Zona Pellucida Proteins, Fibrils, and Matrix.

The zona pellucida (ZP) is an extracellular matrix that surrounds all mammalian oocytes, eggs, and early embryos and plays vital roles during oogenesis, fertilization, and preimplantation development. The ZP is composed of three or four glycosylated proteins, ZP1-4, that are synthesized, processed, secreted, and assembled into long, cross-linked fibrils by growing oocytes. ZP proteins have an immunoglobulin-like three-dimensional structure and a ZP domain that consists of two subdomains, ZP-N and ZP-C, with ZP-N of ZP2 and ZP3 required for fibril assembly. A ZP2-ZP3 dimer is located periodically along ZP fibrils that are cross-linked by ZP1, a protein with a proline-rich N terminus. Fibrils in the inner and outer regions of the ZP are oriented perpendicular and parallel to the oolemma, respectively, giving the ZP a multilayered appearance. Upon fertilization of eggs, modification of ZP2 and ZP3 results in changes in the ZP's physical and biological properties that have important consequences. Certain structural features of ZP proteins suggest that they may be amyloid-like proteins.

Egg's ZP3 structure speaks volumes.

Binding of mammalian sperm to eggs depends in part on ZP3, a glycoprotein in the egg's extracellular coat, the zona pellucida. In this issue, Han et al. (2010) describe the structure of an avian ZP3 homolog, providing insights into ZP3 processing and polymerization and the roles of the ZP3 polypeptide and its carbohydrate in sperm binding.

Female fertility and the mammalian egg's zona pellucida.

All mammalian eggs are surrounded by a relatively thick extracellular matrix (ECM) or zona pellucida (ZP) to which free-swimming sperm bind in a species-restricted manner during fertilization. The ZP consists of either three (e.g., Mus musculus) or four (e.g., Homo sapiens) glycosylated proteins, called ZP1-4. These proteins are unlike those found in somatic cell ECM, are encoded by single-copy genes on different chromosomes, and are well conserved among different mammals. Mammalian ZP proteins are synthesized as polypeptide precursors by growing oocytes that will become ovulated, unfertilized eggs. These precursors are processed to remove a signal-sequence and carboxy-terminal propeptide and are secreted into the extracellular space. Secreted ZP proteins assemble into long, crosslinked filaments that exhibit a structural repeat due to the presence of ZP2-ZP3 dimers every 140 Å or so along filaments. Filaments are crosslinked by ZP1 and are oriented either perpendicular, parallel, or randomly to the plasma membrane of eggs depending on their position in the ZP. Free-swimming mouse sperm recognize and bind to ZP2 or ZP3 that serve as sperm receptors. Acrosome-intact sperm bind to ZP3 oligosaccharides and acrosome-reacted sperm bind to ZP2 polypeptide. ZP filaments fail to assemble in the absence of either nascent ZP2 or ZP3 and results in mouse eggs that lack a ZP and female infertility. Gene sequence variations due to point, missense, or frameshift mutations in genes encoding ZP1-4 results in human eggs that lack a ZP or have an abnormal ZP and female infertility. These and other features of the mouse and human egg's ZP are discussed here.

Female fertility and the zona pellucida.

Fertility in female mammals, including mice and humans, is dependent on the presence of a zona pellucida (ZP) around growing oocytes and unfertilized eggs. A ZP is required to stabilize contacts between oocyte microvilli and follicle cell projections that traverse the ZP to form gap junctions that support the health of growing oocytes and developing follicles. In the absence of a ZP, due to inactivation or mutation of genes encoding ZP proteins, there is a loss of contacts between growing oocytes and neighboring follicle cells and a concomitant reduction in the production of ovulated eggs that results in female infertility.

Mouse zona pellucida proteins as receptors for binding of sperm to eggs.

Fertilization in mammals is initiated by species-restricted binding of free-swimming sperm to the unfertilized egg's thick extracellular matrix, the zona pellucida (ZP). Both acrosome-intact and acrosome-reacted sperm can bind to the ZP, but only the latter can penetrate the ZP, reach the egg's plasma membrane, and fuse with plasma membrane (fertilization) to produce a zygote. Following fertilization, the ZP is modified by cortical granule components such that acrosome-intact and acrosome-reacted sperm are unable to bind to fertilized eggs. Here we review some of the evidence that bears directly on the involvement of two mouse ZP proteins, mZP2 and mZP3, as receptors for binding of mouse sperm to unfertilized eggs and address some contentious issues surrounding this important initial step in the process of mammalian fertilization.

Zona Pellucida Genes and Proteins: Essential Players in Mammalian Oogenesis and Fertility.

All mammalian oocytes and eggs are surrounded by a relatively thick extracellular matrix (ECM), the zona pellucida (ZP), that plays vital roles during oogenesis, fertilization, and preimplantation development. Unlike ECM surrounding somatic cells, the ZP is composed of only a few glycosylated proteins, ZP1-4, that are unique to oocytes and eggs. ZP1-4 have a large region of polypeptide, the ZP domain (ZPD), consisting of two subdomains, ZP-N and ZP-C, separated by a short linker region, that plays an essential role in polymerization of nascent ZP proteins into crosslinked fibrils. Both subdomains adopt immunoglobulin (Ig)-like folds for their 3-dimensional structure. Mouse and human ZP genes are encoded by single-copy genes located on different chromosomes and are highly expressed in the ovary by growing oocytes during late stages of oogenesis. Genes encoding ZP proteins are conserved among mammals, and their expression is regulated by cis-acting sequences located close to the transcription start-site and by the same/similar trans-acting factors. Nascent ZP proteins are synthesized, packaged into vesicles, secreted into the extracellular space, and assembled into long, crosslinked fibrils that have a structural repeat, a ZP2-ZP3 dimer, and constitute the ZP matrix. Fibrils are oriented differently with respect to the oolemma in the inner and outer layers of the ZP. Sequence elements in the ZPD and the carboxy-terminal propeptide of ZP1-4 regulate secretion and assembly of nascent ZP proteins. The presence of both ZP2 and ZP3 is required to assemble ZP fibrils and ZP1 and ZP4 are used to crosslink the fibrils. Inactivation of mouse ZP genes by gene targeting has a detrimental effect on ZP formation around growing oocytes and female fertility. Gene sequence variations in human ZP genes due to point, missense, or frameshift mutations also have a detrimental effect on ZP formation and female fertility. The latter mutations provide additional support for the role of ZPD subdomains and other regions of ZP polypeptide in polymerization of human ZP proteins into fibrils and matrix.

Zona pellucida genes and proteins and human fertility.

The zona pellucida (ZP) is an extracellular matrix (ECM) that surrounds all mammalian oocytes, eggs, and embryos and plays vital roles during oogenesis, fertilization, and preimplantation development. The mouse and human ZP is composed of three or four unique proteins, respectively, called ZP1-4, that are synthesized, processed, and secreted by oocytes during their growth phase. All ZP proteins have a zona pellucida domain (ZPD) that consists of ≈270 amino acids and has 8 conserved Cys residues present as four intramolecular disulfides. Secreted ZP proteins assemble into long fibrils around growing oocytes with ZP2-ZP3 dimers located periodically along the fibrils. The fibrils are cross-linked by ZP1 to form a thick, transparent ECM to which sperm must first bind and then penetrate during fertilization of eggs. Inactivation of mouse ZP1, ZP2, or ZP3 by gene targeting affects both ZP formation around oocytes and fertility. Female mice with eggs that lack a ZP due to inactivation of either ZP2 or ZP3 are completely infertile, whereas inactivation of ZP1 results in construction of an abnormal ZP and reduced fertility. Results of a large number of studies of infertile female patients strongly suggest that gene sequence variations (GSV) in human ZP1, ZP2, or ZP3 due to point, missense, or frameshift mutations have similar deleterious effects on ZP formation and female fertility. These findings are discussed in light of our current knowledge of ZP protein synthesis, processing, secretion, and assembly.

Purified trout egg vitelline envelope proteins VEbeta and VEgamma polymerize into homomeric fibrils from dimers in vitro.

The rainbow trout egg vitelline envelope (VE) is composed of three proteins, called VEalpha ( approximately 58-60kDa Mr), VEbeta ( approximately 52kDa Mr), and VEgamma ( approximately 47kDa Mr). Each of these proteins is related to mouse egg zona pellucida (ZP) glycoproteins, called ZP1, ZP2, and ZP3, and possesses a ZP domain that has been implicated in the polymerization of the proteins into long, interconnected fibrils or filaments. Here, trout egg VEbeta and VEgamma were purified to homogeneity and analyzed under various experimental conditions (SDS-PAGE, Blue Native-(BN-)PAGE, size-exclusion chromatography, and transmission electron microscopy) to determine whether individual VE proteins would polymerize into fibrils in vitro. Such analyses revealed that in the presence of 6M urea each VE protein is present primarily as monomers and as small oligomers (dimers, tetramers, etc.). However, either a reduction in urea concentration or a complete removal of urea results in the polymerization of VEbeta and VEgamma dimers into very large oligomers. Mixtures of VEbeta and VEgamma also give rise to large oligomers. Under these conditions, VE proteins are visualized by transmission electron microscopy as aggregates of long fibrils, with each fibril composed of contiguous beads located periodically along the fibril. The relationship between the behavior of fish egg VE proteins and mouse ZP glycoproteins, as well as other ZP domain-containing proteins, is discussed.

Zona pellucida glycoprotein ZP3 and fertilization in mammals.

An early step in mammalian fertilization is species-restricted binding of sperm to the egg's zona pellucida (ZP), a thick extracellular coat that surrounds eggs. Sperm bind to the ZP of unfertilized eggs, but not to the ZP of fertilized eggs. Shortly after binding to the unfertilized egg ZP, sperm undergo the acrosome reaction, a form of cellular exocytosis that enables sperm to penetrate the ZP. Three glycoproteins, mZP1-3, constitute the mouse egg's ZP and participate in the process of fertilization. For example, sperm exposed to unfertilized egg mZP3 at nanomolar concentrations are inhibited from binding to eggs and undergo the acrosome reaction. Neither mZP1 nor mZP2 has an effect on sperm binding or the acrosome reaction. Furthermore, mZP3 from fertilized eggs has no effect on sperm binding and is unable to induce the acrosome reaction. These and other properties of mZP3 suggest that it is a receptor for sperm and inducer of the acrosome reaction. Mapping of the mZP3 combining-site for sperm suggests that it is located near the C-terminus of the polypeptide, just downstream of the ZP domain, in a region encoded by exon-7 of the mZP3 gene. This region of mZP3 is a site of positive Darwinian selection. When mZP3 exon-7 is fused to the Fc fragment of human IgG and sperm exposed to the chimeric protein, sperm are inhibited from binding to eggs. However, the chimeric protein does not induce the acrosome reaction. Therefore, polypeptide encoded by mZP3 exon-7 is necessary and sufficient for binding of mouse sperm.

Mammalian fertilization: the egg's multifunctional zona pellucida.

The zona pellucida (ZP) is a specialized extracellular coat that surrounds the plasma membrane of mammalian eggs. Its presence is essential for successful completion of oogenesis, fertilization and preimplantation development. The ZP is composed of only a few glycoproteins which are organized into long crosslinked fibrils that constitute the extracellular coat. A hallmark of ZP glycoproteins is the presence of a ZP domain, a region of polypeptide responsible for polymerization of the glycoproteins into a network of interconnected fibrils. The mouse egg ZP consists of only three glycoproteins, called ZP1, ZP2, and ZP3, that are synthesized and secreted exclusively by growing oocytes. One of the glycoproteins, ZP3, serves as both a binding partner for sperm and inducer of sperm exocytosis, the acrosome reaction. Female mice lacking ZP3 fail to assemble a ZP around growing oocytes and are completely infertile. Sperm bind to the carboxy-terminal region of ZP3 polypeptide encoded by ZP3 exon-7 and binding is sufficient to induce sperm to complete the acrosome reaction. Whether sperm recognize and bind to ZP3 polypeptide, oligosaccharide, or both remains an unresolved issue. Purified ZP3 self-assembles into long homomeric fibrils under non-denaturing conditions. Apparently, sperm added to ZP3 bind to the fibrils and are prevented from binding to ovulated eggs in vitro. These, as well as other aspects of ZP structure and function are addressed in this article.

Purified mouse egg zona pellucida glycoproteins polymerize into homomeric fibrils under non-denaturing conditions.

The mouse egg's zona pellucida (ZP) is composed of three glycoproteins, called ZP1, ZP2, and ZP3, that migrate as relatively broad, single bands on SDS-PAGE. The glycoproteins are organized within the ZP as a network of long interconnected fibrils that exhibit a structural periodicity. Here, ZP2 and ZP3 were purified by HPLC to homogeneity and analyzed by Blue Native- (BN-) PAGE and transmission electron microscopy (TEM), as well as by SDS-PAGE. As opposed to SDS-PAGE, BN-PAGE, and TEM permit analysis of ZP2 and ZP3 under non-denaturing conditions. ZP2 and ZP3 migrate on BN-PAGE, not as single bands, but as several discrete oligomers that give rise to larger structures which remain at the origin of the gel. Consistent with this, ZP2 and ZP3 are visualized by TEM as long interconnected fibrils that consist of contiguous beads. Therefore, under non-denaturing conditions both purified ZP2 and ZP3 polymerize into higher order structures. These findings are of interest since purified ZP3 inhibits binding of mouse sperm to eggs and induces sperm to undergo the acrosome reaction in vitro. Results presented here suggest that these biological effects of ZP3 are due to binding of homomeric fibrils of ZP3 to sperm.

Mammalian fertilization is dependent on multiple membrane fusion events.

Successful completion of fertilization in mammals is dependent on three membrane fusion events. These are (1) the acrosome reaction of sperm, (2) the fusion of sperm and egg plasma membranes to form a zygote, and (3) the cortical reaction of fertilized eggs. Extensive research into the molecular basis of each of these events has identified candidate proteins and factors involved in fusion of membranes during the mammalian fertilization process. Some of this information is provided here.

The Fish Egg's Zona Pellucida.

All fish eggs are surrounded by an envelope, called the zona pellucida (ZP), that plays various roles during oogenesis, egg deposition, fertilization, and embryogenesis. The fish egg ZP consists of only a few proteins that are homologs of mammalian ZP proteins ZP1, ZP3, and ZP4. Unlike the situation in mammals, in fishes there are often multiple copies of ZP genes, perhaps a consequence of ancient polyploidization, gene amplification, and mutation. Like mammalian ZP proteins, fish egg ZP1-like proteins exhibit conserved organization with distinct domains and motifs, but unlike mammalian ZP1 and ZP4 have a glutamine (Q)- and/or proline (P)-rich stretch as an N-terminal extension. Such extensions may play a role in assembly of ZP fibrils and/or account for certain properties of the fish egg ZP, such as elasticity. Recent proposals suggest that fish egg ZP proteins can adopt amyloid-like structures, serve as antifreeze proteins in Antarctic icefishes, and protect eggs subjected to desiccating conditions in small shallow pools. In this chapter, these and other aspects of fish egg ZP proteins are presented.

The Mouse Egg's Zona Pellucida.

All mammalian eggs are surrounded by a highly specialized extracellular matrix (ECM), called the zona pellucida (ZP), that functions before, during, and after fertilization. Unlike somatic cell ECM the mouse ZP is composed of three different proteins, ZP1-3, that are synthesized and secreted by growing oocytes and assembled into long interconnected fibrils. ECM or vitelline envelope (VE) that surrounds fish, reptilian, amphibian, and avian eggs also consists of a limited number of proteins all closely related to ZP1-3. Messenger RNAs encoding ZP1-3 are expressed only by growing oocytes at very high levels from single-copy genes present on different chromosomes. Processing at the amino- and carboxy-termini of nascent ZP1-3 permits secretion of mature proteins into the extracellular space and assembly into fibrils and matrix. Structural features of nascent ZP proteins prevent assembly within secretory vesicles of growing oocytes. Homozygous knockout female mice that fail to synthesize either ZP2 or ZP3 are unable to construct a ZP, ovulate few if any eggs, and are infertile. ZP1-3 have a common structural feature, the ZP domain (ZPD), that has been conserved through 600 million years of evolution and is essential for ZP protein assembly into fibrils. The ZPD consists of two subdomains, each with four conserved cysteine residues present as two intramolecular disulfides, and resembles an immunoglobulin (Ig) domain found in a wide variety of proteins that have diverse functions, from receptors to mechanical transducers. ZP2 and ZP3 function as receptors for acrosome-reacted and acrosome-intact sperm, respectively, during fertilization of ovulated eggs, but are inactivated as sperm receptors as a result of fertilization.

The PLAC1-homology region of the ZP domain is sufficient for protein polymerisation.

BACKGROUND: Hundreds of extracellular proteins polymerise into filaments and matrices by using zona pellucida (ZP) domains. ZP domain proteins perform highly diverse functions, ranging from structural to receptorial, and mutations in their genes are responsible for a number of severe human diseases. Recently, PLAC1, Oosp1-3, Papillote and CG16798 proteins were identified that share sequence homology with the N-terminal half of the ZP domain (ZP-N), but not with its C-terminal half (ZP-C). The functional significance of this partial conservation is unknown. RESULTS: By exploiting a highly engineered bacterial strain, we expressed in soluble form the PLAC1-homology region of mammalian sperm receptor ZP3 as a fusion to maltose binding protein. Mass spectrometry showed that the 4 conserved Cys residues within the ZP-N moiety of the fusion protein adopt the same disulfide bond connectivity as in full-length native ZP3, indicating that it is correctly folded, and electron microscopy and biochemical analyses revealed that it assembles into filaments. CONCLUSION: These findings provide a function for PLAC1-like proteins and, by showing that ZP-N is a biologically active folding unit, prompt a re-evaluation of the architecture of the ZP domain and its polymers. Furthermore, they suggest that ZP-C might play a regulatory role in the assembly of ZP domain protein complexes.

A Bespoke Coat for Eggs: Getting Ready for Fertilization.

All eggs have an extracellular coat (EC) that plays unique roles during reproduction and development. ECs are designed to protect eggs and support their growth, regulate fertilization of eggs, and protect early embryos. ECs of mammalian and nonmammalian eggs consist of only a few proteins that are closely related to one another. All these proteins possess regulatory elements and a structural domain responsible for processing and assembly of the proteins into ECs. They also possess regions responsible for their functional roles during and after fertilization. Our essay addresses these and other aspects of EC biology.

Recent aspects of mammalian fertilization research.

Mammalian fertilization has been the subject of intensified research in recent times. Application of recombinant DNA, transgenic and gene targeting technology, in particular, to issues in mammalian fertilization has revolutionized the field. Here, we present some of the latest results coming from application of these and other technologies to four aspects of mammalian fertilization: 1. formation of the egg zona pellucida (ZP) during oocyte growth; 2. species-specific binding of sperm to the egg zona pellucida; 3. induction of the sperm acrosome reaction (AR) by the egg zona pellucida 4. binding of sperm to and fusion with egg plasma membrane. In virtually every instance, new information and new insights have come from relatively recent investigations.

Egg-sperm interactions at fertilization in mammals.

Mammalian eggs are surrounded by a zona pellucida (ZP) that regulates egg-sperm teractions during fertilization. The ZP consists of long filaments composed of two glycoproteins, ZP2 and ZP3, that are crosslinked by a third glycoprotein, ZP1. The presence of both ZP2 and ZP3 is essential for assembling a ZP around growing oocytes, as well as for fertility of females. Acrosome-intact sperm recognize and bind to O-linked oligosaccharides linked to Ser residues at the sperm combining-site of ZP3. Structural differences in oligosaccharides on ZP3 from different species may account for whether or not sperm are able to bind to the ZP. Bound sperm undergo the acrosome reaction, penetrate the ZP, and can then fuse with egg plasma membrane. Following fertilization, sperm are unable to bind to either ZP3 or the ZP of one-cell embryos.

Evolution, structure, and synthesis of vertebrate egg-coat proteins.

All vertebrate eggs are surrounded by an extracellular coat that supports growth of oocytes, protects oocytes, eggs, and early embryos, and participates in the process of fertilization. In mammals (platypus to human beings) the coat is called a zona pellucida (ZP) and in non-mammals (molluscs to birds), a vitelline envelope (VE). The ZP and VE are composed of just a few proteins that are related to one another and possess a common motif, called the zona pellucida domain (ZPD). The ZPD arose more than ~600 million years ago, consists of ~260 amino acids, and has 8 conserved Cys residues that participate in 4 intramolecular disulfides. It is likely that egg-coat proteins are derived from a common ancestral gene. This gene duplicated several times during evolution and gave rise to 3-4 genes in fish, 5 genes in amphibians, 6 genes in birds, and 3-4 genes in mammals. Some highly divergent sequences, N- and C-terminal to the ZPD, have been identified in egg-coat proteins and some of these sequences may be under positive Darwinian selection that drives evolution of the proteins. These and other aspects of egg-coat proteins, including their structure and synthesis, are addressed in this review.