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 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.     

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.     

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.     

Precambrian animal life: probable developmental and adult cnidarian forms from Southwest China

We have previously described the affinity of a pig sperm surface protein, P68, to mammalian zonae pellucidae (ZP). In this report, we identified P68 as arylsulfatase A (AS-A) based on the presence of P68 tryptic peptide sequences in the pig testis AS-A cDNA sequence. Our objective was to demonstrate the presence of AS-A on the sperm surface and to elucidate its role in ZP binding. Immunogold electron microscopy revealed the presence of AS-A on the sperm surface. Furthermore, live pig sperm and the extract of peripheral sperm plasma membrane proteins exhibited AS-A's desulfation activity. Significantly, the role of pig sperm surface AS-A in ZP binding was demonstrated by dose-dependent decreases of sperm-ZP binding upon sperm pretreatment with anti-AS-A IgG/Fab, and by the binding of Alexa-430-conjugated sperm surface AS-A to homologous ZP. ZP pretreatment with anti-pig-ZP3 antibody abolished AS-A binding, suggesting that ZP3, recognized as the pig sperm receptor, was AS-A's binding ligand. This was further confirmed by the ability of exogenous ZP3 to competitively inhibit AS-A-ZP binding. Similarly, purified ZP3alpha, a major sperm receptor component of ZP3, exhibited great inhibitory effect on AS-A-ZP binding. All of these results designated a new function of AS-A in gamete interaction.     

95 kd sperm proteins bind ZP3 and serve as tyrosine kinase substrates in response to zona binding

In the mouse, the zona pellucida (ZP) glycoprotein ZP3 both binds intact sperm and induces acrosomal exocytosis. The subsequent signaling pathway(s) is still uncertain, but Gi-like proteins have been implicated. By analogy with other signal transduction mechanisms, we examined anti-phosphotyrosine antibody reactivity in mouse sperm. Antibodies reacted with three proteins of 52, 75, and 95 kd. Indirect immunofluorescence localized reactivity to the acrosomal region of the sperm head. The 52 kd and 75 kd phosphoproteins are detected only in capacitated sperm, whereas the 95 kd protein is detected in both fresh and capacitated sperm. For the 95 kd protein, the level of immunoreactivity is not related to sperm motility but is enhanced by both capacitation and sperm interaction with solubilized ZP proteins. In addition, binding of radiolabeled whole ZP or purified ZP3 to blots of separated sperm proteins identified two ZP binding proteins of 95 kd and 42 kd. 95 kd sperm proteins that bind to ZP3 also react with anti-phosphotyrosine antibodies (in a ZP concentration-dependent manner), supporting the idea that the same 95 kd sperm protein serves as a ZP3 receptor and as a tyrosine kinase substrate. These findings and our evidence on acrosome reaction triggering via sperm receptor aggregation suggest that a 95 kd protein in the sperm plasma membrane is aggregated by ZP3, which stimulates tyrosine kinase activity leading to acrosomal exocytosis.     

Sulfogalactosylglycerolipid is involved in human gamete interaction.

Recent results from our laboratory have revealed the role of sulfogalactosylglycerolipid (SGG) in mouse sperm-zona pellucida (ZP) binding. In this report, we demonstrated the presence of SGG in Percoll-gradient centrifuged (PGC) human sperm by high performance thin layer chromatography with orcinol and Azure A staining, specific for glycolipids and sulfolipids, respectively. SGG in human PGC sperm was quantified by its affinity to Azure A to be 12-15 mol% of sperm lipids. Indirect immunofluorescence revealed that SGG existed on both live and aldehyde fixed human sperm in the head region. Pretreatment of human PGC sperm with affinity purified antiSGG Fab markedly inhibited sperm binding to the ZP in a concentration dependent manner, without any changes in the spontaneous acrosome rate or sperm motility parameters. Fluorescently labeled SGG liposomes also bound uniformly to isolated human ZP, while fluorescently labeled galactosylglycerolipid (GG, SGG's parental lipid) or phosphatidylserine (PS, negatively charged like SGG) liposomes did not. All of these results suggested the role of human sperm SGG in ZP binding.     

Mouse sperm protein sp56 is a component of the acrosomal matrix

Previously, we identified the guinea pig sperm acrosomal matrix glycoprotein AM67 and demonstrated that it is most closely related to mouse sperm sp56, initially reported to be a cell-surface protein. On the contrary, our studies demonstrated that sp56 is an intra-acrosomal component. Based upon the homology between guinea pig AM67 and mouse sp56, we hypothesized that sp56 was part of the acrosomal matrix, a structure that had yet to be demonstrated to exist in mouse sperm. In this paper, we show that sp56 first appeared in late meiotic cells and accumulated during spermiogenesis, the haploid stage of spermatogenic cell development. Using affinity-purified anti-peptide antisera, we determined that the molecular weight of sp56 in cauda epididymal sperm approximated that of guinea pig AM67 ( approximately 67 000 M:(r)) and that sp56 was present in a high molecular weight, disulfide-linked complex. The forms of sp56 in pachytene spermatocytes and spermatids had higher molecular weights than was found for the sperm form; the size differences were apparently due to alterations in carbohydrate side chains. The sp56 complex could not be solubilized by the nonionic detergent Triton X-100 but remained associated with the dorsal surface of the mouse sperm head, demonstrating that sp56 is a component of the mouse sperm acrosomal matrix.     

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.     

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)     

Immunocytochemical and biochemical characterization of guanine nucleotide-binding regulatory proteins in mammalian spermatozoa

Polyclonal antisera directed against conserved and subtype-specific peptide sequences of the alpha-subunits of guanine nucleotide-binding regulatory proteins (G proteins) were used to characterize the nature of mammalian sperm G proteins and to determine whether their localization was consistent with their proposed roles in mediating ZP3-induced acrosomal exocytosis. Mouse and guinea pig sperm exhibit positive immunofluorescence in the acrosomal region using an antiserum directed against a peptide region common to all alpha-subunits of G proteins (G alpha). The immunofluorescence disappears after sperm have undergone the acrosome reaction, suggesting that the immunoreactive material is associated with the plasma membrane/outer acrosomal membrane region overlying the acrosome. The presence of G proteins in this region is confirmed by the presence of a Mr 41,000 substrate for pertussis toxin (PT)-catalyzed [32P]ADP-ribosylation in purified plasma membrane/outer acrosomal membrane hybrid vesicles obtained from acrosome-reacted guinea pig sperm. Immunoprecipitation and polyacrylamide gel electrophoresis of PT-catalyzed [32P]ADP-ribosylated protein(s) using anti-peptide antisera generated against sequences unique to Gi alpha 1, Gi alpha 2, and Gi alpha 3 confirm the existence of all three Gi subtypes in mouse sperm extracts. Indirect immunofluorescence using an antiserum directed against a peptide region present in Gz alpha, a PT-insensitive G protein, demonstrates positive immunoreactivity in the postacrosomal/lateral face region of the mouse sperm head. This immunoreactivity is retained during acrosomal exocytosis in response to solubilized ZP and then disappears subsequent to this exocytotic event. These data demonstrate that Gi protein alpha-subunits are present in the acrosomal region of mammalian sperm, consistent with their postulated role in regulating ZP3-mediated acrosomal exocytosis, and that PT-insensitive Gz alpha is found in a region of the sperm head distinct from that of the Gi alpha subunits.     

Role of sperm surface arylsulfatase A in mouse sperm-zona pellucida binding

We have previously described the zonae pellucidae (ZP) binding ability of a pig sperm surface protein, P68. Our recent results on peptide sequencing of 3 P68 tryptic peptides and molecular cloning of pig testis arylsulfatase A (AS-A) revealed the identity of P68 as AS-A. In this report, we demonstrate the presence of AS-A on the mouse sperm surface and its role in ZP binding. Using anti-AS-A antibody, we have shown by immunoblotting that AS-A was present in a Triton X-100 extract of mouse sperm. The presence of AS-A on the sperm plasma membrane was conclusively demonstrated by indirect immunofluorescence, immunogold electron microscopy, and AS-A's desulfation activity on live mouse sperm. The AS-A remained on the head surface of in vivo capacitated sperm, as revealed by positive immunofluorescent staining of oviductal/uterine sperm. Significantly, the role of mouse sperm surface AS-A on ZP binding was demonstrated by dose-dependent decreases of sperm-ZP binding on sperm pretreatment with anti-AS-A IgG/Fab. Furthermore, Alexa-430 conjugated AS-A bound to mouse ZP of unfertilized eggs but not to fertilized ones, and this level of binding increased and approached saturation with increasing Alexa-430 AS-A concentrations. Moreover, in vivo fertilization was markedly decreased when mouse sperm pretreated with anti-AS-A IgG were artificially inseminated into females. All of these results designated a new function for AS-A in mouse gamete interaction.     

Protein dynamics in sperm membranes: implications for sperm function during gamete interaction.

A number of mammalian sperm plasma membrane antigens have been implicated as playing a functional role in sperm-egg interaction, by virtue of the fact that antibodies against these antigens interfere with fertilization. Two such mouse sperm plasma membrane antigens are M42, a 200/220 kD glycoprotein doublet, and M5, a 150-160 kD glycoprotein. We show that both of these antigens are concentrated on the posterior region of caudal epididymal and capacitated mouse sperm heads and are relatively diffusible, as determined by fluorescence recovery after photobleaching measurements (D = 3-8 x 10(-9) cm2/s with approximately 23% diffusing). Crosslinking of these antigens with bivalent antibodies causes them to redistribute into the anterior region (acrosomal crescent) of the sperm head. In contrast, we describe a third antigen, P220, which is also localized to the posterior region of the sperm head on caudal epididymal sperm but which exhibits very little diffusion and does not redistribute upon crosslinking. Bivalent anti-M42 blocks the ZP3-induced acrosome reaction. We have found that monovalent Fab fragments of anti-M42 do not block the ZP3-induced acrosome reaction, but that inhibition is restored by addition of a second antibody which crosslinks the Fabs. Thus, crosslinking is required for both inhibition of the acrosome reaction and redistribution. This suggests that redistribution of antigen away from the posterior region of the head may be part of the mechanism of inhibition of the ZP3-induced acrosome reaction.     

Distinct membrane fractions from mouse sperm bind different zona pellucida glycoproteins.

Interactions between sperm and zona pellucida (ZP) during mammalian fertilization are not well characterized at the molecular level. To identify sperm proteins that recognize ligand ZP3, we used sonicated sperm membrane fractions as competitors in a quantitative binding assay. Sonicated membranes were density fractionated into 4 fractions. Bands 1-3 contained membrane vesicles, and band 4 contained axonemal and midpiece fragments. In competitive binding assays, bands 1, 2, and 3 but not band 4 were able to compete with live, capacitated, intact sperm for soluble 125I-ZP binding. Affinity-purified ZP fractions consisting of a ZP3-enriched fraction (125I-ZP3) and a fraction enriched for ligands ZP1 and ZP2 and depleted of ZP3 (125I-ZP1/2) were obtained by antibody affinity purification of ZP3. In competitive binding assays, bands 2 and 3 competed for 125I-ZP3 binding, but band 1 did not interact with enriched 125I-ZP3. None of the membrane fractions competed for 125I-ZP1/2 binding. These results demonstrate that band 2 and band 3 contain sperm components that interact with ZP3 alone and that components in band 1 interact with ZP3 in conjunction with either ZP1 or ZP2. These data indicate that there must be at least 2 unique sperm plasma membrane components that mediate intact sperm interactions with ZP glycoproteins in mouse. Bands 2 and 3 are likely to contain a primary ZP-binding protein because they interacted directly with ZP3, whereas band 1 may contain sperm proteins involved in later interactions with the ZP, perhaps transitional interactions to maintain sperm contact with the ZP during acrosomal exocytosis.     

Capacitation-dependent reorganization of microdomains in the apical sperm head plasma membrane: functional relationship with zona binding and the zona-induced acrosome reaction

For sperm to successfully fertilize an oocyte, it needs to pass through certain steps prior to, during and after initial recognition of the zona pellucida (ZP). During capacitation, the surface of the sperm head becomes remodelled, priming it to bind to the ZP and subsequently to undergo the ZP-induced acrosome reaction. During capacitation, sperm ZP-binding proteins are ordered in functional protein complexes that only emerge at the apical tip of the sperm head plasma membrane; this is also functionally the exclusive sperm surface area involved in primary ZP binding. After primary ZP binding, the same area is probably involved in the induction of the acrosome reaction. A combination of biochemical and proteomic membrane protein techniques have enabled us to dissect and highly purify the apical sperm plasma membrane area from control and capacitated sperm cells. The actual ZP-binding proteins identified predominantly belonged to the sperm membrane-associated family members of spermadhesins (AQN-3) and were present in the aggregating lipid ordered membrane microdomains (lipid rafts) that emerged during in vitro capacitation in the apical ridge area of the sperm head plasma membrane. This clustering of these rafts was dependent on the presence of bicarbonate (involved in protein kinase A activation) and on the presence of albumin (involved in cholesterol removal). Remarkably, cholesterol removal was restricted to the non-raft membrane fraction of the sperm plasma membrane, but did not cause any depletion of cholesterol in the raft membrane fraction. Interestingly, sperm SNARE proteins (both VAMP from the outer acrosomal membrane, as well syntaxin from the apical sperm head plasma membrane) shared lateral redistribution properties, along with the ZP-binding protein complex and raft marker proteins. All of these were recovered after capacitation in detergent-resistant membrane preparations from sperm thought to represent membrane lipid rafts. We inferred that the capacitation-dependent formation of an aggregated lipid ordered apical ridge surface area in the sperm head plasma membrane was not only relevant for ZP-binding, but also for the ZP-induced acrosome reaction.     

Porcine zona pellucida ZP3α glycoprotein mediates binding of the biotin-labeled Mr 55,000 family (ZP3) to boar sperm membrane vesicles

The two M_r 55,000 glycoproteins, ZP3α and ZP3b?, of porcine zona pellucida copurify as a preparation designated ZP3. Gamete binding assays have implicated ZP3α, but not ZP3b?, as participating in sperm-zona recognition events. We now report that boar sperm contain membrane-associated binding sites with specificity for ZP3α. Biotin-labeled (b-) preparations of ZP3 bind to intact boar sperm in a saturable manner, with localization on the anterior head region. Membrane vesicles obtained from capacitated sperm by nitrogen cavitation retain b-ZP3 binding sites as determined by an enzyme-linked method employing alkaline phosphatase-conjugated strepavidin. In competitive binding assays using b-ZP3 (0.1μg/ml) as probe, heat-solubilized zonae and ZP3 were effective competitors, whereas the nonzona molecules fetuin and fucoidin were not. Digestion of ZP3 with endo-b?-galactosidase, an enzyme that trims polylactosamines, enhanced its affinity for membrane receptors. In contrast treatments such as chemical deglycosylation, pronase digestion, or disruption of disulfide bonds abolished the ligand activity of ZP3. Finally, purified ZP3α was an at least 100-fold better antagonist than purified ZP3b?. The results demonstrate that binding of b-ZP3 to isolated boar sperm membranes is mediated by sperm receptors with specificity for the ZP3α macromolecular component and reveal a complex contribution of both carbohydrate and protein moieties toward the ligand activity of this sperm adhesive zona molecule. © 1993 Wiley-Liss, Inc.     

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.     

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.     

Characterization of zona pellucida glycoprotein 3 (ZP3) and ZP2 binding sites on acrosome-intact mouse sperm

There is considerable evidence that mouse fertilization requires the binding of sperm to two of the three glycoproteins that form the zona pellucida (ZP), ZP3 and ZP2. Despite the biologic importance of this binding, no one has demonstrated that sperm express separate, saturable, and specific binding sites for ZP3 and for ZP2. Such a demonstration is a prerequisite for defining the distribution, numbers, affinities, and regulation of function of ZP3 and ZP2 binding sites on sperm. The experiments reported herein used fluorochrome-labeled ZP3 and ZP2 and quantitative image analysis to characterize the saturable binding of ZP3 and ZP2 to distinct sites on living, capacitated, acrosome-intact mouse sperm. Approximately 20% of the ZP3 binding sites were found over the acrosomal cap, and the remaining sites were located over the postacrosomal region of the head. In contrast, ZP2 binding sites were detected only over the postacrosomal region. Saturation analysis estimated numbers and affinities of the binding sites for ZP3 (B(max) approximately 185 000 sites per sperm; K(d) approximately 67 nM) and ZP2 (B(max) approximately 500 000 sites per sperm; K(d) approximately 200 nM). Use of unlabeled ZP3, ZP2, and ZP1 as competitive inhibitors of the binding of fluorochrome-labeled ZP3 and ZP2 demonstrated that ZP3 and ZP2 bound specifically to their respective sites on sperm. Finally, we demonstrate that extracellular calcium as well as capacitation and maturation of sperm are required for these sites to bind their respective ligands.     

Immunocontraception in mice using repeated, multi-antigen peptides: immunization with purified recombinant antigens

Two immunocontraceptive antigens (AgE and AgF) were constructed that included different combinations of highly species-specific peptides from the mouse reproductive antigens SP56, ZP3, ZP2, and ZP1 in the form of multi-antigen peptides (MAPs). Both AgE and AgF contained three tandem repeats each of ZP2 and ZP3 peptide epitopes and a single copy of a ZP1 peptide sequence all of which had previously been demonstrated to individually have immunodominant or contraceptive effects. In addition, AgF contained a single contraceptive peptide derived from SP56, the putative ZP3 receptor protein on sperm. The antigens were expressed and affinity purified as recombinant repeated multi-antigen (polyepitope) peptides using an Escherichia coli maltose binding protein (MBP) expression system. Female BALB/c mice actively immunized with these antigens in Freund's adjuvants produced variable serum antibody responses to the component peptides. Fertility rates for animals immunized with AgE (40%) and AgF (20%) were significantly reduced compared to MBP immunized mice (90%), but the reduction in fertility did not correlate with peptide-specific serum antibody levels. Ovaries from all immunized mice appeared histologically normal with no evidence of oophoritis. These results demonstrate that high levels of immunocontraception can be achieved in mice, without apparent side-effects, using species-specific immunogens that include repeated peptides from proteins involved in fertilization.