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.     

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.     

Embryonal carcinoma cells transfected with ZP3 genes differentially glycosylate similar polypeptides and secrete active mouse sperm receptor

Mouse and hamster sperm receptors, called mZP3 (approximately 83,000 Mr) and hZP3 (approximately 56,000 Mr), respectively, are glycoproteins located in the ovulated egg zona pellucida. Certain of the glycoprotein O-linked oligosaccharides are essential for sperm receptor activity. Here, we transfected mouse embryonal carcinoma (EC) cells with mZP3 and hZP3 genes placed under control of a constitutive promoter. Transfected cells synthesized and secreted large amounts of the glycoproteins, called EC-mZP3 and EC-hZP3. Although the primary structures of mZP3 and hZP3 polypeptides (44,000 Mr) are very similar to one another, EC-mZP3 (approximately 83,000 Mr) and EC-hZP3 (approximately 49,000 Mr) were glycosylated to very different extents, such that they resembled their egg counterparts. Like egg mZP3, EC-mZP3 inhibited binding of sperm to ovulated eggs and induced sperm to acrosome-react in vitro. In addition, large numbers of sperm bound to aggregates of mZP3-transfected EC cells in vitro. On the other hand, unlike egg hZP3, EC-hZP3 did not exhibit either sperm receptor or acrosome reaction-inducing activity, and sperm failed to bind to aggregates of hZP3-transfected EC cells. Thus, transfected EC cells not only express sperm receptor genes, but also discriminate between very similar polypeptides with respect to glycosylation and, in the case of mZP3, add specific oligosaccharides essential for biological activity. In addition, the results demonstrate that EC cells can serve as a source for large amounts of functional mouse sperm receptor.     

Enzymatic dissection of the functions of the mouse egg's receptor for sperm

During the course of sperm-egg interaction in mice, zona pellucida glycoprotein ZP3 (approximately equal to 80 kDa) serves as both receptor for sperm (J. D. Bleil and P. M. Wassarman, 1980c, Cell 20, 873-882) and inducer of the acrosome reaction (J. D. Bleil and P. M. Wassarman, 1983, Dev. Biol. 95, 317-324). In this investigation, small ZP3 glycopeptides (approximately equal to 1.5-6 kDa), obtained by extensive digestion of the purified glycoprotein with insoluble Pronase, were assayed for both sperm receptor and acrosome reaction-inducing activities. While ZP3 glycopeptides were virtually as effective as intact ZP3 in inhibiting binding of sperm to eggs in vitro ("receptor activity"), unlike intact ZP3, they failed to induce sperm to undergo the acrosome reaction. The latter was determined by indirect immunofluorescence using a monoclonal antibody directed against the acrosomal cap region of sperm. These results suggest that the sperm receptor activity of ZP3 is dependent only on its carbohydrate components, whereas acrosome reaction-inducing activity is dependent on the polypeptide chain of ZP3 as well.     

Polypeptide backbone derived from carboxyl terminal of mouse ZP3 inhibits sperm-zona binding

For mammalian organism, fertilization begins with species-specific recognition between sperm and egg, a process depending upon egg zona pellucida glycoproteins and putative sperm interacting protein(s). In mouse, zona pellucida glycoprotein ZP3 is believed to be the primary receptor for sperm and inducer of sperm acrosomal reaction, and its function has been attributed to the specific O-linked oligosaccharides attached to polypeptide backbone. While lots of reports have focused on the role of ZP3's oligosaccharides in fertilization, there are few concerning its polypeptide backbone. To investigate whether mZP3 polypeptide backbone is involved in sperm-egg recognition, three partially overlapping cDNA fragments, together covering entire mouse ZP3, were cloned, expressed and purified under denaturing condition. Although all three refolded proteins possess native conformation, only one derived from the carboxyl terminal showed inhibitory effect to the sperm-zona binding during in vitro fertilization. This phenomenon could not be explained by enhanced acrosomal exocytosis rate, in that the acrosomal reaction assay demonstrated its inability to induce the acrosomal reaction. Our results suggest that the carboxyl terminal of mZP3 polypeptide backbone interacts with sperm and such interaction plays a significant role in sperm-zona binding, ultimately successful fertilization.     

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.     

O-linked oligosaccharides of mouse egg ZP3 account for its sperm receptor activity

Previously, we reported that ZP3, one of three different glycoproteins present in the mouse egg's zona pellucida, serves as a sperm receptor. Furthermore, small glycopeptides derived from egg ZP3 retain full sperm receptor activity, suggesting a role for carbohydrate, rather than polypeptide chain in receptor function. Here, we report that removal of O-linked oligosaccharides from ZP3 destroys its sperm receptor activity, whereas removal of N-linked oligosaccharides has no effect. A specific size class of O-linked oligosaccharides, recovered following mild alkaline hydrolysis and reduction of ZP3, is shown to possess sperm receptor activity and to bind to sperm. The results presented strongly suggest that mouse sperm bind to eggs via O-linked oligosaccharides present on ZP3.     

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.     

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.     

鼠透明带3(ZP3)融合蛋白表达以及抗血清制备

鼠透明带3（mZP3)作为精子的初级受体,是鼠透明带中的一种主要糖蛋白,抗鼠透明带3抗体能够阻断精卵的结合,达到不育的效果,因此mZP3是免疫避孕研究的重要候选抗原。从小鼠卵巢中提取总RNA,分离mRNA,反转录获得cDNA,将cDNA连接到测序载体pUCm-T质粒上,通过序列测定和分析得到正确的mZP3 cDNA。经内切酶EcoR I和XhoI处理,将mZP3 cDNA克隆至融合蛋白表达载体pGEX-4T-1中,在T4 DNA连接酶的作用下构建融合表达载体pGEX-mZP3,转化大肠杆菌BL－21菌株,利用IPTG诱导后获得可溶性的蛋白质产物,经过SDS－PAGE鉴定,表达的融合蛋白GST－mZP3分子质量约为72kD左右,纯化的融合蛋白免疫兔子,获得效价为1：1000的抗血清,Western blot检测抗血清具有针对mZP3融合蛋白的专一性,为进一步开展mZP3的免疫功能检测的研究奠定 了基础。     

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.     

Location of antigenic determinants on primary sequences of subunits of nicotinic acetylcholine receptor by peptide mapping

The binding domains of 28 monoclonal antibodies (mAbs) against the alpha, beta, and delta subunits of the Torpedo acetylcholine receptor were mapped on the primary sequences of these subunits. Small peptide fragments (2000-20,000 daltons) of the purified subunits were obtained by digestion with staphylococcal V8 protease and papain, separated on a discontinuous polyacrylamide gel electrophoretic system, and electroblotted onto diaminophenyl thioether paper. The blots were probed with the various monoclonal antibodies and also with antibodies against carboxy-terminal decapeptides of the alpha, beta, and delta subunits to identify the carboxy-terminal fragments. From inspection of the binding patterns of the various antibodies to the subunits fragments and the molecular weights of these fragments, and by using the carboxy termini of the subunits as reference points, it was possible to deduce the regions on the primary sequence of each subunit in which the antibodies bound and in some cases to order the binding sites within these sequences. mAb 148, which inhibits receptor function by cross-linking receptor molecules on the cytoplasmic side, was mapped to the sequence beta 368-406. The main immunogenic region of the native receptor, which is of pathological importance in the autoimmune disease myasthenia gravis, was mapped by using mAb 210 to within 80 amino acid residues (alpha 46-127). The overall antigenic structure of alpha subunits was examined. Synthetic peptides have been used to locate determinants responsible for 83% of the antibodies in antisera to denatured alpha subunits and 46% of the antibodies to denatured alpha subunits in antisera to intact receptor. Theoretical models of the transmembrane orientation of the subunit polypeptide chains were tested by determining whether mapped monoclonal antibodies bound to the extracellular or intracellular surface of receptor-rich membranes. Our results confirm previous reports that the carboxy termini of the subunits are exposed on the intracellular surface, as is part of the region between a putative channel-forming domain (M5) and a putative membrane-spanning region (M3). However, contrary to current theoretical models, the region between M5 and the putative membrane-spanning sequence M4 also appears to be on the intracellular surface, implying that M4 and M5 are not membrane-spanning domains.(ABSTRACT TRUNCATED AT 400 WORDS)     

Physicochemical characterization of lymphocyte-activating factor (LAF).

The LAF produced by the mouse macrophage cell line, P388D1, is a single polypeptide chain of m.w. 12,000 to 16,000 daltons. Native LAF was destroyed by Streptomyces griseus protease, but not by trypsin, chymotrypsin, and papain, although in the presence of 8 M urea, papain completely destroyed LAF activity. LAF did not bind to concanavalin A-Sepharose, suggesting that LAF does not contain significant amounts of mannosyl or glycosyl residues. Since LAF activity was not inactivated by a treatment of reduction and alkylation the active conformation of LAF does not appear to be dependent on disulfide linkages. LAF was not irreversibly denatured by 8 M urea or 0.1 to 0.5% SDS. On SDS-polyacrylamide gels, the m.w. of LAF was 12,000 daltons, as compared to a value of 16,000 daltons, as determined by gel filtration. The isoelectric point of LAF was 5.0 to 5.4 as determined on 7.5% acrylamide gels (pH 3 to 10). On the basis of these results it appears that the P388D1 cell line-derived LAF is a relatively stable molecule that shares several physicochemical properties with normal human and mouse macrophage-derived LAF.     

Isolation of a glycopeptide fraction from the surface of the sea urchin egg that inhibits sperm-egg binding and fertilization

The role of cell surface glycoproteins of the sea urchin egg in binding sperm has been examined by studying the biological activity of glycopeptides derived from these glycoproteins. Glycopeptides were produced from egg surface glycoproteins by Pronase digestion. After fractionation by gel filtration the glycopeptides were tested for their ability to inhibit the binding of sperm to eggs, presumably by competing with the egg surface glycoproteins for binding sites on the sperm. One glycopeptide fraction with an apparent molecular weight of approximately 6,000 was found to be a potent inhibitor of sperm-egg binding, as well as fertilization, even at nanomolar concentrations. This activity was heat stable and exerted its effect against the sperm and not the egg. Experiments with a radiolabeled form of the glycopeptide fraction directly demonstrated that at least one component of it bound to sperm. Specific binding of the radiolabeled glycopeptide occurred only to acrosome-reacted sperm. Because the isolated glycopeptide fraction has many of the characteristics that one would expect of a biologically active fragment of an egg surface receptor for sperm, these findings are consistent with the idea that one or more glycoconjugates on the surface of the egg are involved in sperm binding.     

An activity from Escherichia coli membranes responsible for the modification of nitrate reductase to its precursor form

An enzymatic activity which modifies nitrate reductase has been identified in the cytoplasmic membrane of Escherichia coli. This activity changes subunit B to a form with a slightly greater electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels (B'). The B' polypeptide produced by this modifying enzyme was compared to an apparently identical polypeptide identified in the precursor form of nitrate reductase which can be found in the cytoplasm of all strains and in the membrane of mutants defective in the insertion of nitrate reductase. These B' polypeptides were all identical with respect to mobility on gradient sodium dodecyl sulfate gels and peptides produced by limited digests using trypsin, papain, and Staphylococcus aureus V8 protease. When compared to subunit B, the proteolytic gel maps of B' polypeptides showed minor differences. From the identity of the modified B' with precursor B', the ability to convert B into B' in vitro and the in vivo nature of B' as a precursor of B, it was concluded that the modification of B to B' is a reversible process and is due to the removal of one or more small nonprotein molecules.     

Subsite mapping of an acidic amino acid-specific endopeptidase from Streptomyces griseus, GluSGP, and protease V8.

The substrate specificities of an acidic amino acid-specific endopeptidase of Streptomyces griseus, GluSGP, and protease V8 [EC 3.4.21.19] were investigated with peptide p-nitroanilide substrates which have a Glu residue at the P1 position. GluSGP and protease V8 favored Pro and Leu residues at S2, respectively, while the S3 subsite of GluSGP preferred Phe over either Ala or Leu. The S3 subsite of protease V8 preferred Leu over either Ala or Phe. The best substrates for GluSGP and for protease V8 were Boc-Ala-Phe-Pro-Glu-pNA with a Km value of 0.41 mM (0.1 M Tris-HCl, pH 8.8) and Boc-Ala-Leu-Leu-Glu-pNA with a Km value of 0.25 mM (0.1 M phosphate, pH 7.8), respectively. The kcat/Km values for these substrates obtained with GluSGP were about one hundred to twenty thousand times larger than those obtained with protease V8. Protease V8 exhibited a single optimal pH of around 8 for the hydrolysis of Boc-Ala-Ala-Leu-Glu-pNA and Boc-Ala-Leu-Leu-Asp-pNA.     

Differential O-glycosylation of a conserved domain expressed in murine and human ZP3

Murine sperm initiate fertilization by binding to the zona pellucida (mZP), the specialized extracellular matrix of their homologous eggs. O-Glycans occupying two highly conserved vicinal glycosylation sites (Ser-332 and Ser-334) on the mZP glycoprotein designated mZP3 were previously implicated in this interaction. However, recent biophysical analyses confirm that neither site is occupied, implying that an alternate O-glycosylation domain may be operational in native mZP3. Since human ZP3 (huZP3) can substitute for mZP3 in rescue mice to mediate sperm binding, the site specificity of O-glycosylation in both native mZP3 and huZP3 was analyzed using ultrasensitive mass spectrometric techniques. Two O-glycosylation sites in native mZP3, one at Thr-155 and the other within the glycopeptide at positions 161-168 (ATVSSEEK), are conserved in huZP3 derived from transgenic mice. Thus, there is a specific O-glycosylation domain within native mZP3 expressing two closely spaced O-glycans that is very well conserved in an evolutionarily related glycoprotein. In native mZP3, core 2 O-glycans predominate at both sites. However, in huZP3 derived from rescue mice, the O-glycans associated with Thr-156 (analogous to Thr-155 in mZP3) are exclusively core 1 and related Tn sequences, whereas core 2 O-glycans predominate at the other conserved site. This unique restriction of O-glycan expression suggests that sequence differences in the conserved O-glycosylation domains of mZP3 and huZP3 affect the ability of core 2 N-acetylglucosaminyltransferase(s) to extend the core 1 sequence. However, this difference in O-glycosylation at Thr-156 does not affect the fertility or the sperm binding phenotype of eggs derived from female huZP3 rescue mice.