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.     

Sonication of mouse sperm membranes reveals distinct protein domains.

Molecular interactions between sperm and zona pellucida (ZP) during mammalian fertilization are not well characterized. To begin to characterize sperm components that are involved in sperm-ZP interactions, we isolated and density fractionated sperm membranes. The membrane fractions recovered from a density fractionation protocol were characterized, and sonication was compared with vortexing for preparation of sperm membranes by examining the distribution of proteins in the membrane fractions obtained from these 2 protocols. Biochemical and microscopic analyses were used to determine the composition of the sonicated membrane fractions, and immunoblotting was used to identify fractions containing some of the previously suggested ZP3 receptors. Transmission electron microscopy revealed that bands 1-3 contained membrane vesicles and band 4 contained axonemal and midpiece fragments. SDS-PAGE revealed that bands 1 and 2 shared many proteins, but band 3 contained a number of unique proteins. Surface labeling with 125I demonstrated that bands 1 and 2 contained the majority of the sperm surface protein markers, whereas band 3 contained minor amounts of surface markers. Lectin-binding characteristics of sperm membrane glycoproteins were used to compare the relative distribution of glycosylated proteins in vortexed or sonicated membrane preparations. These characterizations indicate that sonication enhanced the differential distribution of sperm membrane proteins among the density fractions and suggests that this method is preferable for preparation of membrane fractions to be used for identification of proteins that mediate sperm-egg interactions.     

Generation of a mouse sperm membrane fraction with zona receptor activity.

To identify mouse sperm components involved in primary sperm binding to zonae pellucidae, a mouse sperm plasma membrane-enriched fraction was generated using a vortex method. The crude membrane fraction recovered after vortexing was resolved into three bands and a pellet by centrifugation on a discontinuous sucrose gradient. Ultrastructural analysis indicated that Bands 2 and 3 were composed predominantly of membranes, although Band 3 was contaminated with mitochondria; Band 1 and the gradient pellet contained insufficient material and were unsuitable for ultrastructural analysis. To determine where plasma membranes migrate in the gradient, sperm were labeled vectorially with 125I; subsequently, membrane fractions were analyzed by SDS-PAGE and autoradiography. Band 2 was enriched threefold in radiolabel when compared with Band 3. Examination of intact and vortexed sperm stained with regionally distributed anti-mouse sperm monoclonal antibodies revealed that vortexing removed anterior head plasma membrane preferentially. Bioactivity, defined as the ability to inhibit primary sperm binding to the zona pellucida in a concentration-dependent manner, was contained in the crude membrane fraction and Band 2 exclusively, with inhibition of 53% and 44%, respectively, at the maximum concentration tested. Band 3 exhibited no significant bioactivity. We conclude from these results that a plasma membrane-enriched fraction, Band 2, isolated from mouse cauda epididymal sperm, exhibits zona pellucida receptor activity.     

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.     

Defining oligosaccharide specificity for initial sperm-zona pellucida adhesion in the mouse

The identity of the sperm surface protein(s) responsible for sperm-zona pellucida binding in the mouse, as well as the characteristics of the oligosaccharide groups on zona pellucida glycoprotein 3 (ZP3) having ligand activity toward this receptor, remain controversial. Conflicting results from several groups have made interpretation of the current data difficult. By developing a quantitative binding assay to evaluate the molecular interactions between mammalian sperm and the zona pellucida during initial gamete interactions, we directly quantified sperm-ZP binding interactions at the molecular level for the first time. The ZP binding assay demonstrated that live, capacitated mouse sperm bind solubilized ¹²⁵I-labeled ZP glycoproteins in a concentration-dependent manner characterized by a rapid forward rate constant of 3.0 × 10⁷ M⁻¹ min⁻¹. Following the initial characterization, the binding assay was used to examine the roles of the sperm surface enzymes galactosyltransferase (GalTase) and fucosyltransferase (FucTase) in sperm-zone pellucida binding in the mouse. These data indicate that substrates for FucTase, but not for GalTase, inhibit sperm-ZP binding, in contrast to earlier reports in which GalTase substrates significantly inhibited sperm binding to intact ZPs. A model is presented which resolves conflicting results between assays using intact ZPs and the results obtained here using soluble ¹²⁵I-ZPs. Assuming a complex binding/recognition site, monosaccharides that could occupy part of the binding site would have a dramatic effect on sperm-ZP binding to the intact ZP, since they need only occupy the binding sites for a short time (∼ 100 msec) to disrupt binding. The current results suggest that the sperm ZP3 receptor binding site minimally recognizes the galβ1,3GlcNAc moiety also recognized by FucTases. The current data do not exclude the possibility that additional sugar residues form part of the ligand oligosaccharide group and are recognized by a yet-to-be-identified sperm surface protein which serves as the ZP3 receptor. © 1996 Wiley-Liss, Inc.     

Preparation of a plasma membrane-rich fraction from rat spermatozoa

Monoclonal antibodies to antigens located on surface and intracellular membranes of spermatozoa from the rat cauda epididymidis have been used as probes to assess the purity of putative plasma membrane fractions. Spermatozoa were demembranated by shearing forces generated on a vortex-mixer. Immunofluorescence and ultrastructural analysis of vortex-mixed spermatozoa showed that they were denuded of approximately 90% of surface membrane. Areas of acrosomal membranes were also removed. Crude plasma membranes were recovered in low-speed wash fluids and fractionated on a 13-23% Nycodenz density gradient. Three bands containing membrane vesicles were resolved. Absorption curves and direct binding assays using monoclonal antibodies specific for acrosomal membranes, mitochondrial membranes and fibrous sheath showed relatively strong binding to bands 1 and 2 but weak binding to band 3. Conversely a monoclonal antibody specific for a surface membrane antigen bound strongly to band 3 and weakly to bands 1 and 2. Identification on immunoblots of the antigens recognized by the monoclonal antibodies revealed that band 3 was positive for surface membrane antigens but gave no reaction for intracellular antigens. However, bands 1 and 2 were strongly positive for intracellular components. The results suggest that vortex-mixing is a simple and efficient means of removing the plasma membrane from spermatozoa and that a membrane fraction can be recovered from a Nycodenz density gradient that is enriched 40- to 50-fold in surface antigens.     

Interaction of sperm with purified native chicken ZP1 and ZPC proteins

The avian perivitelline membrane (PVM) is the site of initial contact between sperm and egg. It consists of only two major components, which are both homologues of the mammalian zona pellucida (ZP) proteins, and belong to the ZP1 and ZPC families, respectively. We have established a method to isolate large quantities of both native avian ZP proteins and have used these preparations to investigate their sperm-binding capacities. Chicken ZPC forms multimeric structures of defined size and binds to an approximately 180-kDa protein complex present in rooster sperm extracts. Based on experiments using both PVM and isolated proteins, we show that chicken ZP1 is proteolytically degraded by a sperm-associated protease but that chicken ZPC remains intact. An antiserum directed against chicken ZP1 is capable of inhibiting sperm binding to the PVM. Taken together, these data suggest that ZP1, in addition to ZPC, plays a major role in the initial interactions between sperm and egg.     

Evidence for the presence of high-mannose/hybrid oligosaccharide chain(s) on the mouse ZP2 and ZP3.

Previous studies from this laboratory have identified a novel alpha-D-mannosidase on plasma membranes of rat, mouse, hamster, and human spermatozoa [Tulsiani et al. J Cell Biol 1989; 109:1257; Biol Reprod 1990; 42:843]. Inhibition of the mouse sperm surface alpha-D-mannosidase inhibits sperm-egg binding in vitro, suggesting that the sperm enzyme may have a receptor-like role in binding to the complementary molecules (presumably mannose-containing oligosaccharide [OS] chains) on the mouse zona pellucida (ZP) glycoconjugates [Cornwall et al. Biol Reprod 1991; 44:913]. In the studies reported here, we demonstrate the presence of high-mannose/hybrid-type OS on mouse zona components. Zona-intact eggs, prepared from superovulated mice, were radioiodinated, and the individual zona components (ZP1, ZP2, and ZP3) were isolated by electrophoresis followed by electroelution. The purified ZP components, when resolved by immobilized concanavalin A column chromatography, showed the following results: 1) Nearly all of the ZP1 applied to the immobilized lectin eluted in the column flow-through (effluent) fractions, and no radioactivity eluted with alpha-methyl mannoside, suggesting that ZP1 may not contain high-mannose/hybrid OS. 2) A significant amount of both ZP2 and ZP3 bound to the immobilized lectin, and nearly 16% and 8% of the two components, respectively, were repeatedly eluted with alpha-methyl mannoside.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of a Gi protein in digitonin/cholate-solubilized membrane preparations of mouse sperm by the Zona pellucida,an egg-specific extracellular matrix

Mammalian sperm possess guanine nucleotide-binding regulatory proteins (G proteins) that are involved in signal transduction pathways leading to zona pellucida (ZP)-mediated acrosomal exocytosis. We have previously examined ZP-G protein dynamics in mouse sperm homogenates, as well as cell-free membrane preparations, and our data support the existence of ZP receptor-G protein complexes in sperm membranes. However, the composition of this complex has not been identified due to experimental limitations of the membrane preparations. In the present study, a detergent-solubilized preparation from mouse sperm membranes that retained the signaling properties of cell homogenates and cell-free membrane preparations was developed using buffers containing digitonin and cholate. GTPγS, a poorly hydrolyzable analogue of GTP, bound to these solubilized preparations in a specific and concentration-dependent fashion that reached saturation at 100 nM. Incubation of this solubilized membrane preparation with heat-solubilized ZP resulted in an increase in specific GTPγS binding in a concentration-dependent manner, with a maximal response at 4-6 ZP/μl. Mastoparan (50 μM) increased GTPγS binding to levels similar to that seen with solubilized ZP. Mastoparan plus ZP stimulated GTPγS binding to the same extent as mastoparan or ZP alone. Pertussis toxin completely inhibited ZP-stimulated GTPγS binding and decreased mastoparan-stimulated GTPγS binding by 50–60%. Purified ZP3, the ZP component that possesses quantitatively all of the sperm binding and acrosomal exocytosis-inducing activities of the intact ZP, stimulated GTPγS binding to an extent similar to that of solubilized ZP. The properties of this solubilized membrane preparation are similar to those found in the cell homogenates and cell-free membrane preparations, suggesting that the components involved in ZP3-mediated signal transduction are effectively solubilized and are responsive to the ZP3 ligand. © 1995 Wiley-Liss, Inc.     

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.     

Involvement of fodrin-binding proteins in the structure of the neuronal postsynaptic density and regulation by phosphorylation

Novel polypeptides with Mr values about 140,000 bind fodrin and spectrin and are enriched in the postsynaptic density (PSD) compared to other tissues or subcellular fractions. 125I-fodrin binding to these polypeptides is competed for by unlabeled spectrin. These polypeptides are distinct from ankyrin and its proteolytic fragments and from band 4.1 which also bind fodrin. Phosphorylation of PSDs by the endogenous calmodulin-dependent protein kinase markedly reduces 125I-fodrin binding to the transblotted preparation. Such an event may play a regulatory role in governing protein-protein interactions among elements of the PSD.     

Reassessing the molecular biology of sperm-egg recognition with mouse genetics

The zona pellucida is an extracellular coat that surrounds mammalian eggs and early embryos. This insoluble matrix separates germ from somatic cells during folliculogenesis and plays critical roles during fertilization and early development. The mouse and human zona pellucida contain three glycoproteins (ZP1 or ZPB, ZP2, ZP3), the primary structures of which have been deduced by molecular cloning. Targeted mutagenesis of endogenous mouse genes and transgenesis with human homologues provide models to investigate the roles of individual zona components. Collectively, the genetic data indicate that no single mouse zona pellucida protein is obligatory for taxon-specific sperm binding and that two human proteins are not sufficient to support human sperm binding. An observed post-fertilization persistence of mouse sperm binding to "humanized" zona pellucida correlates with uncleaved ZP2. These observations are consistent with a model for sperm binding in which the supramolecular structure of the zona pellucida necessary for sperm binding is modulated by the cleavage status of ZP2.     

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.     

Activation of a Gi protein in mouse sperm membranes by solubilized proteins of the zona pellucida, the egg's extracellular matrix.

Zona pellucida (ZP)-induced acrosomal exocytosis in mammalian spermatozoa is thought to be mediated by signal transduction cascades similar to those found in hormonally responsive cells. In order to characterize this process further, we have examined the role of GTP-binding regulatory proteins (G proteins) in coupling sperm-ZP interaction to intracellular second messenger systems in mouse sperm. An in vitro signal transduction assay was developed to assess ZP-G protein dynamics in sperm membrane preparations. Guanosine 5'-3-O-(thio)triphosphate (GTP gamma S), a poorly hydrolyzable analogue of GTP, bound to these membranes in a specific and concentration-dependent fashion which reached saturation at 100 nM. Incubation of the membrane preparations with heat-solubilized ZP resulted in a significant increase in specific GTP gamma S binding in a concentration-dependent fashion with a half-maximal response at 1.25-2 ZP/microliters. Solubilized ZP also caused a significant increase in high affinity GTPase activity in the membranes over basal levels. Mastoparan increased specific GTP gamma S binding to the sperm membranes and stimulated high-affinity membrane GTPase activity to levels consistently greater than that seen with the solubilized ZP. Mastoparan, together with solubilized ZP, gave the same level of stimulation of GTP gamma S binding as mastoparan alone. Pertussis toxin completely inhibited the ZP-stimulated GTP gamma S binding, but only decreased mastoparan-stimulated GTP gamma S binding by 70-80%. Purified ZP3, the ZP component which possesses quantitatively all of the acrosomal exocytosis-inducing activity of the intact ZP, stimulated GTP gamma S binding to the same level as solubilized ZP; ZP1 and ZP2 did not stimulate GTP gamma S binding. ZP from fertilized eggs (ZPf), which does not possess acrosome reaction-inducing activity, also failed to stimulate GTP gamma S binding to sperm membranes. These data demonstrate the direct activation of a Gi protein in sperm membrane preparations in response to the ZP glycoprotein, ZP3, that induces the acrosome reaction. These data imply that Gi protein activation is an early event in the signal sequence leading to sperm acrosomal exocytosis.     

Localization and functional importance of a conserved zona pellucida 2 protein domain in the human and bovine ovary using monoclonal anti-ZP2 peptide antibodies

In mammals, gamete recognition and sperm binding to the oocyte are mediated by the zona pellucida (ZP), an acellular coat surrounding the plasma membrane of the oocyte that consists of particular ZP proteins. The ZP2 protein mediates secondary sperm binding to the ZP. Its primary structures are highly conserved as revealed by cDNA cloning. In the present study, we investigated the localization of ZP2 in human and bovine ovaries and oocytes and the influence of monoclonal anti-ZP2 peptide antibodies upon bovine sperm-egg interactions. We generated a monoclonal anti-ZP2 synthetic peptide antibody, mAb ZP2-20, against a sequence that is strongly conserved in the mammalian ZP2 amino acid sequence. Specificity of mAb ZP2-20 was determined by ELISA and immunoblotting, respectively. Our results show that mAb ZP2-20 specifically detected the peptide used as an antigen and reacted with its corresponding protein antigen in human and bovine ovaries. In order to elucidate effects of mAb ZP2-20 upon bovine sperm-ZP binding, we used the competitive hemizona assay (cHZA) and found that the antibodies clearly inhibit sperm binding to the ZP. We conclude that (i). monoclonal antibodies against ZP2 peptides react with ZP proteins present in bovine and human ovaries and can be used as a specific marker for ZP2; and that (ii). mAb ZP2-20 detects a ZP2 epitope that is of functional relevance for sperm-ZP interactions.     

Isolation of antiSLIP1-reactive boar sperm P68/62 and its binding to mammalian zona pellucida

Single-step purification of boar sperm P68/62 that is cross-reactive with a polyclonal antibody against sulfolipidimmobilizing protein 1 (SLIP1) was achieved by chromatofocusing. This method is useful for obtaining P68/62 in quantity. The two proteins, P68 and P62, were antigenically related, since the antibody generated specifically against the 68-kDa band reacted with both the 68- and 62-kDa bands. Like rat testis SLIP1, purified boar sperm P68/62 bound to sulfogalactosylglycerolipid (SGG) and inhibited sperm-egg binding in a dose-dependent manner when added exogenously to sperm-egg coincubates. This inhibitory effect occurred at the level of the zona pellucida (ZP), and further studies showed that biotinylated boar sperm P68/62 bound to the ZP of unfertilized mouse eggs. Furthermore, biotinylated boar sperm P68/62 bound to isolated ZP of unfertilized eggs from other species, including pig, rat, cat, dog, and human, as well as to ZP of intact fertilized mouse eggs and preimplantation embryos of various developmental stages, although the degree of its binding to the ZP of intact eight-cell embryos, morulae, and blastocysts was much lower than that of fertilized eggs and two-cell embryos. These results suggest that P68/62 of capacitated sperm must act together with other sperm surface proteins/molecules that regulate zona binding specificity within homologous species and in unfertilized eggs. Together with our previous findings, we suggest that rather than being a true ZP receptor, sperm P68/62 may be involved in the initial step of sperm-ZP binding that is adhesive in nature. Mol. Reprod. Dev. 49:203–216, 1998 © 1998 Wiley-Liss, Inc.     

Four universal forms of chlorophyll a

We have matched the red absorption band measured at −196 C in a variety of chloroplast preparations with four major component curves representing forms of chlorophyll a having peaks at 661.6, 669.6, 677.1, and 683.7 nanometers. Chloroplast fractions enriched in one or the other of the two photochemical systems both contain these four major components, but system 1 preparations contain relatively more chlorophyll a 684. Chlorophyll a 677 and chlorophyll a 684 have greater bandwidths in system 1. Bands at longer wavelengths near 693 and 704 nanometers also often occur, but with far smaller heights than the above major bands. The longer wavelength bands are more common in system 1 than in system 2. In system 1 the half-widths of the four major bands in typical spectra average 11.3, 10.0, 10.3, and 10.8 nanometers while in system 2 they are 11.6, 9.8, 9.4, and 9.6 nanometers. Some spectra with sharper and some with wider bands were found, but the wavelengths were identical.     

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.     

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.     

Sperm surface hyaluronan binding protein (HABP1) interacts with zona pellucida of water buffalo (Bubalus bubalis) through its clustered mannose residues

Sperm-oocyte interaction during fertilization is multiphasic, with multicomponent events, taking place between zona pellucida (ZP) glycoproteins and sperm surface receptor. d-mannosylated glycoproteins, the major constituents of ZP are considered to serve as ligands for sperm binding. The presence of hyaluronan binding protein 1 (HABP1) on sperm surface of different mammals including cattle and its possible involvement in sperm function is already reported. Recently, we have demonstrated the specificity of clustered mannose as another ligand for HABP1 (Kumar et al., 2001: J Biosci 26:325-332). Here, we report that only N-linked mannosylated zona-glycoproteins bind to sperm surface HABP1. Labeled HABP1 interacts with ZP of intact oocyte of Bubalus bubalis, which can be competed with unlabeled HABP1 or excess d-mannosylated albumin (DMA). This data suggests the specific interaction of HABP1 with ZP, through clustered mannose residues. In order to examine the physiological significance of such an interaction, the capacity of sperm binding to oocytes under in vitro fertilization plates was examined either in presence of DMA alone or in combination with HABP1. The number of sperms, bound to oocytes was observed to reduce significantly in presence of DMA, which could be reversed by the addition of purified recombinant HABP1 (rHABP1) in the same plate. This suggests that sperm surface HABP1 may act as mannose binding sites for zona recognition.