Involvement of an acrosin-like enzyme in the acrosome reaction of sea urchin sperm

Extracts of the jelly coat of eggs of several marine invertebrates are known to induce in homologous sperm morphological changes known as the acrosome reaction. When sperm of the sea urchin Strongylocentrotus purpuratus are treated with low concentrations (0.2 μg fucose/ml) of egg jelly coat or 30 mM CaCl₂ in artificial seawater the acrosome reaction does not occur. However, either of these treatments causes the exposure of an acrosin-like enzyme to exogenous substrate and inhibitors. Subsequent addition of jelly coat to 3.7 μg fucose/ml to sperm in this “initial stage” induces the acrosome reaction (as judged by the appearance of an acrosomal filament). This concentration is also effective for untreated sperm. If inhibitors of the enzyme (diisopropylphosphofluoridate or phenylmethanesulfonyl fluoride) are added to sperm in the initial stage, no acrosomal filaments are observed when the high concentration of jelly coat is added. Whether other morphological changes occur in these sperm has not been examined. If phenylmethanesulfonyl fluoride is added 4 sec after the jelly coat, the acrosomal filaments are observed, but the sperm still fail to fertilize eggs. These results suggest a dual role for the acrosin-like enzyme(s), first in the mechanism of the acrosomal filament formation and then in a subsequent event in the fertilization process.     

Human teratoma cells share antigens with mouse teratoma cells.

Cytochalasin B inhibits the penetration of sperm nuclei into Urechis eggs without inhibiting sperm-induced egg activation. The acrosome reaction appears normal, and plasma membranes of the acrosomal tubule and egg become closely apposed. It is uncertain whether or not the drug blocks fusion of these membranes; however, sperm penetration cone formation is inhibited.     

Antibody to a sperm surface glycoprotein inhibits the egg jelly-induced acrosome reaction of sea urchin sperm

When the surface of sea urchin (Strongylocentrotus purpuratus) sperm is radioiodinated, 75% of the protein-incorporated radioactivity is associated with two glycoproteins of M_r 84,000 (84K) 64,000 (64K) (Lopo and Vacquier 1980). Antibodies were prepared against these two components by separating a Triton X-100 extract of sperm on SDS-polyacrylamide gels, cutting out the band containing the glycoprotein and injecting the homogenized gel into rabbits. Both anti-84K and anti-64K sera agglutinate sperm. Light and EM immunoperoxidase localization show both antigens are distributed over the entire sperm surface. By the immunoperoxidase technique there is some degree of cross-reactivity of both antisera with sperm of other Strongylocentrotus species, but not with those of other genera. Living sperm incubated with anti-84K Fab fragments are completely inhibited from undergoing the egg jelly-induced acrosome reaction and fertilizing eggs. Anti-64K Fab fragments have no effect on the ability of the sperm to undergo the acrosome reaction or fertilize eggs. Sperm incubated in anti-84K or anti-64K Fab fragments undergo the acrosome reaction in response to the Ca²⁺ ionophore A23187, or when the extracellular pH is increased to 9.2 with NH₄OH, indicating that the inhibition of the egg jelly-induced acrosome reaction results from the binding of the anti-84K Fab to an external molecule involved in the initiation or propagation of the acrosome reaction. The 84K glycoprotein is the first sperm surface component identified that might have a role in the induction of the acrosome reaction.     

A reevaluation of the fertilizin hypothesis of sperm agglutination and the description of a novel form of sperm adhesion

The classical isoagglutination of sea urchin sperm by egg jelly is not an agglutination of cells, as proposed by the fertilizin-antifertilizin hypothesis. Sperm motility is required to obtain the isoagglutination of Strongylocentrotus purpuratus sperm, and the sperm do not adhere to each other in the isoagglutination clusters, which cannot be fixed for microscopy and which disperse rapidly into individual cells when sperm motility is inhibited. These observations suggest that isoagglutination is the swarming of freely moving sperm to a common focus and is quite distinct from the agglutination of sperm by known crosslinking agents (antibodies or lectins).A previously unrecognized form of sperm agglutination is described which follows induction of an acrosome reaction by egg jelly, ammonia, or the ionophore A23187 in a suspension of sea urchin or sand dollar sperm. The sperm form rosettes of up to 100 cells in which the newly extended acrosomal processes adhere to each other. Rosettes can form containing sperm of different species, in which the acrosomal processes adhere without species preference.As observed by transmission electron microscopy, the acrosomal process of Lytechinus pictus sperm consists of an acrosomal tubule covered by a sheath of extracellular material. Rosette formation results from attachment between the extracellular materials of adjacent sperm.Less frequently, the acrosomal process of one sperm adheres to the midpiece of another by fusion of the acrosomal tubule and midpiece plasma membranes.     

A study of factors involved in induction of the acrosomal reaction in sperm of the sea urchin, Arbacia punctulata.

Several factors involved in induction of the acrosomal reaction in sperm of the sea urchin, Arbacia punctulata, have been investigated quantitatively using a simple substrate film technique to monitor extension of the acrosomal process by electron microscopy. Verification of typical acrosomal process formation has been accomplished using thin sections. Sperm were found to undergo the acrosomal reaction in artificial sea water in the absence of egg jelly coat at pH values above 9.6. In the presence of egg jelly a high percentage of sperm react at pH 8.6. At this pH, the fraction of sperm that undergo the acrosomal reaction is directly proportional to the concentration of egg jelly. The Ca²⁺ ionophore A23187 induces the acrosomal reaction in the absence of egg jelly at pH 8.6. The proportion of sperm that react is dependent on the concentration of ionophore and on the concentration of Ca²⁺ in the medium. Pretreatment of sperm with low levels of La³⁺ ion, which is known to be a Ca²⁺ ion antagonist, results in inhibition of egg jelly induction of the acrosomal reaction. These findings suggest that there are marked similarities between the acrosomal reaction in sea urchin sperm and membrane fusion dependent secretory processes in other cell types.     

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 G_i, 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 G_i proteins, bind to the ZP of mouse eggs but are inhibited from undergoing acrosomal exocytosis. We have measured high-affinity GTPase activity and GTPγ[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 G_i protein. An increase in GTP hydrolysis (～50% over basal activity) and GTPγ[³⁵S] binding (～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 M_r = 41,000 sperm G_i protein, suggesting that the increase in GTPase activity and GTPγ[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.     

Studies on the interactions of sperm with the surface of the sea urchin egg

We have examined the relationship between sperm adhesion and fertilization in the cross species insemination of Arbacia punctulata eggs by Strongylocentrotus purpuratus sperm. As previously reported (Kinsey et al., 1980) the addition of S. purpuratus egg jelly results in induction of the acrosome reaction in sperm and significant numbers of S. purpuratus sperm adhere to A. punctulata eggs. However, in the absence of S. purpuratus egg jelly, S. purpuratus sperm fail to bind to A. punctulata eggs. Although at least 200 S. purpuratus sperm bind to an A. punctulata egg in the presence of S. purpuratus jelly, less than 8% of the eggs are fertilized. The adhesion of S. purpuratus sperm meets the same functional criteria as homologous A. punctulata sperm-egg adhesion. Electron microscopy shows that S. purpuratus sperm that have undergone the acrosome reaction adhere to A. punctulata eggs by their bindin-coated acrosomal process in a manner that is morphologically identical to that observed with homologous A. punctulata sperm. We have also compared the ability of S. purpuratus and A. punctulata sperm to fuse and fertilize with A. punctulata eggs after removal of the vitelline layer. Using high levels of sperm of either species, heterologous as well as homologous fertilization is readily detectable. Under these conditions, where stable binding is not demonstrable, there is no difference in the ability of S. purpuratus and A. punctulata sperm to fertilize A. punctulata eggs. These observations suggest that the failure of S. purpuratus sperm to fertilize A. punctulata eggs under normal conditions may be due to their inability to penetrate the vitelline layer so that they can fuse with the egg plasma membrane. In relation to the possible mechanism of vitelline layer penetration, we have also investigated the mode of action of chymostatin, an inhibitor of chymotrypsin that has been reported to inhibit fertilization of sea urchin eggs (Hoshi et al., 1979). Our findings suggest that the fertilization inhibitory activity of chymostatin is not related to its antichymotrypsin activity. Rather, it appears that this inhibition is due to the induction of an abnormal acrosome reaction in sperm that precludes formation of the acrosome process.     

Voltage clamp studies of fertilization in sea urchin eggs: I. Effect of clamped membrane potential on sperm entry,activation, and development

To analyze the role of the activation potential (a positive shift of the membrane potential which occurs following sperm attachment) in fertilization and development of the sea urchin egg, unfertilized Lytechinus variegatus eggs were voltage clamped at membrane potentials (E_m) from +20 to ?90 mV, and then inseminated. Either a fast two electrode voltage clamp, or a single electrode switched voltage clamp was used. The clamp was maintained for 3 to 15 min after initiation of a conductance increase. At E_m more positive than +18 mV, even though many sperm may attach, the egg remains completely inert (Jaffe, Nature (London)261, 68–71, 1976). At E_m from +17 to ?90 mV, all inseminated eggs elevate normal fertilization envelopes, although substantially increased concentrations of sperm are required at E_m from +17 to +12 mV. Whether cleavage occurs depends on the clamped E_m. When clamped at E_m from +17 to ?25 mV, 100% of activated eggs cleave. However, when clamped at E_m from ?26 to ?75 mV the percentage of activated eggs which cleave progressively decreases. At clamped E_m between ?76 and ?90 mV, none of the activated eggs cleave. All monospermic voltage clamped eggs that cleave develop to normal swimming blastulae. In all eggs that fail to cleave (clamped at E_m more negative than ?30 mV), sperm penetration is blocked, the sperm is lifted off the egg surface as the fertilization envelope rises, and a sperm aster never forms. Preventing formation of the fertilization envelope by prior disruption of the vitelline layer with dithiothreitol does not promote entry of the sperm. In conclusion, preventing the depolarization normally associated with fertilization suppresses sperm entry in the sea urchin egg, yet activation proceeds. Present evidence suggests an effect of the electrical field across the plasma membrane in suppressing sperm entry.     

Microtubule proteins in axolotl eggs and developing embryos

Tubulin from eggs and embryos of the Mexican axolotl was characterized by electrophoresis and colchicine binding. In urea-polyacrylamide gel electrophoresis, soluble axolotl egg tubulin migrated as two bands, identical to tubulins from sea urchin sperm and Drosophila eggs. However, in SDS-containing gels, on which the α and β subunits of standard tubulins were well resolved, axolotl egg tubulin migrated as a single band with an apparent molecular weight of 53,500. The method of disruption of the eggs affected both yield of tubulin from vinblastine sulfate precipitates and stability of the colchicine binding activity. The colchicine binding activity of soluble tubulin from gently disrupted eggs was specific and of high affinity, with properties similar to those reported for other tubulins. The tubulin pool in unfertilized eggs was determined to be approximately 2 μg/egg; the level decreased 20% after initiation of cleavage and then remained constant through development to postneurula stages. The colchicine binding activity of soluble tubulin from embryos was much less stable than that of unfertilized eggs and decreased further during development. No differences were found in properties of tubulin from eggs of several strains of normally pigmented axolotls; however, tubulin from albino eggs showed slightly different properties in both electrophoresis and colchicine binding. The colchicine binding activity of soluble tubulin accounts for only half the total activity in axolotl eggs; they possess, in addition, a particulate nontubulin colchicine binding activity.     

Phosphorylation state of protamines 1 and 2 in human spermatids and spermatozoa

The basic nuclear proteins of a fraction of elongating spermatids from human tests and of a fraction of motile spermatozoa from the ejaculate, separated by ion-exchange chromatography, were compared. Analysis by acetic acid-urea polyacrylamide gel electrophoresis (PAGE) showed that, in both fractions, four proteins of lower mobility were coeluted with protamine 1 by 23% guanidinium chloride (GuCI) while protamine 2 alone was eluted by 50% GuCI. Treatment with alkaline phosphatase identified those four proteins as phosphorylated protamines, and cyanogen bromide (CNBr) treatment of the dephosphorylated protamines distinguished them as variants of protamine 2 and not of protamine 1. Thus far, phosphorylated forms of protamine 1 have not been detected in either spermatids or spermatozoa. Those observations indicate that protamine 2 functions in the cycle of phosphorylation-dephosphorylation, which is essential to the process of sperm chromatin condensation, while the role of protamine 1 in human spermiogenesis is not yet defined. The presence of phosphorylated protamine in motile, presumably mature spermatozoa appears to be characteristic of human sperm but not of the sperm of other mammals and is probably the basis for the heterogeneity of chromatin condensation frequently observed in human spermatozoa.     

The molecular evolution of sperm bindin in six species of sea urchins (Echinoida: Strongylocentrotidae)

The acrosomal protein bindin attaches sperm to eggs during sea urchin fertilization. Complementary to ongoing functional biochemical studies, I take a comparative approach to explore the molecular evolution of bindin in a group of closely related free-spawning echinoid species. Two alleles of the mature bindin gene were sequenced for each of six species in the sea urchin family Strongylocentrotidae. The nucleotide sequences diverged by at least 1% per Myr at both silent and replacement sites. Two short sections flanking the conserved block show an excess of nonsynonymous substitutions. Each is homologous to a region that had been identified as a target of selection in other sea urchin comparisons. A large proportion of the bindin-coding sequence consists of a highly variable repeat region. Bindin sequences, even including the large intron, could not resolve the branching order among five of the species.      

Protamines in plant sperm

Sperm protamines have been isolated from representatives of three major plant groups: algae (Chara corallina ), bryophytes ( Marchantia polymorpha), and ferns ( Marsilea vestitia ). We previously reported the complete displacement of histones by protamines in Marchantia (Reynolds W F & Wolfe, S L, Exp cell res 116 (1978) 269 [8] ). Marchantia protamines appear as four components on acid-urea gels, whereas Chara and Marsilea protamines comigrate as a single band with a mobility comparable to salmon protamine. The amino acid compositions of the plant protamines show these to be arginine-rich, highly basic (35-42%) proteins which display overall similarity in amino acid composition (84-91%). The molecular weights of Chara and Marsilea protamines are approx. 4700-5300 D.     

Studies on the specificity of sperm binding in echinoderm fertilization

Using gametes from the sea urchins Arbacia punctulata and Strongylocentrotus purpuratus, we have evaluated the role of the acrosome reaction and the sperm-egg binding process in the block to interspecific fertilization among echinoids. The results indicate that sperm preinduced to undergo the acrosomal reaction by two different methods still bind to homologous eggs in a species specific manner. These results, taken in conjunction with an earlier study on species specificity of jelly coat induction of the acrosomal reaction (SeGall and Lennarz 1978), indicate that both the acrosome reaction and the sperm binding process contribute to the species specificity of fertilization in S. purpuratus and A. punctulata.     

Reactions of the sea urchin oocyte to foreign spermatozoa

Spermatozoa of the mussel Cyprina islandica and the nemertine Malacobdella grossa have been adddd to oocytes and mature eggs of the sea urchin Psammechinus miliaris. No spermatozoa were found to attach to the surface of the mature eggs which also remained unactivated throughout the experiments. Spermatozoa of both species were found to reach the oocyte surface and to become attached there. The interaction between egg and sperm was different in the two species and different from the situation of a sea urchin sperm on the sea urchin oocyte. The nemertine sperm was found to penetrate the cortex of the oocyte in a fashion resembling phagocytosis. The mussel sperm was partly surrounded by thin protrusions from the sea urchin oocyte which extended along a major portion of the sperm head.     

Radioiodination and characterization of the plasma membrane of sea urchin sperm

Two methods were used to radioiodinate sea urchin sperm: lactoperoxidase-glucose oxidase and Iodo-Gen. Following iodination the sperm are viable, they undergo the acrosome reaction, and they fertilize eggs. Of the radioactivity associated with the labeled sperm, 28–50% is presumed to be free ¹²⁵I^?, 37–47% is incorporated in lipid, and 8–15% is in trypsin-digestible material believed to be protein. Digestion of the labeled, living sperm with trypsin removes 95.6–99.5% of the macromolecular label (the cells are alive after digestion) suggesting that almost all the protein label is on the external surface of the cell. Thin-layer chromatography of the lipid fraction shows that the major membrane phospholipids and cholesterol are labeled. SDS-PAGE analysis shows the protein-incorporated ¹²⁵I is distributed among four glycoproteins of >250K, 84K, 64K, and 52K dalton apparent molecular weight. Twenty-eight percent of the total protein (trypsin-digestible) label is in the 84K component and 46% in the 64K band. Although both molecules contain much of the label, they are relatively minor components of the TX-100 extract of sperm. The methods outlined will be useful in determining the role of sperm surface components in fertilization.     

Involvement of an acrosinlike proteinase in the sulfhydryl-induced degradation of rabbit sperm nuclear protamine

Previous studies demonstrated that proteolytic activity is associated with isolated rabbit sperm nuclei and is responsible for the degradation of nuclear protamine that occurs during thiol-induced in vitro decondensation of the nuclei (Zirkin and Chang, 1977; Chang and Zirkin, 1978). In this study, we present the results of experiments designed to characterize this proteolytic activity. Basic protein isolated from rabbit sperm nuclei incubated with 5 mM dithiothreitol (DTT) and 1 percent Triton X-100 for increasing periods of time exhibited progressively faster migrating bands on acid-urea polyacrylamide gels, reflection the progressive degradation of protamine. Ultimately, a specific and characteristic peptide banding pattern resulted. When sperm nuclei were treated with the esterase inhibitor nitrophenyl-p-guanidino benzoate (NPGB) to inhibit the nuclear-associated proteolytic activity and then incubated with one of several exogenous proteinases in addition to DTT and Triton X-100, characteristic peptide banding patterns were seen for each exogenous proteinase employed. For trypsin, chymotrypsin, pronase, and papain, the peptide banding patterns differed from one another and from the pattern characteristic of protamine degradation by the nuclear-associated proteinase. By contrast, when rabbit acrosin served as the exogenous proteinase, the peptide banding pattern seen was identical to the pattern characteristic of the nuclear-associated proteinase. These results demonstrate directly that the proteinase associated with rabbit sperm nuclei and involved in sperm nuclear decondensation in vitro is acrosinlike.     

Certain Strongylocentrotus purpuratus sperm mitochondrial proteins co-purify with low density detergent-insoluble membranes and are PKA or PKC-substrates possibly involved in sperm motility regulation

Background

Sea urchin sperm motility is regulated by Speract, a sperm-activating peptide (SAP) secreted from the outer egg coat. Upon binding to its receptor in the sperm flagellum, Speract induces a series of ionic and metabolic changes in Strongylocentrotus purpuratus spermatozoa that regulate their motility. Among these events, protein phosphorylation is one of the most relevant and evidence indicates that some proteins of the Speract signaling cascade localize in low density detergent-insoluble membranes (LD-DIM).

Methods

LD-DIM-derived proteins from immotile, motile or Speract-stimulated S. purpuratus sperm were resolved in 2-D gels and the PKA and PKC substrates detected with specific antibodies were identified by LC–MS/MS.

Results

Differential PKA and PKC substrate phosphorylation levels among the LD-DIM isolated from sperm in different motility conditions were found and identified by mass spectrometry as: ATP synthase, creatine kinase, NADH dehydrogenase (ubiquinone) flavoprotein 2, succinyl-CoA ligase and the voltage-dependent anion channel 2 (VDAC2), which are mitochondrial proteins, as well as, the cAMP-dependent protein kinase type II regulatory (PKA RII) subunit, Tubulin β chain and Actin Cy I changed their phosphorylation state.

Conclusions

Some mitochondrial proteins regulated by PKA or PKC may influence sea urchin sperm motility.

General significance

The fact that a high percentage (66%) of the PKA or PKC substrates identified in LD-DIM are mitochondrial proteins suggests that the phosphorylation of these proteins modulates sea urchin sperm motility via Speract stimulation by providing sufficient energy to sperm physiology. Those mitochondrial proteins are indeed PKA- or PKC-substrates in the sea urchin spermatozoa.     

THE POLYMERIZATION OF ACTIN: ITS ROLE IN THE GENERATION OF THE ACROSOMAL PROCESS OF CERTAIN ECHINODERM SPERM

When Asterias or Thyone sperm come in contact with egg jelly, a long process which in Thyone measures up to 90 µm in length is formed from the acrosomal region. This process can be generated in less than 30 s. Within this process is a bundle of microfilaments. Water extracts prepared from acetone powders of Asterias sperm contain a protein which binds rabbit skeletal muscle myosin forming a complex whose viscosity is reduced by ATP. Within this extract is a protein with the same molecular weight as muscle actin. It can be purified either by collecting the pellet produced after the addition of Mg⁺⁺ or by reextracting an acetone powder of actomyosin prepared by the addition of highly purified muscle myosin to the extract. The sperm actin can be polymerized and by electron microscopy the polymer is indistinguishable from muscle F-actin. The sperm actin was shown to be localized in the microfilaments in the acrosomal processes by: (a) heavy meromyosin binding in situ, (b) sodium dodecyl sulfate (SDS) gel electrophoresis of the isolated acrosomal processes and a comparison to gels of flagella which contain no band corresponding to the molecular weight of actin, and (c) SDS gel electrophoresis of the extract from isolated acrosomal caps. Since the precursor for the microfilaments in the unreacted sperm appears amorphous, we suspected that the force for the generation of the acrosomal process is brought about by the polymerization of the sperm actin. This supposition was confirmed, for when unreacted sperm were lysed with the detergent Triton X-100 and the state of the actin in the sperm extract was analyzed by centrifugation, we determined that at least 80% of the actin in the unreacted sperm was in the monomeric state.     

MORPHOLOGY OF GAMETE MEMBRANE FUSION AND OF SPERM ENTRY INTO OOCYTES OF THE SEA URCHIN

Sea urchin gametes predominate in molecular studies of fertilization, yet relatively little is known of the subcellular aspects of sperm entry in this group. Accordingly, it seemed desirable to make a detailed examination of sperm entry phenomena in sea urchins with the electron microscope. Gametes of the sea urchins Arbacia punctulata and Lytechinus variegatus were used in this study. Samples of eggs containing 2 to 8 per cent oocytes were selected and fixed with osmium tetroxide in sea water at various intervals after insemination. Fixed specimens were embedded in Epon 812, sectioned, and examined with an electron microscope. An apical vesicle was observed at the anterior end of the acrosome. The presence of this structure, together with other observations, suggested that initiation of the acrosome reaction in sea urchin sperm involves dehiscence of the acrosomal region with the subsequent release of the acrosomal granule. Contact and initial fusion of gamete membranes was observed in mature eggs and oocytes and invariably involved the extended acrosomal tubule of the spermatozoon. Only one spermatozoon normally enters the mature egg. The probability of locating such a sperm in ultrathin sections is exceedingly low. Several sperm do normally enter oocytes. Consequently, observations of sperm entry were primarily restricted to the latter. The manner of sperm entry into oocytes did not resemble phagocytosis. Organelles of the spermatozoon were progressively divested of their plasma membrane as they entered the ground cytoplasm of the oocyte fertilization cone. Initiation of the acrosome reaction, contact and initial fusion of gamete membranes, and sperm entry into oocytes of sea urchins conform to the Hydroides-Saccoglossus pattern of early fertilization events as described by Colwin and Colwin (13).     

Identification,isolation, and properties of a plasma membrane protein involved in the adhesion of boar sperm to the porcine zona pellucida

Boar sperm plasma membranes contain an integral protein (Mr 55 kDa) that apparently functions in the adhesion of sperm to the zona pellucida (Peterson and Hunt: J Cell Biol 105:170a, 1987.) In experiments described in this report, the protein is identified after additional steps of purification involving lectin affinity chromatography and preparative PAGE. An active form of the adhesion protein (AP_z) develops or becomes first exposed in the corpus epididymis and is fully active in the cauda epididymis; a significant portion of this conformationally labile protein, while integral to the plasma membrane, cannot be solubilized by nonionic detergents and may be associated with the membrane skeleton. AP_z does not exhibit enzymatic properties thought possibly to be involved in sperm-zona interaction in this and other species. Galactosyltransferase substrates and inhibitors and anliproteases including soybean trypsin inhibitor, pepstatin, leupeptin, and p-aminoben-zamidine failed to block sperm from binding to porcine eggs. Boar sperm proacrosin and antiproacrosin antibody failed to inhibit sperm-egg binding. When plasma membranes or fractions containing AP_z that bind to dextran sulfate agarose were chromatographed on L-fucose agarose, a sugar which binds proacrosin, plasma membrane proteins that bound to the column failed to absorb anti-AP_z antibody, Anti-AP_z was absorbed by fractions that did not contain proacrosin. These data indicate that AP_z is not proacrosin. Since anti-AP_z monovalcnt antibody raised from whole cauda or corpus sperm plasma membranes or from chromatographic fractions containing AP_z completely block capacitated sperm from binding to eggs, and since the ability of this antibody to be absorbed develops as sperm become capable of binding to eggs, we view AP, to be the major and perhaps only plasma membrane protein involved in the adhesion of capacitated boar sperm to eggs prior to the acrosome reaction.