro to metaphase II before incubation with capacitated epididymal spermatozoa for 12 h at 39 degrees C. Parthenogenetic eggs were also prepared by stimulation of the mature eggs with an electric pulse. The zonae were solubilized with 0.2% pronase/phosphate-buffered saline (PBS) or lactic acid/PBS. The time taken for solubilization was 30-40% shorter than for unfertilized eggs, indicating that zona hardening was induced during fertilization. At the same time, the sperm receptor activity of the zona was reduced. Electrophoretic analyses of zona glycoproteins from the ovarian, mature and fertilized eggs revealed that the amount of 90 kDa proteins decreased substantially during fertilization. This fraction could barely be detected in the zonae from parthenogenetic eggs. However, modification with a fluorescent probe showed that the general architecture of the zona remained unchanged during fertilization. These results suggest that the minor 90 kDa proteins are specifically degraded by the protease(s) released from the oocyte at fertilization, thereby leading to the block to polyspermy.