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.     

Recombinant mouse ZP3 inhibits sperm binding and induces the acrosome reaction.

Mammalian fertilization involves interactions of sperm surface receptors with ligands of the zona pellucida, an extracellular matrix surrounding the ovulated egg. In mouse, the zona is composed of three glycoproteins. One of them, ZP3, participates in primary sperm binding and in the subsequent triggering of the sperm's acrosome reaction. Considerable evidence suggests that carbohydrate determinants of ZP3 are responsible for binding to sperm and may be important for acrosomal exocytosis. A full-length cDNA encoding mouse ZP3 was assembled and cloned into expression vectors that contained either a cytomegalovirus (CMV) or a vaccinia (P11) promoter. Mouse L-929 cells were stably transformed with the pZP3-CMV constructs, and green monkey CV-1 cells were infected with a recombinant vaccinia virus containing ZP3. rZP3 was affinity purified from culture media and detected on Western blots as a single 60- to 70-kDa band, which differed in molecular weight from native ZP3 (mean, 83 kDa). Nevertheless, rZP3 is biologically active. rZP3 decreases sperm-zona binding with a potency equivalent to that of native zona pellucida and, like native ZP3, rZP3 triggers acrosomal exocytosis in capacitated mouse sperm. Thus, rZP3 isolated from both rodent and primate cells appears to contain those carbohydrate and protein structures necessary for ZP3's dual role in fertilization.     

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.     

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.     

Mouse gamete adhesion molecules.

Complementary adhesion molecules are located on the surface of mouse eggs and sperm. These molecules support species-specific interactions between sperm and eggs that lead to gamete fusion (fertilization). Modification of these molecules shortly after gamete fusion assists in prevention of polyspermic fertilization. mZP3, an 83,000-Mr glycoprotein located in the egg extracellular coat, or zona pellucida, serves as primary sperm receptor. Gamete adhesion in mice is carbohydrate-mediated, since sperm recognize and bind to certain mZP3 serine/threonine- (O-) linked oligosaccharides. As a consequence of binding to mZP3, sperm undergo the acrosome reaction, which enables them to penetrate the zona pellucida and fertilize the egg. A 56,000-Mr protein called sp56, which is located in plasma membrane surrounding acrosome-intact mouse sperm heads, is a putative primary egg-binding protein. It is suggested that sp56 recognizes and binds to certain mZP3 O-linked oligosaccharides. Acrosome-reacted sperm remain bound to eggs by interacting with mZP2, a 120,000-Mr zona pellicida glycoprotein. Thus, mZP2 serves as secondary sperm receptor. Perhaps a sperm protease associated with inner acrosomal membrane, possibly (pro)acrosin, serves as secondary egg-binding protein. These and, perhaps, other egg and sperm surface molecules regulate fertilization in mice. Homologous molecules apparently regulate fertilization in other mammals.     

Autoradiographic visualization of the mouse egg's sperm receptor bound to sperm

The extracellular coat, or zona pellucida, of mammalian eggs contains species-specific receptors to which sperm bind as a prelude to fertilization. In mice, ZP3, one of only three zona pellucida glycoproteins, serves as sperm receptor. Acrosome-intact, but not acrosome-reacted, mouse sperm recognize and interact with specific O- linked oligosaccharides of ZP3 resulting in sperm-egg binding. Binding, in turn, causes sperm to undergo the acrosome reaction; a membrane fusion event that results in loss of plasma membrane at the anterior region of the head and exposure of inner acrosomal membrane with its associated acrosomal contents. Bound, acrosome-reacted sperm are able to penetrate the zona pellucida and fuse with the egg's plasma membrane (fertilization). In the present report, we examined binding of radioiodinated, purified, egg ZP3 to both acrosome intact and acrosome reacted sperm by whole-mount autoradiography. Silver grains due to bound 125I-ZP3 were found localized to the acrosomal cap region of heads of acrosome-reacted sperm. Under the same conditions, 125I-fetuin bound at only bacKground levels to heads of both acrosome-intact and - reacted sperm, and 125I-ZP2, another zona pellucida glycoprotein, bound preferentially to acrosome-reacted sperm. These results provide visual evidence that ZP3 binds preferentially and specifically to heads of acrosome intact sperm; properties expected of the mouse egg's sperm receptor.     

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.     

Function of the acrosomal matrix: zona pellucida 3 receptor (ZP3R/sp56) is not essential for mouse fertilization

In mammalian fertilization, sperm-zona pellucida binding is considered to be a critical aspect of gamete interaction. In this study, we examine the mouse sperm acrosomal matrix protein zona pellucida 3 receptor (ZP3R; formerly called sp56) because of our interest in defining the function of the acrosomal matrix, the particulate compartment within the sperm secretory acrosome. Using targeted deletion of the Zp3r gene by homologous recombination, we examined the fertility of nullizygous animals. Our experiments showed that males and females homozygous for the affected gene exhibited no differences in litter sizes compared to wild-type and heterozygous animals. Testis weights of nullizygous males were equivalent to those of wild-type and heterozygous males, and no differences in the number of sperm produced by mice of three genotypes were found. In vitro fertilization rates using cumulus-intact and cumulus-free oocytes were also equivalent. Examination of sperm-binding zonae of unfertilized eggs and the ability of the sperm to undergo acrosomal exocytosis in response to calcium ionophore A23187 displayed no differences between wild-type, heterozygous, and nullizygous mouse sperm. These results provide further evidence that either ZP3R is not involved in sperm-zona pellucida binding or this process might be functionally redundant, involving multiple proteins for gamete interactions.     

Characterization of a novel ZP3-independent sperm-binding ligand that facilitates sperm adhesion to the egg coat

During mammalian fertilization, sperm adhere to the extracellular coat of the egg, or zona pellucida, in a species-specific manner. In mouse, evidence suggests that sperm recognize and bind to specific oligosaccharide ligands within the zona pellucida glycoprotein, ZP3, via beta1,4-galactosyltransferase I (GalT I), a lectin-like receptor on the sperm surface. Although in vitro experiments using isolated gametes lend support to this model, recent in vivo studies of genetically altered mice question whether ZP3 and/or GalT I are solely responsible for sperm-egg binding. In this regard, sperm from GalT I-null mice bind poorly to ZP3 and fail to undergo a zona-induced acrosome reaction; however, they still bind to the ovulated egg coat in vitro. In this report, we characterize a novel ZP3- and GalT I-independent mechanism for sperm adhesion to the egg coat. Results show that the ovulated zona pellucida contains at least two distinct ligands for sperm binding: a ZP3-independent ligand that is peripherally associated with the egg coat and facilitates gamete adhesion; and a ZP3-dependent ligand that is present in the insoluble zona matrix and is recognized by sperm GalT I to facilitate acrosomal exocytosis. The ZP3-independent ligand is not a result of contamination by egg cortical granules, nor is it the mouse homolog of oviduct-specific glycoprotein. It behaves as a 250 kDa, WGA-reactive glycoprotein with a basic isoelectric point, distinguishing it from the acidic glycoproteins that form the insoluble matrix of the egg coat. When eluted from isoelectric focusing gels, the acidic matrix glycoproteins possess sperm-binding activity for wild-type sperm, but not for GalT I-null sperm, whereas the basic glycoprotein retains sperm-binding activity for both wild-type and GalT I-null sperm. Thus, GalT I-null sperm are able to resolve gamete recognition into at least two distinct binding events, leading to the characterization of a novel, peripherally associated, sperm-binding ligand on the ovulated zona pellucida.     

Transgenic mice with reduced numbers of functional sperm receptors on their eggs reproduce normally.

To initiate fertilization in mice, free-swimming sperm bind to mZP3, an approximately 83-kDa glycoprotein present in the ovulated egg zona pellucida (ZP). mZP3 is located periodically along the filaments that constitute the ZP. Sperm recognize and bind to specific oligosaccharides linked to one or more of five Ser residues clustered in the carboxy-terminal one-third of the mZP3 polypeptide. When all five Ser residues are converted to nonhydroxy amino acids by site-directed mutagenesis of the mZP3 gene, an inactive form of mZP3, called mZP3[ser], is secreted by embryonal carcinoma cells stably transfected with the mutated gene. Here, seven independent transgenic mouse lines were established that harbor the mutated mZP3 gene. In all lines, the mutant gene is expressed by growing oocytes and mZP3[ser] is synthesized, secreted, and incorporated into the ZP. Purified mZP3[ser] prepared from ovaries of transgenic mice, like mZP3[ser] from transfected embryonal carcinoma cells, is inactive in sperm binding assays in vitro. On the other hand, the presence of mZP3[ser] in the ZP does not significantly affect either the binding of sperm to ovulated eggs in vitro or the reproduction of the mice, i.e., the transgenic mice are fertile, breed at normal intervals, and produce litters of normal sizes. These results indicate that the number of functional sperm receptors in the ZP can be reduced by more than 50% without adversely affecting fertilization of eggs in vivo.     

Genomic organization and polypeptide primary structure of zona pellucida glycoprotein hZP3, the hamster sperm receptor

During the course of fertilization in mammals, free-swimming sperm bind tightly to receptors located in the egg extracellular coat, or zona pellucida. Recently, the hamster sperm receptor, a 56,000 Mr zona pellucida glycoprotein called hZP3, was identified and partially characterized (C. C. Moller et al., (1990). Dev. Biol. 137, 276-286). Here, we describe genomic cloning of hZP3, certain organizational features of the hZP3 gene, and primary structures of hZP3 mRNA and polypeptide. The findings are compared with reported results of comparable analyses of the mouse sperm receptor, an 83,000 Mr zona pellucida glycoprotein called mZP3. Such comparisons reveal a high degree of conservation of genomic organization and polypeptide structure for the two mammalian sperm receptors, despite the considerable difference in their Mrs. These findings are of interest in view of the extremely restricted expression of the ZP3 gene during development and the important role of ZP3 oligosaccharides in gamete adhesion.     

Aggregation of beta-1,4-galactosyltransferase on mouse sperm induces the acrosome reaction

beta-1,4-Galactosyltransferase (GalTase) is present on the surface of mouse sperm, where it functions during fertilization by binding to oligosaccharide residues in the egg zona pellucida. The specific oligosaccharide substrates for sperm GalTase reside on the glycoprotein ZP3, which possesses both sperm-binding and acrosome reaction-inducing activity. A variety of reagents that perturb sperm GalTase activity inhibit sperm binding to the zona pellucida, including UDP-galactose, N-acetylglucosamine, alpha-lactalbumin, and anti-GalTase Fab fragments. However, none of these reagents are able to cross-link GalTase within the membrane nor are they able to induce the acrosome reaction. On the other hand, intact anti-GalTase IgG blocks sperm-zona binding as well as induces the acrosome reaction. Anti-GalTase IgG induces the acrosome reaction by aggregating GalTase on the sperm plasma membrane, as shown by the inability of anti-Gal-Tase Fab fragments to induce the acrosome reaction unless cross-linked with goat anti-rabbit IgG. These data suggest that zona pellucida oligosaccharides induce the acrosome reaction by clustering GalTase on the sperm surface.     

Polypeptide encoded by mouse ZP3 exon-7 is necessary and sufficient for binding of mouse sperm in vitro

Fertilization in mice is initiated by species-specific binding of sperm to mZP3, one of three mouse zona pellucida (ZP) glycoproteins. At nanomolar concentrations, purified egg mZP3 binds to acrosome-intact sperm heads and inhibits binding of sperm to eggs in vitro. Although several reports suggest that sperm recognize and bind to a region of mZP3 encoded by mZP3 exon-7 (so-called, sperm combining-site), this issue remains controversial. Here, exon-swapping and an IgG(Fc) fusion construct were used to further evaluate whether mZP3 exon-7 is essential for binding of sperm to mZP3. In one set of experiments, hamster ZP3 (hZP3) exon-6, -7, and -8 were individually replaced with the corresponding exon of mZP3. Stably transfected embryonal carcinoma (EC) cell lines carrying the recombinant genes were produced and secreted recombinant glycoprotein was purified and assayed for the ability to inhibit binding of sperm to eggs. While EC-hZP3, a recombinant form of hZP3 made by EC cells, is unable to inhibit binding of mouse sperm to eggs in vitro, the results suggest that substitution of mZP3 exon-7 for hZP3 exon-7, but not mZP3 exon-6 or -8, can impart inhibitory activity to EC-hZP3. In this context, a fusion construct consisting of human IgG(Fc) and mZP3 exon-7 and -8 was prepared, an EC cell line carrying the recombinant gene was produced, and secreted chimeric glycoprotein, called EC-huIgG(Fc)/mZP3(7), was purified and assayed. It was found that the chimeric glycoprotein binds specifically to plasma membrane overlying sperm heads to a similar extent as egg mZP3 and, at nanomolar concentrations, inhibits binding of mouse sperm to eggs in vitro. Collectively, these observations provide new evidence that sperm recognize and bind to a region of mZP3 polypeptide immediately downstream of its ZP domain that is encoded by mZP3 exon-7. The implications of these findings are discussed.     

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.     

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)     

The Biological and Functional Significance of the Sperm Acrosome and Acrosomal Enzymes in Mammalian Fertilization

The mammalian spermatozoon undergoes continuous modifications during spermatogenesis, maturation in the epididymis, and capacitation in the female reproductive tract. Only the capacitated spermatozoa are capable of binding the zona-intact egg and undergoing the acrosome reaction. The fertilization process is a net result of multiple molecular events which enable ejaculated spermatozoa to recognize and bind to the egg's extracellular coat, the zona pellucida (ZP). Sperm–egg interaction is a species-specific event which is initiated by the recognition and binding of complementary molecule(s) present on sperm plasma membrane (receptor) and the surface of the ZP (ligand). This is a carbohydrate-mediated event which initiates a signal transduction cascade resulting in the exocytosis of acrosomal contents. This step is believed to be a prerequisite which enables the acrosome reacted spermatozoa to penetrate the ZP and fertilize the egg. This review focuses on the formation and contents of the sperm acrosome as well as the mechanisms underlying the induction of the acrosome reaction. Special emphasis has been laid on the synthesis, processing, substrate specificity, and mechanism of action of the acid glycohydrolases present within the acrosome. The hydrolytic action of glycohydrolases and proteases released at the site of sperm-zona binding, along with the enhanced thrust generated by the hyperactivated beat pattern of the bound spermatozoon, are important factors regulating the penetration of ZP. We have discussed the most recent studies which have attempted to explain signal transduction pathways leading to the acrosomal exocytosis.     

Contribution of mouse egg zona pellucida glycoproteins to gamete recognition during fertilization

For sperm to fertilize eggs, they must first bind to the thick zona pellucida (ZP) that surrounds the plasma membrane of all unfertilized mammalian eggs. An extensive literature suggests that mouse sperm recognize and bind to a specific ZP glycoprotein called mZP3. However, the role of individual ZP glycoproteins in binding of mouse sperm to eggs has been called into question by recent transgenic experiments with null mice. Results of such experiments have been interpreted to mean that binding of sperm depends on the supramolecular structure of the ZP, not on an individual ZP glycoprotein. Here, it is argued that results of these transgenic experiments actually are consistent with the prevailing view of gamete recognition that implicates a specific ZP glycoprotein in both binding of mouse sperm to eggs and induction of the acrosome reaction.     

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.     

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.     

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.