Egg extracellular coat proteins: from fish to mammals.

The extracellular coat surrounding fish (vitelline envelope; VE) and mammalian (zona pellucida; ZP) eggs is composed of long, interconnected filaments. Fish VE and mammalian ZP proteins that make up the filaments are highly conserved groups of proteins that are related to each other, as well as to their amphibian and avian egg counterparts. The rainbow trout (O. mykiss) egg VE is composed of 3 proteins, called VEalpha (approximately 58 kDa), VEbeta (approximately 54 kDa), and VEgamma (approximately 47 kDa). The mouse (M. musculus) egg ZP also is composed of 3 proteins, called ZP1 (approximately 200 kDa), ZP2 (approximately 120 kDa), and ZP3 (approximately 83 kDa). Overall, trout VE and mouse ZP proteins share approximately 25% sequence identity and have features in common; these include an N-terminal signal sequence, a ZP domain, a consensus furin cleavage-site, and a C-terminal tail. VEalpha, VEbeta, and ZP1 also have a trefoil or P-type domain upstream of the ZP domain. VEalpha and VEbeta are very similar in sequence (approximately 65% sequence identity) and are related to ZP1 and ZP2, whereas VEgamma is related to ZP3 (approximately 25% sequence identity). Mouse ZP proteins are synthesized and secreted exclusively by growing oocytes in the ovary. Trout VE proteins are synthesized by the liver under hormonal control and transported in the bloodstream to growing oocytes in the ovary. The trout VE is assembled from VEalpha/gamma and VEbeta/gamma heterodimers. The mouse ZP is assembled from ZP2/3 heterodimers and crosslinked by ZP1. Despite approximately 400 million years separating the appearance of trout and mice, and the change from external to internal fertilization and development, trout VE and mouse ZP proteins have many common structural features; as do avian and amphibian egg VE proteins. However, the site of synthesis of trout and mouse egg extracellular coat proteins has changed over time from the liver to the ovary, necessitating some changes in the C-terminal region of the polypeptides that regulates processing, secretion, and assembly of the proteins.

Mouse zona pellucida genes and glycoproteins.

The zona pellucida (ZP) is a thick extracellular coat that surrounds all mammalian eggs. The ZP plays important roles during oogenesis, fertilization, and preimplantation development. The mouse ZP consists of only three glycoproteins, called ZP1, ZP2, and ZP3. All three glycoproteins are essential structural components of the ZP. Additionally, ZP3 serves as a primary sperm receptor and acrosome reaction-inducer, and ZP2 serves as a secondary sperm receptor during fertilization. ZP1, ZP2, and ZP3 are encoded by single-copy genes present on three different chromosomes. The genes are expressed exclusively by mouse oocytes as they grow and the cellular specificity can be ascribed to cis-acting sequences close to the site of transcription initiation and to certain trans-acting factors. Concomitantly, ZP polypeptides are synthesized, modified with N- and O-linked oligosaccharides, secreted, and assembled into crosslinked filaments that exhibit a structural repeat. Nascent ZP glycoproteins are incorporated into large secretory vesicles that fuse with the oocyte plasma membrane and deposit nascent ZP glycoproteins into the innermost layer of the thickening ZP. Each ZP polypeptide possesses several characteristic features, including an N-terminal signal sequence, a ZP domain, a consensus furin cleavage site, and a C-terminal transmembrane domain. The latter is required for assembly of nascent ZP polypeptides into a ZP, cleavage at the consensus furin cleavage site is required for secretion, and the ZP domain supports protein:protein interactions during ZP assembly. At ovulation, when meiotic maturation of oocytes occurs and chromosomes condense into bivalents, expression of the three ZP genes ceases. Using "knockout mice", in the absence of either ZP2 or ZP3 expression, a ZP fails to assemble around growing oocytes and females are infertile. There is no effect on males. In the absence of ZP1 expression, a disorganized ZP assembles around growing oocytes and females exhibit reduced fertility. These observations are consistent with the current model for ZP structure in which ZP2 and ZP3 form long Z filaments crosslinked by ZP1.

Multiple functions of mouse zona pellucida glycoprotein mZP3, the sperm receptor.

Zona pellucida glycoprotein mZP3 is a structural glycoprotein, a sperm receptor, and an acrosome reaction-inducer. The latter two functions are mediated, at least in part, by mZP3 oligosaccharides. mZP3 is unique to mammalian eggs, from mice to humans, although related glycoproteins are found in vitelline envelopes of a variety of eggs, from fish to birds.

Association of egg zona pellucida glycoprotein mZP3 with sperm protein sp56 during fertilization in mice.

Purified mouse sperm receptor, a zona pellucida glycoprotein called mZP3, binds to plasma membrane overlying acrosome-intact sperm heads (P.M. Wassarman, 1999, Cell 96, 175-183). Some evidence suggests that mZP3 binds to sp56, a protein reported to be associated peripherally with the plasma membrane of acrosome-intact sperm heads (J.D. Bleil and P.M. Wassarman, 1990, Proc. Natl. Acad. Sci., USA 87, 7215-7219; A. Cheng et al., 1994, J. Cell Biol. 125, 867-878). Here, we report that membrane vesicles prepared from acrosome-intact sperm contain sp56. When these vesicles are incubated with eggs they inhibit binding of sperm to eggs in vitro (ID50 approximately 50-100 microg protein/ml). On the other hand, a monoclonal antibody directed against sp56 relieves the inhibition of binding of sperm to eggs by membrane vesicles. As expected, incubation of intact sperm with the antibody directed against sp56 inhibits binding of the sperm to eggs. Results of immunoprecipitation of sperm extracts incubated with mZP3, by either a polyclonal antibody directed against mZP3 or a monoclonal antibody directed against sp56, suggest that mZP3 is specifically associated with sp56. Results of laser scanning confocal microscopy of fixed sperm probed with antibodies directed against either sp56 or a approximately 155 kDa acrosomal protein, suggest that the two proteins are present in the acrosome, but with different distributions. Furthermore, confocal images of sperm, fixed after exposure to purified mZP3 and probed with antibodies against mZP3 and sp56, reveal overlap between mZP3 and sp56 at the surface of the sperm head. The possible implications of these results are discussed in the context of mammalian fertilization.

A profile of fertilization in mammals.

Fertilization is defined as the process of union of two gametes, eggs and sperm. When mammalian eggs and sperm come into contact in the female oviduct, a series of steps is set in motion that can lead to fertilization and ultimately to development of new individuals. The pathway begins with species-specific binding of sperm to eggs and ends a relatively short time later with fusion of a single sperm with each egg. Although this process has been investigated extensively, only recently have the molecular components of egg and sperm that participate in the mammalian fertilization pathway been identified. Some of these components may participate in gamete adhesion and exocytosis, whereas others may be involved in gamete fusion. Here we describe selected aspects of mammalian fertilization and address some of the latest experimental evidence that bears on this important area of research.

Secretion of mouse ZP3, the sperm receptor, requires cleavage of its polypeptide at a consensus furin cleavage-site.

The mouse egg extracellular coat, or zona pellucida, consists of three glycoproteins, called mZP1-3. Each glycoprotein possesses a consensus sequence recognized by the furin family of proprotein convertases. Previously, it was reported that mZP2 and mZP3 are cleaved at their consensus furin cleavage-sites located near the C-terminus of the polypeptides [Litscher, E. S., Qi, H., and Wassarman, P. M. (1999) Biochemistry 38, 12280-12287]. Here, use of site-directed mutagenesis of the mZP3 gene and a specific inhibitor of furin-like enzymes revealed that secretion of nascent mZP3 from transfected cells is dependent on cleavage of mZP3 at its consensus furin cleavage-site. The dependence of secretion on cleavage represents a novel function for furin family enzymes.

Towards the molecular basis of sperm and egg interaction during mammalian fertilization.

During the past 2 decades, a number of genes have been cloned from mammals which encode polypeptides that participate in the process of fertilization. Among these are glycoproteins ZP1-3 that constitute the zona pellucida of eggs from mice to human beings. In mice, one of these glycoproteins, mZP3, acts as a primary sperm receptor and acrosome reaction-inducer. The evidence suggests that acrosome-intact sperm recognize and bind to a specific class of mZP3 oligosaccharides present on two serine residues (O-linked) located near the carboxy-terminus of the polypeptide. Mutagenesis of either of these residues results in the synthesis of an inactive form of the receptor. Therefore, mammalian fertilization is a carbohydrate-mediated event. It is possible that changes in the structure of these oligosaccharides (e.g., composition, sequence, linkages, modifications, etc.) could account for species-specific binding of sperm to eggs. Stably transfected somatic cells, null mutant animals, and DNA constructs are now available to test this possibility both in vivo and in vitro.

Mouse zona pellucida glycoproteins mZP2 and mZP3 undergo carboxy-terminal proteolytic processing in growing oocytes.

The extracellular coat, or zona pellucida, of the mouse egg consists of three glycoproteins, called mZP1-3. The glycoproteins are synthesized and secreted concomitantly by growing oocytes during their 2-3-week growth phase. Each of the glycoproteins has a consensus furin cleavage site (-Arg-X-Lys/Arg-Arg-) near the C-terminus of their polypeptide. Here, several approaches were employed to determine whether nascent mZP2 and mZP3 are cleaved at the consensus sites, -Arg-Ser-Lys-Arg- and -Arg-Asn-Arg-Arg-, respectively, prior to secretion. Molecular mass determinations of deglycosylated mZP2 and mZP3 suggest that their polypeptides are approximately 9 and approximately 7 kDa smaller, respectively, than predicted from exon sequences. Two-dimensional thin-layer chromatographic analyses were also carried out to identify amino acids released from the C-terminus of mZP2 and mZP3 by carboxypeptidase B. On the basis of exon sequences, there are no Arg residues at the predicted C-terminus of the mature glycoproteins. However, for both mZP2 and mZP3, Arg residues were released by carboxypeptidase B, consistent with processing at the consensus furin cleavage site. Furthermore, an antiserum raised against an mZP3 peptide, located downstream of the consensus furin cleavage site, failed to label purified mZP3 on Western immunoblots. The antiserum also failed to label the zona pellucida of oocytes examined by laser scanning confocal microscopy. Collectively, these results strongly suggest that mZP2 and mZP3 are processed at their consensus furin cleavage site prior to secretion and incorporation into the zona pellucida.

Fertilization in animals.

Fertilization in mammalian and nonmammalian organisms has many features in common. These features include a final maturation phase for sperm and eggs, species-specific binding of sperm to eggs, penetration by sperm of one or more extracellular coats surrounding eggs, fusion of sperm and eggs, and activation of eggs. Implicit in this are a variety of basic molecular events, including receptor-ligand interactions, signalling cascades, specific proteolysis, and nuclear transformations. Here, several of these events are addressed for fertilization in animals as diverse as starfish and mice.

Secretion of zona pellucida glycoprotein mZP2 by growing oocytes from mZP3(+/+) and mZP3(-/-) mice.

The mouse egg extracellular coat, or zona pellucida (ZP), is composed of three glycoproteins, called mZP1-3, which are synthesized and secreted concomitantly by growing oocytes. Disruption of the mZP3 gene by targeted mutagenesis yields mice that are homozygous nulls (mZP3(-/-)). Growing oocytes from mZP3(-/-) mice do not synthesize mZP3 mRNA or protein and, as a result, do not assemble a ZP. Here, we examined secretion of mZP2 by growing oocytes and eggs from mZP3(-/-) mice, as well as incorporation of mZP2 into the ZP of oocytes from mZP3(+/+) mice. Laser scanning confocal microscopy (LSCM) of antibody-labeled samples showed that, indeed, mZP2 was synthesized and secreted by oocytes isolated from mZP3(-/-) mice and cultured in vitro. Nascent mZP2 was found in the culture medium, associated with the surface of the plasma membrane of growing oocytes, and in the oocyte cytoplasm. By contrast, mZP2 was barely detectable at any of these sites when ovulated eggs from mZP3(-/-) mice were examined. Examination of oocytes from wild-type (mZP3(+/+)) mice showed that, while a portion of nascent mZP2 was assembled into the ZP (approximately 40%), here too a significant fraction was secreted into the culture medium (approximately 60%). Similar results also were obtained when intact pre-antral follicles were isolated from mZP3(+/+) mice and cultured in vitro. Several of these observations are consistent with previous results obtained with oocytes from heterozygous null mice (mZP3(+/-)). Furthermore, the results suggest that ZP assembly from nascent glycoproteins may be a stochastic process that requires the presence of both mZP2 and mZP3 and occurs completely outside the growing oocyte.

The Parkes Lecture. Zona pellucida glycoprotein mZP3: a versatile player during mammalian fertilization.

All mammalian eggs are surrounded by a relatively thick extracellular coat, the zona pellucida (ZP), which facilitates fertilization of eggs by a single spermatozoon. The mouse egg ZP is constructed of only three glycoproteins, termed mZP1-3. Each of these glycoproteins consists of a unique polypeptide that is heterogeneously glycosylated with both asparagine-(N-)linked and serine/threonine-(O-)linked oligosaccharides. Polypeptides of ZP glycoproteins are highly conserved among mammalian species and are similar to polypeptides of egg vitelline envelope glycoproteins of fish, birds and amphibians. One of the mouse ZP glycoproteins, mZP3, serves as both a receptor for spermatozoa and an inducer of the acrosome reaction during fertilization. Free-swimming acrosome-intact spermatozoa recognize and bind to certain serine-(O-)linked oligosaccharides located close to the carboxy terminus of mZP3 polypeptide and, after binding, undergo the acrosome reaction (cellular exocytosis). In this review, in addition to the background information presented, results of recent experiments using homologous recombination to produce mZP3 null mice and site-directed mutagenesis to inactivate mZP3 as a sperm receptor and inducer of the acrosome reaction are presented and discussed.

Zona pellucida glycoprotein mZP3 produced in milk of transgenic mice is active as a sperm receptor, but can be lethal to newborns.

Mouse egg zona pellucida glycoprotein mZP3 (approximately 83 kDa M(r)) serves as a species-specific sperm receptor and acrosome reaction-inducer during fertilization in mice. These biological activities are dependent on certain mZP3 serine/threonine- (O-) linked oligosaccharides present at the combining-site for sperm. In an attempt to produce large amounts of biologically active mZP3, we generated several transgenic mouse lines carrying the full-length mZP3 gene fused to the beta-casein gene promoter and transcription termination sequence. We found that different transgenic mouse lines have different amounts of recombinant mZP3 (approximately 63 kDa M(r)) in milk of lactating females, from approximately 0.3 to 3.5 micrograms/microliter of milk. In all cases, purified milk-mZP3 is active as a sperm receptor and acrosome reaction-inducer in vitro. Unexpectedly, we also found that development of litters from these transgenic mice is related to the amount of mZP3 in the mother's milk. In the most extreme case, litters from the highest expressers fail to live beyond about day-7 post partum unless placed immediately after birth with surrogate wild-type mothers. Litters from lower expressers initially display a complex phenotype that includes effects on hair and body growth, but some of the mice survive and, in time, are restored to a wild-type phenotype. These results demonstrate that relatively large amounts of biologically active mZP3 can be produced in transgenic mouse milk for structural and other studies, but that the presence of mZP3 in milk has dramatic developmental effects on litters, ranging from retarded hair and body growth to death of newborn pups.

Inactivation of the mouse sperm receptor, mZP3, by site-directed mutagenesis of individual serine residues located at the combining site for sperm.

To initiate fertilization, mouse sperm bind to Ser- (O-) linked oligosaccharides located at the sperm combining site of zona pellucida glycoprotein mZP3. Apparently, the oligosaccharides are present on one or more of five Ser residues clustered in the carboxyl-terminal region of the mZP3 polypeptide. Here, each of the Ser residues, as well as an intervening Asn residue, was converted to a small, nonhydroxy amino acid by site-directed mutagenesis. Mouse embryonal carcinoma (EC) cells were then stably transfected with the wild-type and mutated mZP3 genes. In each case, transfected cells synthesized and secreted recombinant EC-mZP3 into the culture medium. The glycoproteins were partially purified and assayed for their ability to inhibit binding of sperm to ovulated eggs in vitro. As compared with wild-type EC-mZP3, mutations of Ser-329, Ser-331, or Ser-333 had no effect on sperm receptor activity. Mutation of Asn-330, a potential N-linked glycosylation site, also had no effect on sperm receptor activity. On the other hand, mutation of either Ser-332 or Ser-334, or mutation of Ser-332, Ser-333, and Ser-334, resulted in complete inactivation of EC-mZP3 as a sperm receptor. These results suggest that Ser-332 and Ser-334, residues conserved in mouse, hamster, and human ZP3, are essential for sperm receptor activity.

Ovarian development in mice bearing homozygous or heterozygous null mutations in zona pellucida glycoprotein gene mZP3.

The plasma membrane of all mammalian eggs is surrounded by a thick extracellular coat, the zona pellucida (ZP), whose paramount function is to regulate species-specific fertilization. The mouse egg ZP is composed of only three glycoproteins, mZP1-3, that are synthesized and secreted exclusively by oocytes during their 2-3 week growth phase. Disruption of the mZP3 gene by targeted mutagenesis in embryonic stem (ES) cells yields mice heterozygous (mZP3 +/-) or homozygous (mZP3-/-) for the null mutation. As expected, male mice bearing the null mutation are indistinguishable from wild-type males with respect to viability and fertility. Female mZP3 +/- mice are as fertile as wild-type animals, but their eggs have a thin ZP (approximately 2.7 microns thick) as compared to the ZP (approximately 6.2 microns thick) of eggs from wild-type animals. On the other hand, female mZP3-/- mice are infertile and their eggs lack a ZP. The infertility apparently is due to the lack of a sufficient number of eggs in oviducts of superovulated mZP3-/- females. Light micrographs reveal that development of ovarian follicles is often retarded in mZP3-/- mice as compared to wild-type animals. This is manifested as reduced ovarian weights, reduced numbers of Graafian follicles, and reduced numbers of fully-grown oocytes in mZP3-/- females. It seems likely that the pleiotropic effects of the homozygous null mutation on ovarian development may be due, at least in part, to disruption of intercellular communication between growing oocytes and their surrounding follicle cells.

Zona pellucida glycoprotein mZP3 bioactivity is not dependent on the extent of glycosylation of its polypeptide or on sulfation and sialylation of its oligosaccharides.

During fertilization in mice, free-swimming sperm bind to mZP3, one of three egg zona pellucida glycoproteins. Sperm recognize and bind to specific serine/threonine-linked (O-linked) oligosaccharides located at the mZP3 combining site for sperm. Shortly after binding to mZP3, sperm undergo the acrosome reaction, a form of cellular exocytosis. Here, we examined the influence of extent of glycosylation, sulfation, and sialylation of mZP3 (M(r) approximately 65,000-100,000) on its bioactivity; i.e. its ability to inhibit binding of sperm to eggs and to induce the acrosome reaction in vitro. Low (av. M(r) approximately 70,000), medium (av. M(r) approximately 82,000), and high (av. M(r) approximately 94,000) M(r) fractions of mZP3 were purified and shown to vary in extent of asparagine-linked (N-linked) glycosylation. All three size-fractions exhibited bioactivity, suggesting that the ability of mZP3 to inhibit binding of sperm to eggs is not related to the extent of glycosylation of its polypeptide (M(r) approximately 44,000). Digestion of mZP3 by neuraminidase decreased its average M(r) from approximately 83,000 to approximately 77,000 and increased its average pI from approximately 4.7 to approximately 6.0, but did not significantly affect mZP3 bioactivity. Terminal sialic acid largely accounts for the glycoprotein's acidic nature, but is not an essential element of the mZP3 combining site for sperm. Experiments with stably transfected embryonal carcinoma (EC) cells that secrete bioactive EC-mZP3 revealed that, of the sulfate present, approximately 70-75% was located on N-linked and approximately 25-30% on O-linked oligosaccharides. EC-mZP3 devoid of sulfate inhibited binding of sperm to eggs and induced the acrosome reaction to the same extent as sulfated EC-mZP3. These results suggest that sulfation of EC-mZP3 oligosaccharides is not essential for bioactivity. Overall, these findings contrast with those reported for certain other glycoproteins involved in cellular adhesion that require sulfate and/or sialic acid for bioactivity.

Mutant female mice carrying a single mZP3 allele produce eggs with a thin zona pellucida, but reproduce normally.

The mouse egg zona pellucida (ZP) is composed of three glycoproteins, called mZP1-3. Disruption of the mZP3 gene by targeted mutagenesis yields mice that are homozygous (mZP3-/-) for the null mutation; although the mutant mice are viable, females are infertile and their eggs lack a ZP. On the other hand, females heterozygous (mZP3+/-) for the mutation are fertile and their eggs have a ZP. Here, we examined fully grown oocytes from mZP3+/- females and found that, although they have a ZP, it is less than half the width (approximately 2.7 microns; volume, approximately 56 pl) of the ZP of oocytes from wild-type (mZP3+/+) mice (approximately 6.2 microns; volume, approximately 145 pl). Oocyte ZP were purified from ovarian homogenates by gradient centrifugation. Immunostaining of purified ZP on Western gels permitted an estimate to be made of the relative amounts of mZP3 and mZP2 present in the ZP of oocytes from mZP3+/+ and mZP3+/- mice. We found that the ZP from mZP3+/- mice contained, on average, 55 +/- 15% of the mZP3 and 44 +/- 8% of the mZP2 present in the ZP of mZP3+/+ mice; a result quite consistent with the observed widths and calculated volumes of the ZP. Despite the presence of a relatively thin ZP surrounding their eggs, reproduction of female mZP3+/- mice was indistinguishable from female mZP3+/+ mice. These results strongly suggest that, when a single mZP3 allele is present, approximately half the wild-type amount of mZP3 and approximately half the wild-type amount of mZP2 is assembled into a ZP. While this produces a relatively thin ZP, it apparently has no affect on reproduction. Furthermore, these results are consistent with the current molecular model for ZP structure.

Recombinant hamster sperm receptors that exhibit species-specific binding to sperm.

Previous studies have shown that mouse sperm bind to hamster eggs and hamster sperm bind to mouse eggs in vitro. Furthermore, sperm receptor glycoprotein isolated from the zona pellucida of unfertilised hamster (hZP3) and mouse (mZP3) eggs binds to sperm from the heterologous species. Here, we expressed the hZP3 gene, under control of a constitutive promoter (pgk-1), in mouse embryonal carcinoma (EC) cells and Chinese hamster ovary (CHO) cells stably transfected with the hZP3 gene. In both cases, recombinant hZP3 (EC-hZP3 and CHO-hZP3) secreted into the culture medium was partially purified by high-performance liquid chromatography on a size-exclusion column and assayed for bioactivity using mouse and hamster gametes. Unlike hamster egg hZP3, which binds to both mouse and hamster sperm, EC-hZP3 and CHO-hZP3 exhibits species-specific binding to hamster sperm and induce hamster sperm, but not mouse sperm, to undergo the acrosome reaction in vitro. These results provide further evidence that species-specific binding of sperm to eggs in mammals is carbohydrate-mediated. Furthermore, the results suggest that recombinant forms of mammalian sperm receptors may be useful in assessing the molecular basis of species-specific fertilisation in mammals.

Targeted disruption of the mZP3 gene results in production of eggs lacking a zona pellucida and infertility in female mice.

Mammalian eggs are surrounded by a thick extracellular coat, the zona pellucida, that plays important roles during early development. The mouse egg zona pellucida is constructed of three glycoproteins, called mZP1, mZP2, and mZP3. The gene encoding mZP3 is expressed only by growing oocytes during a 2- to 3-week period of oogenesis. Here, the mZP3 gene was disrupted by targeted mutagenesis using homologous recombination in mouse embryonic stem cells. Viable female mice homozygous for the mutated mZP3 allele (mZP3-/-) were obtained. These mice are indistinguishable in appearance from wild-type (mZP3+/+) and heterozygous (mZP3+/-) littermates. However, although ovaries of juvenile and adult mZP3-/- females possess growing and fully grown oocytes, the oocytes completely lack a zona pellucida. Consistent with this observation, eggs recovered from oviducts of superovulated, adult mZP3-/- females also lack a zona pellucida. Thus far, mZP3-/- females mated with wild-type males have failed to become pregnant.