Secretion and assembly of zona pellucida glycoproteins by growing mouse oocytes microinjected with epitope-tagged cDNAs for mZP2 and mZP3

The zona pellucida (ZP) is a highly organized extracellular coat that surrounds all mammalian eggs. The mouse egg ZP is composed of three glycoproteins, called mZP1-3, that are synthesized, secreted, and assembled into a ZP exclusively by growing oocytes. Here, we microinjected epitope-tagged (Myc and Flag) cDNAs for mZP2 and mZP3 into the germinal vesicle (nucleus) of growing oocytes isolated from juvenile mice. Specific antibodies and laser scanning confocal microscopy were used to follow nascent, recombinant ZP glycoproteins in both permeabilized and nonpermeabilized oocytes. When such cDNAs were injected, epitope-tagged mZP2 (Myc-mZP2) and mZP3 (Flag-mZP3) were synthesized, packaged into large intracellular vesicles, and secreted by the vast majority of oocytes. Secreted glycoproteins were incorporated into only the innermost layer of the thickening ZP, and the amount of nascent glycoprotein in this region increased with increasing time of oocyte culture. Consistent with prior observations, the putative transmembrane domain at the C terminus of mZP2 and mZP3 was missing from nascent glycoprotein incorporated into the ZP. When the consensus furin cleavage site near the C terminus of mZP3 was mutated, such that it should not be cleaved by furin, secretion and assembly of mZP3 was reduced. On the other hand, mZP3 incorporated into the ZP lacked the transmembrane domain downstream of the mutated furin cleavage site, suggesting that some other protease(s) excised the domain. These results strongly suggest that nascent mZP2 and mZP3 are incorporated into only the innermost layer of the ZP and that excision of the C-terminal region of the glycoproteins is required for assembly into the oocyte ZP.     

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.     

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.     

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. Dev. Genet. 25:95–102, 1999. © 1999 Wiley‐Liss, Inc.     

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.     

Mutation of a conserved hydrophobic patch prevents incorporation of ZP3 into the zona pellucida surrounding mouse eggs

Three glycoproteins (ZP1, ZP2, and ZP3) are synthesized in growing mouse oocytes and secreted to form an extracellular zona pellucida that mediates sperm binding and fertilization. Each has a signal peptide to direct it into a secretory pathway, a "zona" domain implicated in matrix polymerization and a transmembrane domain from which the ectodomain must be released. Using confocal microscopy and enhanced green fluorescent protein (EGFP), the intracellular trafficking of ZP3 was observed in growing mouse oocytes. Replacement of the zona domain with EGFP did not prevent secretion of ZP3, suggesting the presence of trafficking signals and a cleavage site in the carboxyl terminus. Analysis of linker-scanning mutations of a ZP3-EGFP fusion protein in transient assays and in transgenic mice identified an eight-amino-acid hydrophobic region required for secretion and incorporation into the zona pellucida. The hydrophobic patch is conserved among mouse zona proteins and lies between a potential proprotein convertase (furin) cleavage site and the transmembrane domain. The cleavage site that releases the ectodomain from the transmembrane domain was defined by mass spectrometry of native zonae pellucidae and lies N-terminal to a proprotein convertase site that is distinct from the hydrophobic patch.     

Transgenic mouse eggs with functional hamster sperm receptors in their zona pellucida.

Sperm receptors are located in the mammalian egg extracellular coat, or zona pellucida. Mouse and hamster sperm receptor glycoproteins, mZP3 (83 x 10(3) M(r)) and hZP3 (56 x 10(3) M(r)), respectively, have very similar polypeptides (44 x 10(3) M(r); 81% identical) that are glycosylated to different extents. Purified mZP3 and hZP3 can bind to mouse sperm, prevent them from binding to eggs and induce them to undergo exocytosis, the acrosome reaction, in vitro. A DNA construct that placed the hZP3 gene under the control of mZP3 gene 5'-flanking sequence was used in this report to produce two mouse lines that harbored the foreign sperm receptor transgene. In both lines, the transgene was expressed only by growing oocytes, at a level comparable to that of the endogenous mZP3 gene, and the developmental pattern of transgene expression resembled that of the mZP3 gene. In addition to mZP3, transgenic mouse oocytes synthesized and secreted a glycoprotein indistinguishable from hZP3, and incorporated both glycoproteins into a mosaic zona pellucida. Importantly, hZP3 purified from such zonae pellucidae exhibited both sperm receptor and acrosome reaction-inducing activities in vitro and, following fertilization of transgenic mouse eggs, was inactivated. These results demonstrate that a biologically active foreign sperm receptor can be synthesized and secreted by transgenic mouse oocytes, assembled into a mosaic zona pellucida, and inactivated following fertilization as part of the secondary block to polyspermy.     

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.     

Differential binding of gold-labeled zona pellucida glycoproteins mZP2 and mZP3 to mouse sperm membrane compartments.

Egg zona pellucida glycoproteins mZP3 and mZP2 serve as primary and secondary sperm receptors, respectively, during initial stages of fertilization in mice [Wassarman (1988) A. Rev. Biochem. 57, 415-442]. These receptors interact with complementary egg-binding proteins (EBPs) located on the sperm surface to support species-specific gamete adhesion. Results of whole-mount autoradiographic experiments suggest that purified egg mZP3 and mZP2 bind preferentially to acrosome-intact (AI) and acrosome-reacted (AR) sperm heads, respectively [Bleil and Wassarman (1986) J. Cell Biol. 102, 1363-1371]. Here, we used purified egg mZP2, egg mZP3 and fetuin, which were coupled directly to colloidal gold ('gold-probes'), to examine binding of these glycoproteins to membrane compartments of AI and AR sperm by transmission electron microscopy. mZP3 gold-probes were found associated primarily with plasma membrane overlying the acrosomal and post-acrosomal regions of AI sperm heads. They were also found associated with plasma membrane overlying the post-acrosomal region of AR sperm heads. mZP2 gold-probes were found associated primarily with inner acrosomal membrane of AR sperm heads, although some gold was associated with outer acrosomal membrane of AI sperm that had holes in plasma membrane overlying the acrosome. Fetuin gold-probes, used to assess background levels of binding, were bound at relatively low levels to plasma membrane and inner acrosomal membrane of AI and AR sperm, respectively. None of the gold-probes exhibited significant binding to sperm tails, or to red blood cells and residual bodies present in sperm preparations. These results provide further evidence that mZP2 and mZP3 bind preferentially to heads of AR and AI sperm, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a region of mouse zona pellucida glycoprotein mZP3 that possesses sperm receptor activity.

The ability of mouse zona pellucida glycoprotein ZP3 (mZP3) to function as a sperm receptor is attributable to certain of its oligosaccharides, not to its polypeptide (P. M. Wassarman, 1990. Development 108, 1-17). Here, purified, radioiodinated mZP3 was digested by either papain or V8 protease, and the glycopeptides produced were fractionated by HPLC and assayed for sperm receptor activity in vitro. Each proteolytic digest of mZP3 contained a heavily glycosylated peptide, approximately 55,000 apparent M(r), that exhibited sperm receptor activity in vitro. To determine the region of mZP3 polypeptide from which the active glycopeptides were derived, Western gel immunoblotting, employing an antiserum directed against a specific mZP3 peptide epitope, and automated amino-terminal amino acid sequencing were employed. Results of these experiments strongly suggest that the active glycopeptides produced by digestion of mZP3 with either papain or V8 protease are derived from the same region of the carboxy-terminal half of the mZP3 polypeptide. These and other findings are discussed in terms of mZP3 structure and function.     

Molecular models for mouse sperm-oocyte binding

Binding of sperm to egg in the mouse has been proposed to depend primarily on interactions between lectin-like egg-binding proteins on the sperm plasma membrane and complementary carbohydrates on the specialized extracellular matrix of the egg known as the zona pellucida. An alternative model posits that initial sperm-egg binding depends on the interaction of a sperm surface protein with a supramolecular complex of the three mouse zona pellucida glycoproteins (mZP1, mZP2, mZP3); the role of carbohydrate recognition in this paradigm is thought to be minimal (Gahlay G, Gauthier L, Baibakov B, Epifano O,Dean J. 2010. Gamete recognition in mice depends on the cleavage status of an egg's zona pellucida protein. Science.329:216-219). This perspective reviews these recent findings,and considers them in light of evidence favoring a major role for lectin-like interactions. An alternative model, the domain specific model for mammalian gamete binding, could reconcile some of the conflicting observations.     

Identification of the carboxyl termini of porcine zona pellucida glycoproteins ZPB and ZPC

The extracellular matrix surrounding mammalian oocytes plays important roles in fertilization and is known as the zona pellucida (ZP). The ZP consists of three glycoproteins, ZPA, ZPB, and ZPC, which contain homologous regions known as ZP domains. The ZP domain is also found in many other secretory glycoproteins. Putative transmembrane domains present at the C-termini of ZP glycoprotein precursors are removed as the proteins proceed through the secretory pathway. However, the details of this processing have been unclear. In particular, the precise locations of the C-termini of mammalian zona proteins have not yet been determined. In this study, the C-terminal residues of porcine ZPB and ZPC were identified as Ala-462 and Ser-332, respectively, by mass spectrometry of C-terminal polypeptide fragments of these proteins. These results suggest that ZPB is processed at its furin consensus site, whereas ZPC is processed N-terminal to the furin consensus site. In addition, the analyses of porcine ZPB and ZPC fragments revealed that disulfide bonds within the ZP domains are divided into two groups, suggesting that the ZP domain consists of two subdomains.     

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.     

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

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

Mouse granulosa cells contribute more to the mRNA synthesis of mZP2 than oocyte does

In order to elucidate the biogenesis of mouse zona pellucida 2 (mZP2) protein, RT-PCR, and in situ hybridization were carried out to localize the expression of mouse ZP2 mRNA. Cumulus cells of the OCC (Oocyte-Cumulus cell Complex) were isolated from the oocytes after superovulation for the RNA extraction. The frozen sections of ovaries from adolescent and aged mice were prepared to hybridize with RNA probe of mouse ZP2. mRNA of ZP2 was detected in isolated cumulus cells by RT-PCR. Results of in situ hybridization showed that the mRNA of ZP2 was synthesized in both oocyte and granulosa cells at different folliculogenesis stages; and the expression of ZP2 mRNA in granulosa cells was stronger than that in oocyte; much weaker expression of mZP2 was detected in the follicles of aged mouse. These suggest that the entire amount of ZP2 mRNA generated in the granulosa cells layer should be much more than that in oocyte. Therefore, we think that the granulosa cells contribute more to the mZP2 mRNA synthesis than oocyte does.     

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

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

Mammalian fertilization: the strange case of sperm protein 56

During mammalian fertilization sperm bind to the egg's zona pellucida (ZP) after undergoing capacitation. Capacitated mouse sperm bind to mZP3 (one of three ZP glycoproteins), undergo the acrosome reaction, penetrate the ZP, and fuse with egg plasma membrane. Sperm protein 56 (sp56), a member of the C3/C4 superfamily of binding proteins, was identified nearly 20 years ago as a binding partner for mZP3 by photoaffinity cross‐linking of acrosome‐intact sperm. However, subsequent research revealed that sp56 is a component of the sperm's acrosomal matrix and, for sperm with an intact acrosome, should be unavailable for binding to mZP3. Recently, this dilemma was resolved when it was recognized that some acrosomal matrix (AM) proteins, including sp56, are released to the sperm surface during capacitation. This may explain why uncapacitated mammalian sperm are unable to bind to the unfertilized egg ZP.     

Influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease.

Many viruses have membrane glycoproteins that are activated at cleavage sites containing multiple arginine and lysine residues by cellular proteases so far not identified. The proteases responsible for cleavage of the hemagglutinin of fowl plague virus, a prototype of these glycoproteins, has now been isolated from Madin-Darby bovine kidney cells. The enzyme has a mol. wt of 85,000, a pH optimum ranging from 6.5 to 7.5, is calcium dependent and recognizes the consensus sequence R-X-K/R-R at the cleavage site of the hemagglutinin. Using a specific antiserum it has been identified as furin, a subtilisin-like eukaryotic protease. The fowl plague virus hemagglutinin was also cleaved after coexpression with human furin from cDNA by vaccinia virus vectors. Peptidyl chloroalkylketones containing the R-X-K/R-R motif specifically bind to the catalytic site of furin and are therefore potent inhibitors of hemagglutinin cleavage and fusion activity.