Mouse Egg Zona Pellucida Research Articles

Fucose, Mannose, and β-N-Acetylglucosamine Glycopolymers Initiate the Mouse Sperm Acrosome Reaction through Convergent Signaling Pathways

The sperm acrosome reaction (AR), an essential exocytosis step in mammalian fertilization, is mediated by a species-specific interaction of sperm surface molecules with glycans on the egg. Previous studies indicate that a subset of terminal carbohydrates on the mouse egg zona pellucida (ZP) trigger the AR by cross-linking or aggregating receptors on the sperm membrane. However, the exact role of those carbohydrates in AR has not been identified and the mechanism underlying the AR still needs further investigation. To study this process, a series of glycopolymers was synthesized. The glycopolymers are composed of a multivalent scaffold (norbornene), a functional ligand (previously identified ZP terminal monosaccharides), and a linker connecting the ligand and the scaffold. The polymers were tested for their ability to initiate AR and through which signaling pathways AR induction occurred. Our data demonstrate that mannose, fucose, and β-N-acetylglucosamine 10-mers and 100-mers initiate AR in a dose-dependent manner, and the 100-mers are more potent on a per monomer basis than the 10-mers. Although nearly equipotent in inducing the AR at the optimal concentrations, their AR activation kinetics are not identical. Similar to mouse ZP3, all 100-mer-activated AR are sensitive to guanine-binding regulatory proteins (G-proteins), tyrosine kinase, protein kinase A, protein kinase C, and Ca2+-related antagonists. Thus, the chemotypes of synthetic glycopolymers imitate the physiologic AR-activation agents and provide evidence that occupation of one of at least three different receptor binding sites is sufficient to initiate the AR.

Influence of the zona pellucida of the mouse egg on folliculogenesis and fertility

All mammalian eggs are surrounded by a relatively thick extracellular coat, called the zona pellucida (ZP), that plays vital roles during oogenesis, fertilization, and preimplantation development. The mouse egg ZP consists of three glycoproteins, called mZP1-3, that are synthesized solely by oocytes during their 2-to-3 week growth phase. The ZP is seen initially as isolated extracellular deposits of nascent ZP fibrils that coalesce to form a thickening matrix. Elimination of ZP glycoprotein synthesis by targeted mutagenesis yields mice that are heterozygous or homozygous for the null mutations. Homozygous null males are unaffected by the mutations and heterozygous females are as fertile as wild-type females. On the other hand, eggs from mZP2-/- and mZP3-/- females lack a ZP and the mice are completely infertile due to a severely reduced number of ovulated eggs in their oviducts. Development of ovarian follicles is retarded in homozygous null females and manifested as reduced ovarian weight, number of Graafian follicles, and number of ovulated eggs. Eggs from mZP1-/- females have a distorted ZP and, although the mice are fertile, they exhibit reduced fertility due to early embryonic loss. Potential relationships between ZP biogenesis, folliculogenesis, and fertility are addressed.

Zona pellucida glycoprotein ZP3 and fertilization in mammals

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

Mammalian fertilization:the eggs multifunctional zona pellucida

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

Purified trout egg vitelline envelope proteins VEβ and VEγ polymerize into homomeric fibrils from dimers in vitro

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

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

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

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.

Biochemical and molecular studies of the proacrosin/acrosin system in patients with unexplained infertility

Biochemical and molecular studies of the proacrosin/acrosin system in patients with unexplained infertility

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.

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.

Developmental regulation of apoptosis in the early bovine embryo

Sperm-egg interaction in mammals is initiated by binding of sperm to the zona pellucida, an acellular coat completely surrounding the plasma membrane of unfertilized eggs. Zonae pellucidae of mouse eggs are composed of three different glycoproteins, designated ZP1, ZP2 and ZP3, having apparent molecular weights of 200,000, 120,000 and 83,000, respectively Bleil and Wassarman, 1978, Bleil and Wassarman, 1980a, Bleil and Wassarman, 1980b. In this investigation, ZP1, ZP2 and ZP3 were purified from zonae pellucidae isolated individually from unfertilized mouse eggs and 2-cell embryos. Each of the glycoproteins was then tested for its ability to interfere with the binding of sperm to eggs in vitro. Solubilized zonae pellucidae isolated from unfertilized eggs, but not from 2-cell embryos, reduced binding of sperm to as little as 10% of control values. Similarly, ZP3 purified from zonae pellucidae of unfertilized eggs reduced the binding of sperm to eggs in vitro to an extent comparable to that observed with solubilized zonae pellucidae. On the other hand, ZP3 purified from zonae pellucidae of 2-cell embryos had no significant effect on the extent of sperm binding, consistent with the inability of solubilized zonae pellucidae from 2-cell embryos to affect sperm binding. In no case did purified ZP1 and ZP2 interfere significantly with the binding of sperm to eggs in vitro. These results suggest that ZP3 possesses the receptor activity responsible for the binding of sperm to zonae pellucidae of unfertilized mouse eggs. Fertilization apparently results in modification of ZP3 such that it can no longer serve as a receptor for sperm.

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.

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.

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.

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.

Inhibition of fertilization by a monoclonal antibody recognizing the oligosaccharide sequence Ga1NAcβ1→4Ga1β1→4 on the mouse zona pellucida

Mouse eggs and pre-implantation stage embryos express on their surfaces a carbohydrate epitope, TEC-2, defined by an IgM monoclonal antibody, TEC-02. The TEC-2 epitope involves the oligosaccharide sequence Ga1NAcβ1→4Ga1β1→4 that is expressed on the plasma membrane and zona pellucida of mouse eggs and on a very limited number of other cell types. In this study we addressed the question whether or not the binding of TEC-02 antibody to the mouse eggs would interfere with their fertilization. Our data showed that the TEC-2 epitope is carried by two zona pellucida glycoproteins, ZP2 and ZP3. Binding of TEC-02 antibody to mouse eggs inhibited specifically and in a dose-dependent manner their fertilization in vitro. The inhibitory effect of TEC-02 antibody was dependent on the presence of an intact zona pellucida. Direct radioantibody binding assays indicated that the TEC-02 antibody completely inhibited fertilization at a concentration at which one quarter of all available TEC-2 binding sites was occupied. Binding of TEC-02 antibody to an egg did not interfere with initial attachment of the sperm to the egg but inhibited maintenance of sperm binding to the zona pellucida, the secondary binding. The combined data indicate that TEC-2, which is a well-defined zona pellucida specific carbohydrate epitope, might be a part of the secondary sperm receptor.

Activation of a G protein in mouse sperm by the zona pellucida, an egg-associated extracellular matrix.

Mammalian sperm possess a guanine nucleotide-binding regulatory protein (G protein), with properties similar to Gi, that appears to be involved in the signal transduction pathway required for zona pellucida (ZP)-mediated acrosomal exocytosis. Mouse sperm treated with pertussis toxin (PT), a toxin that functionally inactivates Gi proteins, bind to the ZP of mouse eggs but are inhibited from undergoing acrosomal exocytosis. We have measured high-affinity GTPase activity and GTP gamma [35S] binding in mouse sperm homogenates incubated in the absence and presence of ZP glycoproteins isolated from either ovulated eggs or from ovarian homogenates to determine whether this extracellular matrix can activate the sperm-associated Gi protein. An increase in GTP hydrolysis (approximately 50% over basal activity) and GTP gamma [35S] binding (approximately 25-60% over basal activity) is observed when sperm homogenates are incubated in the presence of solubilized ZP glycoproteins, and the increase in GTPase activity is dependent on the concentration of ZP added to the homogenates. Accompanying this increase is a reduction in the ability of PT to catalyze in vitro [32P]ADP-ribosylation of a Mr = 41,000 sperm Gi protein, suggesting that the increase in GTPase activity and GTP gamma [35S] binding is associated with the activation of a PT-sensitive sperm G protein(s). The ability of the ZP to stimulate high-affinity GTPase activity in these homogenates appears to be dependent on the capacitation state of the sperm from which the homogenates are prepared. These data suggest that a component(s) of the ZP may function in a manner similar to that of other ligands by binding to a sperm surface-associated receptor and subsequently activating a G protein coupled to an intracellular signal transduction cascade(s) required for induction of acrosomal exocytosis.

Rescue of murine androgenones to the blastocyst stage by injection of normal embryonic cytoplasm

Sperm-egg interaction in mammals is initiated by binding of sperm to the zona pellucida, an acellular coat completely surrounding the plasma membrane of unfertilized eggs. Zonae pellucidae of mouse eggs are composed of three different glycoproteins, designated ZP1, ZP2 and ZP3, having apparent molecular weights of 200,000, 120,000 and 83,000, respectively Bleil and Wassarman, 1978, Bleil and Wassarman, 1980a, Bleil and Wassarman, 1980b. In this investigation, ZP1, ZP2 and ZP3 were purified from zonae pellucidae isolated individually from unfertilized mouse eggs and 2-cell embryos. Each of the glycoproteins was then tested for its ability to interfere with the binding of sperm to eggs in vitro. Solubilized zonae pellucidae isolated from unfertilized eggs, but not from 2-cell embryos, reduced binding of sperm to as little as 10% of control values. Similarly, ZP3 purified from zonae pellucidae of unfertilized eggs reduced the binding of sperm to eggs in vitro to an extent comparable to that observed with solubilized zonae pellucidae. On the other hand, ZP3 purified from zonae pellucidae of 2-cell embryos had no significant effect on the extent of sperm binding, consistent with the inability of solubilized zonae pellucidae from 2-cell embryos to affect sperm binding. In no case did purified ZP1 and ZP2 interfere significantly with the binding of sperm to eggs in vitro. These results suggest that ZP3 possesses the receptor activity responsible for the binding of sperm to zonae pellucidae of unfertilized mouse eggs. Fertilization apparently results in modification of ZP3 such that it can no longer serve as a receptor for sperm.

Identification of a ZP3-binding protein on acrosome-intact mouse sperm by photoaffinity crosslinking.

During the process of fertilization in mammals, sperm bind in a relatively species-specific manner to the zona pellucida (ZP) of ovulated eggs. ZP3, a glycoprotein found in the mouse egg zona pellucida, serves as receptor for sperm during gamete adhesion. We report here that a Mr 56,000 protein found on mouse sperm has properties expected for a sperm component that recognizes and binds to ZP3. This sperm protein is radiolabeled preferentially by a photoactivatable heterobifunctional crosslinker ("Denny-Jaffee reagent") covalently linked to purified ZP3, binds very tightly to ZP3-affinity columns, and is localized to heads of acrosome-intact but not acrosome-reacted sperm. These and other findings suggest that this protein may be a "ZP3-binding protein" that, together with the sperm receptor, supports species-specific binding of mouse sperm to unfertilized eggs.

Endpoint of first stage of zona pellucida-induced acrosome reaction in mouse spermatozoa characterized by acrosomal H+ and Ca2+ permeability: population and single cell kinetics.

The acrosome reaction induced by the mouse egg's zona pellucida in mouse sperm has been shown to proceed in two stages as characterized empirically by sequential changes in patterns of chlortetracycline fluorescence on the sperm plasma membrane surfaces. The chlortetracycline fluorescence pattern characteristic of fully intact sperm is designated B; in sperm bound to structurally intact zonae that induce the acrosome reaction, the B pattern changes first to an intermediate pattern S and then to a terminal pattern AR characteristic of the completed acrosome reaction. In the same study, it was shown, using a 9-amino acridine fluorescent pH probe, that completion of the first stage was characterized by increase in H+ permeability such that the H+ gradient between sperm head and medium was dissipated. In this study, we show that the fluorescent pH probe 9-N-dodecylamino acridine and the intracellular Ca2+ fluorescent probe fura-2 are both localized to the anterior part of the sperm head encompassing the acrosomal compartment in intact sperm, and the fluorescence associated with each probe is lost as the first stage of the acrosome reaction is completed. Loss of the pH probe fluorescence, pattern N, corresponds to onset of H+ permeability, and loss of fura-2 fluorescence, pattern F, corresponds to onset of Ca2+ permeability. Localization of intracellular fura-2 fluorescence to the acrosomal compartment required extracellular Mn2+ to quench surface-bound fura-2 AM, the tetra-acetoxymethyl ester of fura-2 used to load the cells. Loss of acrosomal fura-2 fluorescence is due to quenching by tracer Mn2+ accompanying Ca2+. Onset of membrane permeability to both H+ and Ca2+, as seen by loss of patterns N and F, occurred in synchrony in populations of sperm bound to isolated, structurally intact zonae, with an overall time course of 210 min postbinding. The loss of pattern N in individual sperm cells bound to zonae was rapid, with a half time of 2.1 min. Concomitant with this rapid loss of pattern N was a shift in the amplitude of flagellar motion from large to small. The lag times to pattern N loss in 50 individual cells ranged from 30 to 140 min. The variable lag times determine the population kinetics; the rate of the endpoint reaction seen in the individual cells is rapid and constant. Dissipation of the H+ gradient with immediate loss of pattern N was readily achieved by addition of nigericin with no change in the time course of the onset of Ca2+ permeability of the membranes enclosing the acrosome.(ABSTRACT TRUNCATED AT 400 WORDS)