ZP2 and ZP3 traffic independently within oocytes prior to assembly into the extracellular zona pellucida

The extracellular zona pellucida surrounds mammalian eggs and mediates taxon-specific sperm-egg recognition at fertilization. In mice, the zona pellucida is composed of three glycoproteins, but the presence of ZP2 and ZP3 is sufficient to form a biologically functional structure. Each zona pellucida glycoprotein is synthesized in growing oocytes and traffics through the endomembrane system to the cell surface, where it is released from a transmembrane domain and assembled into the insoluble zona pellucida matrix. ZP2 and ZP3 colocalize in the endoplasmic reticulum and in 1- to 5-microm post-Golgi structures comprising multivesicular aggregates (MVA), but a coimmunoprecipitation assay does not detect physical interactions. In addition, ZP2 traffics normally in growing oocytes in the absence of ZP3 or if ZP3 has been mutated to prevent incorporation into the zona pellucida matrix, complementing earlier studies indicating the independence of ZP3 secretion in Zp2 null mice. N glycosylation has been implicated in correct protein folding and intracellular trafficking of secreted proteins. Although ZP3 contain five N-glycans, enhanced green fluorescent protein-tagged ZP3 lacking N glycosylation sites is present in MVA and is incorporated into the zona pellucida matrix of transgenic mice. Thus, ZP2 secretion is seemingly unaffected by ZP3 lacking N-glycans. Taken together, these observations indicate that ZP2 and ZP3 traffic independently through the oocyte prior to assembly into the zona pellucida.     

Relevance of glycosylation of human zona pellucida glycoproteins for their binding to capacitated human spermatozoa and subsequent induction of acrosomal exocytosis

To delineate the functional aspects of zona pellucida (ZP) glycoproteins during fertilization in human, in the present study, fluorochrome-conjugated Escherichia coli (E. coli)- and baculovirus-expressed recombinant human ZP glycoprotein-2 (ZP2), -3 (ZP3), and -4 (ZP4) were employed. In an immunofluorescence assay, capacitated human sperm exhibited binding of the baculovirus-expressed recombinant ZP3 as well as ZP4 to either acrosomal cap or equatorial region whereas acrosome-reacted sperm failed to show any binding to the acrosomal cap. Using double labeling experiments, simultaneous binding of ZP3 and ZP4 to the acrosomal cap was observed suggesting the possibility of different binding sites of these proteins on the sperm surface. No binding of ZP2 was observed to the capacitated sperm. However, acrosome-reacted sperm (20.00 +/- 1.93%) showed binding of ZP2 that was restricted to only equatorial region. Interestingly, E. coli-expressed recombinant human zona proteins also showed very similar binding profiles. Competitive inhibition studies with unlabeled recombinant human zona proteins revealed the specificity of the above binding characteristics. Binding characteristics have been further validated by an indirect immunofluorescence assay using native human heat solubilized isolated zona pellucida. Employing baculovirus-expressed recombinant ZP3 and ZP4 with reduced N-linked glycosylation and respective E. coli-expressed recombinant proteins, it was observed that glycosylation is required for induction of acrosomal exocytosis but its absence may not compromise on their binding ability. These studies have revealed the binding profile of individual human zona protein to spermatozoa and further strengthened the importance of glycosylation of zona proteins for acrosomal exocytosis in spermatozoa.     

Human sperm bind to the N-terminal domain of ZP2 in humanized zonae pellucidae in transgenic mice

Fertilization requires taxon-specific gamete recognition, and human sperm do not bind to zonae pellucidae (ZP1-3) surrounding mouse eggs. Using transgenesis to replace endogenous mouse proteins with human homologues, gain-of-function sperm-binding assays were established to evaluate human gamete recognition. Human sperm bound only to zonae pellucidae containing human ZP2, either alone or coexpressed with other human zona proteins. Binding to the humanized matrix was a dominant effect that resulted in human sperm penetration of the zona pellucida and accumulation in the perivitelline space, where they were unable to fuse with mouse eggs. Using recombinant peptides, the site of gamete recognition was located to a defined domain in the N terminus of ZP2. These results provide experimental evidence for the role of ZP2 in mediating sperm binding to the zona pellucida and support a model in which human sperm-egg recognition is dependent on an N-terminal domain of ZP2, which is degraded after fertilization to provide a definitive block to polyspermy.     

Characteristics of monoclonal antibodies against porcine zona pellucida-3 and their functional relevance.

Seven monoclonal antibodies (MAs) against 55 kDa glycoprotein family of porcine zona pellucida (ZP3) reacting with either ZP3 alpha (MA-7, MA-27, MA-28) or ZP3 beta (MA-1, MA-2, MA-11, MA-30) have been described. MA-1, -2, -27, -28 and -30 do not recognize carbohydrate determinants as shown by their reactivity to the deglycosylated (DG) ZP3 alpha and ZP3 beta. Indirect immunoperoxidase studies showed that all MAs reacted with zona pellucida from porcine and monkey ovaries. Only MA-1 and -27 reacted with ZP from rabbit ovary as well, while none of the MAs recognised mouse ZP, MA-7, -11, -27, -28 and -30 inhibited in vitro, the zona lysis by trypsin as well as the binding of ZP3 to sperm membrane vesicle as investigated by ELISA.     

Egg-induced modifications of the zona pellucida of mouse eggs: effects of microinjected inositol 1,4,5-trisphosphate

Mouse eggs microinjected with physiological concentrations of inositol 1,4,5-trisphosphate (IP3) do not emit the second polar body, form a pronucleus, or display a fertilization-associated set of changes in the pattern of protein synthesis. IP3-injected eggs, however, display a conversion of the zona pellucida glycoprotein ZP2 to ZP2f. The effect is concentration-dependent with an EC50 (effective concentration, 50%) of 5-10 nM and also occurs in the presence of reduced levels of extracellular calcium. The egg-induced zona pellucida modification is not elicited by several other inositol phosphates that are not able to release calcium from intracellular stores in other systems. Analysis of individual eggs microinjected with IP3 reveals a strong correlation between a reduced binding of sperm to the zona pellucida and the ZP2 to ZP2f conversion. In addition, solubilized zonae pellucidae isolated from IP3-injected eggs possess reduced levels of acrosome reaction-inducing activity. These egg-induced modifications of the zona pellucida--reduced sperm receptor and acrosome reaction-inducing activities and the ZP2 to ZP2f conversion--elicited by microinjected-IP3 are similar to those that occur following fertilization. Results of these experiments suggest that IP3 generated in response to fertilization may play a role in the egg-induced modifications of the zona pellucida that result in the polyspermy block.     

Mouse egg extracellular coat is a matrix of interconnected filaments possessing a structural repeat

As the result of a combined biochemical and electron microscopic investigation, hitherto unrecognized structural features of the mouse egg extracellular coat, or zona pellucida, have been revealed. Specimens were prepared for electron microscopy by spraying individually isolated zonae pellucidae onto a substrate and were observed by both rotary shadowing and negative staining techniques. Results of these experiments suggest that the three zona pellucida glycoproteins, ZP1 (200,000 Mr), ZP2 (120,000 Mr) and ZP3 (83,000 Mr), are organized into long filaments. Negatively stained zona pellucida filaments resemble "beads-on-a-string", with each bead (9.5 nm in diameter) located every 17 nm or so (center-to-center distance) along the axis of the filament. The filaments, in turn, appear to be interconnected by one of the three zona pellucida glycoproteins, ZP1, giving rise to a three-dimensional matrix. Proteolysis of ZP1 by chymotrypsin or reduction of intermolecular disulfides of ZP1 by dithiothreitol results in both solubilization of zonae pellucidae and disruption of interconnections between individual zona pellucida filaments. These observations suggest that the zona pellucida, which plays important roles both during and after fertilization of mammalian eggs, is a highly organized extracellular coat in which glycoproteins are assembled into filaments possessing a recognizable structural repeat.     

Conserved furin cleavage site not essential for secretion and integration of ZP3 into the extracellular egg coat of transgenic mice

The extracellular zona pellucida surrounding mammalian eggs is formed by interactions of the ZP1, ZP2, and ZP3 glycoproteins. Female mice lacking ZP2 or ZP3 do not form a stable zona matrix and are sterile. The three zona proteins are synthesized in growing oocytes and secreted prior to incorporation into the zona pellucida. A well-conserved furin site upstream of a transmembrane domain near the carboxyl terminus of each has been implicated in the release of the zona ectodomains from oocytes. However, mutation of the furin site (RNRR --> ANAA) does not affect the intracellular trafficking or secretion of an enhanced green fluorescent protein (EGFP)-ZP3 fusion protein in heterologous somatic cells. After transient expression in growing oocytes, normal EGFP-ZP3 and mutant EGFP-ZP3 associate with the inner aspect of the zona pellucida, which is distinct from the plasma membrane. These in vitro results are confirmed in transgenic mice expressing EGFP-ZP3 with or without the mutant furin site. In each case, EGFP-ZP3 is incorporated throughout the width of the zona pellucida and the transgenic mice are fertile. These results indicate that the zona matrix accrues from the inside out and, unexpectedly, suggest that cleavage at the furin site is not required for formation of the extracellular zona pellucida surrounding mouse eggs.     

Reassessing the molecular biology of sperm-egg recognition with mouse genetics

The zona pellucida is an extracellular coat that surrounds mammalian eggs and early embryos. This insoluble matrix separates germ from somatic cells during folliculogenesis and plays critical roles during fertilization and early development. The mouse and human zona pellucida contain three glycoproteins (ZP1 or ZPB, ZP2, ZP3), the primary structures of which have been deduced by molecular cloning. Targeted mutagenesis of endogenous mouse genes and transgenesis with human homologues provide models to investigate the roles of individual zona components. Collectively, the genetic data indicate that no single mouse zona pellucida protein is obligatory for taxon-specific sperm binding and that two human proteins are not sufficient to support human sperm binding. An observed post-fertilization persistence of mouse sperm binding to "humanized" zona pellucida correlates with uncleaved ZP2. These observations are consistent with a model for sperm binding in which the supramolecular structure of the zona pellucida necessary for sperm binding is modulated by the cleavage status of ZP2.     

Evaluation of ZP2 domains of functional importance with antisera against synthetic ZP2 peptides

The mouse zona pellucida protein ZP2 plays an important role in the process of fertilization by mediating secondary sperm binding to mammalian oocytes. ZP2 primary structures are highly conserved as revealed by cDNA cloning. The aim of the study was to identify ZP2 domains of functional relevance. Antisera were raised against synthetic peptides that are either conserved in the structure of ZP2 from different mammalian species (AS ZP2-20) or present in the human ZP2 but not in the mouse ZP2 amino acid sequence (AS ZP2-26). Antibody binding to zona pellucida proteins was assessed by assaying the antisera with human hemizonae. Using human zonae pellucidae, we demonstrated that anti-ZP2 common antibodies and anti-ZP2 human peptide antibodies react with human zona pellucida antigens. For the first time, ZP2 domains of functional relevance for human sperm-oocyte interaction could be identified applying the competitive hemizona assay. Antiserum AS ZP2-20 significantly inhibited binding of spermatozoa to test hemizonae, whereas treatment of hemizonae with AS ZP2-26 did not influence sperm-oocyte interaction. These results show that antibodies against synthetic ZP2 peptides react with ZP2 protein and that AS ZP2-20 identifies a linear ZP2 epitope that is of possible functional importance for sperm-oocyte interaction.     

Proteolytic processing of human zona pellucida proteins.

Formation of the egg's extracellular matrix, the zona pellucida, is critical for fertilization and development of growing embryos. Zona pellucida glycoproteins, ZP1, ZP2, and ZP3, are secreted to form an insoluble extracellular matrix surrounding mammalian eggs. All cloned mammalian zona pellucida sequences contain a furin consensus cleavage site, RX(K)/(R)R, upstream of a putative transmembrane domain, which suggests processing by an endoprotease of the furin-proprotein-convertase family. Recombinant expression of human (h) ZP1, ZP2, and ZP3 produces glycoproteins that are secreted and have migration patterns in SDS-PAGE identical to those of native human zona pellucida proteins. Because a C-terminal epitope tag that is present in the cell-associated zona proteins is, however, absent from the secreted zona proteins, secreted recombinant zona pellucida proteins lack their C-terminal regions. Three different strategies were used to explore processing events in the C-terminal region: site-directed mutagenesis of the furin cleavage site, treatment with a competitive inhibitor of all furin family members, and interference with Golgi modifications by Brefeldin A. All treatments altered the SDS-PAGE migration of recombinant hZP3, concordant with cleavage by a furin family member and Golgi glycosylation of secreted hZP3. Furthermore, cleavage of cell-associated hZP3 by exogenous furin converts the migration of cell-associated hZP3 to that of secreted hZP3. To determine whether a similar cleavage pattern exists in zona pellucida proteins that are assembled in the zona matrix, "hZP3 rescue" mouse zonae pellucidae were employed. Immunoblotting experiments revealed that hZP3, assembled and functional in the "hZP3 rescue" mouse zona pellucida, lacks the furin cleavage site, supporting the hypothesis that formation of the zona pellucida matrix involves regulated proteolysis by a member of the furin convertase family.     

Recombinant mouse sperm ZP3-binding protein (ZP3R/sp56) forms a high order oligomer that binds eggs and inhibits mouse fertilization in vitro

Many candidates have been proposed as zona pellucida-binding proteins. Without precluding a role for any of those candidates, we focused on mouse sperm protein ZP3R/sp56, which is localized in the acrosomal matrix. The objective of this study was to analyze the role of ZP3R/sp56 in mouse fertilization. We expressed recombinant ZP3R/sp56 as a secreted protein in HEK293 cells and purified it from serum-free, conditioned medium. In the presence of reducing agents, the recombinant ZP3R/sp56 exhibited a molecular weight similar to that observed for the native ZP3R/sp56. Reminiscent of the native protein, recombinant ZP3R/sp56 formed a high molecular weight, disulfide cross-linked oligomer consisting of six or more monomers under non-reducing conditions. Recombinant ZP3R/sp56 bound to the zona pellucida of unfertilized eggs but not to 2-cell embryos, indicating that the changes that take place in the zona pellucida at fertilization affected the interaction of this protein with the zona pellucida. The extent of in vitro fertilization was reduced in a dose-dependent manner when unfertilized eggs were preincubated with recombinant ZP3R/sp56 (74% drop at the maximum concentrations assayed). Eggs incubated with the recombinant protein showed an absence of or very few sperm in the perivitelline space, suggesting that the reduction in the fertilization rate is caused by the inhibition of sperm binding and/or penetration through the zona pellucida. These results indicate that sperm ZP3R/sp56 is important for sperm-zona interactions during fertilization and support the concept that the acrosomal matrix plays an essential role in mediating the binding of sperm to the zona pellucida.     

Fetuin inhibits zona pellucida hardening and conversion of ZP2 to ZP2f during spontaneous mouse oocyte maturation in vitro in the absence of serum.

The zona pellucida of mouse oocytes becomes resistant to chymotrypsin digestion, or "hardened", when spontaneous maturation occurs in serum-free medium (De Felici and Siracusa, Gam Res 1982; 6:107). The hardened zona pellucida is refractory to sperm penetration, thus preventing fertilization. Conversion of the zona pellucida glycoprotein ZP2 to ZP2f by a protease from precociously released oocyte cortical granules appears to be a major contributory factor of zona pellucida hardening (Ducibella et al., Dev Biol 1990; 137:46). Fetal bovine serum (FBS) prevents zona hardening and the ZP2 to ZP2f conversion during oocyte maturation in vitro (Downs et al., Gam Res 1986; 15:115; Ducibella et al., Dev Biol 1990; 137:46). This study was conducted to determine whether fetuin, a major glycoprotein constituent of FBS and a protease inhibitor, could prevent zona pellucida hardening during murine oocyte maturation in serum-free medium. Commercially available preparations of fetuin purified by three different methods were all active in inhibiting zona pellucida hardening in a concentration-dependent manner. Further chromatographic purification of one of these preparations indicated that the activity preventing zona pellucida hardening was associated specifically with fetuin. Fetuin also inhibited the conversion of ZP2 to ZP2f in a concentration-dependent manner during oocyte maturation in serum-free medium. Moreover, oocytes that matured in serum-free medium containing fetuin could be fertilized and could undergo preimplantation development to the blastocyst stage. These results indicate that fetuin, a component of FBS, inhibits zona pellucida hardening during oocyte maturation, and suggest that fetuin acts by preventing the proteolytic conversion of ZP2 to ZP2f by precociously released cortical granules.     

Defective zonae pellucidae in Zp2-null mice disrupt folliculogenesis, fertility and development

All vertebrate eggs are surrounded by an extracellular matrix. This matrix is known as the zona pellucida in mammals and is critically important for the survival of growing oocytes, successful fertilization and the passage of early embryos through the oviduct. The mouse zona pellucida is composed of three glycoproteins (ZP1, ZP2 and ZP3), each encoded by a single copy gene. Using targeted mutagenesis in embryonic stem cells, Zp2-null mouse lines have been established. ZP1 and ZP3 proteins continue to be synthesized and form a thin zona matrix in early follicles that is not sustained in pre-ovulatory follicles. The abnormal zona matrix does not affect initial folliculogenesis, but there is a significant decrease in the number of antral stage follicles in ovaries isolated from mice lacking a zona pellucida. Few eggs are detected in the oviduct after stimulation with gonadotropins, and no two-cell embryos are recovered after mating Zp2-null females with normal male mice. The structural defect is more severe than that observed in Zp1-null mice, which have decreased fecundity, but not quite as severe as that observed in Zp3-null mice, which never form a visible zona pellucida and are sterile. Although zona-free oocytes matured and fertilized in vitro can progress to the blastocyst stage, the developmental potential of blastocysts derived from either Zp2- or Zp3-null eggs appears compromised and, after transfer to foster mothers, live births have not been observed. Thus, in addition to its role in fertilization and protection of early embryos, these data are consistent with the zona pellucida maintaining interactions between granulosa cells and oocytes during folliculogenesis that are critical to maximize developmental competence of oocytes.     

How the egg regulates sperm function during gamete interaction: facts and fantasies

Although details of the molecular mechanism are not yet clear, considerable evidence suggests that the egg-specific extracellular matrix component ZP3 regulates an essential event of sperm function, the acrosome reaction. Spatial control of this exocytotic event appears to be exerted by immobilization of the triggering ligand, ZP3, in the zona pellucida matrix surrounding the egg. Our data suggest that the signal transduction pathway in sperm activated by this ligand involves highly conserved components that are involved in many other eukaryotic signalling events. Recent experiments indicate that the murine zona pellucida glycoprotein ZP3 regulates acrosomal exocytosis by aggregating its corresponding receptors (ZP3-Rs) located in the mouse sperm plasma membrane. In other experiments, we have identified a putative ZP3-R of mouse sperm with Mr 95,000. Indirect immunofluorescence localizes this ZP3-R, termed p95, to the acrosomal region of the mouse sperm head, which is the anticipated location for ZP3-Rs. Membrane fractionation studies indicate that p95 cofractionates with a plasma membrane-enriched preparation from sperm that contains zona pellucida-receptor activity. In addition to its role as a ZP3-R, p95 also serves as a substrate for a tyrosine kinase in response to zona pellucida binding. On the basis of the data presented here, and borrowing heavily from findings for other signalling systems, we have formulated two testable hypotheses that are compatible with the available data: either p95 is itself a protein tyrosine kinase receptor, or p95 serves as a ZP3 receptor and is separate from a protein tyrosine kinase that is activated during gamete interaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a secondary sperm receptor in the mouse egg zona pellucida: role in maintenance of binding of acrosome-reacted sperm to eggs

During fertilization in mice, acrosome-intact sperm bind via plasma membrane overlying their head to a glycoprotein, called ZP3, present in the egg extracellular coat or zona pellucida. Bound sperm then undergo the acrosome reaction, which results in exposure of inner acrosomal membrane, penetrate through the zona pellucida, and fuse with egg plasma membrane. Thus, in the normal course of events, acrosome-reacted sperm must remain bound to eggs, despite loss of plasma membrane from the anterior region of the head and exposure of inner acrosomal membrane. Here, we examined maintenance of binding of sperm to the zona pellucida following the acrosome reaction. We found that polyclonal antisera and monoclonal antibodies directed against ZP2, another zona pellucida glycoprotein, did not affect initial binding of sperm to eggs, but inhibited maintenance of binding of sperm that had undergone the acrosome reaction on the zona pellucida. On the other hand, polyclonal antisera and monoclonal antibodies directed against ZP3 did not affect either initial binding of acrosome-intact sperm to eggs or maintenance of binding following the acrosome reaction. We also found that soybean trypsin inhibitor, a protein reported to prevent binding of mouse sperm to eggs, did not affect initial binding of sperm to eggs, but, like antibodies directed against ZP2, inhibited maintenance of binding of sperm that had undergone the acrosome reaction on the zona pellucida. These and other observations suggest that ZP2 serves as a secondary receptor for sperm during the fertilization process in mice and that maintenance of binding of acrosome-reacted sperm to eggs may involve a sperm, trypsin-like proteinase.     

Sperm binding to the zona pellucida is not sufficient to induce acrosome exocytosis

At fertilization, spermatozoa bind to the zona pellucida (ZP1, ZP2, ZP3) surrounding ovulated mouse eggs, undergo acrosome exocytosis and penetrate the zona matrix before gamete fusion. Following fertilization, ZP2 is proteolytically cleaved and sperm no longer bind to embryos. We assessed Acr3-EGFP sperm binding to wild-type and huZP2 rescue eggs in which human ZP2 replaces mouse ZP2 but remains uncleaved after fertilization. The observed de novo binding of Acr3-EGFP sperm to embryos derived from huZP2 rescue mice supports a ;zona scaffold' model of sperm-egg recognition in which intact ZP2 dictates a three-dimensional structure supportive of sperm binding, independent of fertilization and cortical granule exocytosis. Surprisingly, the acrosomes of the bound sperm remain intact for at least 24 hours in the presence of uncleaved human ZP2 regardless of whether sperm are added before or after fertilization. The persistence of intact acrosomes indicates that sperm binding to the zona pellucida is not sufficient to induce acrosome exocytosis. A filter penetration assay suggests an alternative mechanism in which penetration into the zona matrix initiates a mechanosensory signal transduction necessary to trigger the acrosome reaction.     

Abnormal zonae pellucidae in mice lacking ZP1 result in early embryonic loss.

All vertebrates have an egg shell that surrounds ovulated eggs and plays critical roles in gamete recognition. This extracellular matrix is known as the zona pellucida in eutherian mammals and consists of three glycoproteins, ZP1, ZP2 and ZP3 in the mouse. To investigate the role of ZP1 in fertilization and early development, we have used targeted mutagenesis in embryonic stem cells to create mouse lines (Zp1(tm/tm)) lacking ZP1. Although a zona pellucida composed of ZP2 and ZP3 was formed around growing Zp1(tm/tm) oocytes, the matrix was more loosely organized than zonae around normal oocytes. In some Zp1 null follicles, this structural abnormality resulted in ectopic clusters of granulosa cells, lodged between the zona matrix and the oolemma, that perturbed normal folliculogenesis. Comparable numbers of eggs were ovulated from Zp1 null females and normal females following hormonal stimulation. However, after mating with males, fewer two-cell embryos were recovered from Zp1 null females, and their litters were significantly smaller than those produced by normal mice. Therefore, although mouse ZP1 is not essential for sperm binding or fertilization, it is required for the structural integrity of the zona pellucida to minimize precocious hatching and reduced fecundity.