The complete primary structure of the spermadhesin AWN, a zona pellucida-binding protein isolated from boar spermatozoa

AWN is a boar protein which originates in secretions of the male accessory glands and which becomes sperm surface-associated upon ejaculation. It is one of the components thought to mediate sperm adhesion to the egg's zona pellucida through a carbohydrate-recognition mechanism. AWN may, thus, participate in the initial events of fertilization in the pig. In this report we describe its complete primary structure by combination of protein-chemical and mass spectrometric methods. AWN exists as two isoforms, AWN-1 and AWN-2, which differ in that AWN-2 is N-terminally acetylated. The amino acid sequence of AWN contains 133 amino acid residues and two disulphide bridges between nearest-neighbour cysteine residues. Analysis of the amino acid sequence of the AWN proteins showed significant similarity only to AQN-1 and AQN-3, two other boar spermadhesins.     

Features of a seminal proteinase inhibitor- zona pellucida-binding component on murine spermatozoa

Murine cauda epididymal sperm contain sites on the plasma membrane over the apical portion of the acrosome that recognize proteinase inhibitors and the homologous zona pellucida. Ten times more of the component can be extracted from cauda and ductus sperm than from equal numbers of caput and corpus sperm. Likewise, few sperm from the upper epididymal regions are able to bind seminal inhibitor, while the majority of sperm from the cauda and ductus do bind. Cauda epididymal and ductus sperm lose little of their ability to bind inhibitor after a 4-hour in vitro incubation in either a capacitating or a noncapacitating medium. The percentage of naturally inseminated sperm with the seminal inhibitor bound to their surface decreases to about 10 after 4 hours in utero. Approximately 80% of these sperm show positive fluorescence when given the opportunity to rebind the inhibitor, and these sperm do have an intact plasma membrane over the apical portion of the acrosome. Furthermore, after 4 hours in utero, the inhibitor bound in the same region of the sperm head as it did on freshly ejaculated sperm. The seminal inhibitor inhibits the binding of sperm to the zona if added during the first 15 minutes of incubation but has no effect on attachment. The data indicate that sperm gain the ability to bind the seminal inhibitor during the epididymal sojourn. Furthermore, this binding capacity is not lost during in vitro or in utero incubation. The site is not involved in sperm-zona attachment but does participate in the binding of sperm to the zona.     

Isolation, characterization, and localization of the zona pellucida binding proteins of boar sperm

In order to isolate the putative zona pellucida-binding proteins (ZPBPs) from boar spermatozoa, a new, simple method has been developed. The new isolation strategy made the most of the highly specific interactions between the components of the gametes. Detergent-extracted boar sperm proteins were submitted to affinity chromatography on a ZP-Sepharose column. SDS-PAGE analyses of the retained fraction under reducing conditions revealed that in addition to a component of Mr 38,000, the predominant ZPBPs contained at least three low molecular weight proteins (Mr less than 20 kDa). The isolated ZPBPs were effective in blocking sperm-oocyte binding in vitro. Using immunofluorescence microscopy, the ZPBPs were shown to be localized primarily in the sperm head, especially in the acrosomal cap region.     

Characterization of low Mr zona pellucida binding proteins from boar spermatozoa and seminal plasma.

A group of low Mr (16 kDa-23 kDa) glycoproteins on ejaculated boar spermatozoa have been shown to have high affinity for homologous zona pellucida glycoproteins (ZPGPs). These ZPGP binding proteins are derived from seminal plasma as shown by their absence from epididymal spermatozoa and their presence in seminal plasma as identified by N-terminal amino acid sequence analysis. They bind to ZPGPs by a polysulphate recognition mechanism similar to that found for proacrosin-ZPGP interactions. The haemagglutination activity of boar seminal plasma is also associated with these low Mr glycoproteins. It is suggested that they play a role in regulating the rate of sperm capacitation and survival in the female reproductive tract.     

Isolation and characterization of a ganglioside containing fucose from boar testis.

A ganglioside containing fucose (fucoganglioside) was obtained from boar testis and purified by silicic acid and DEAE-cellulose column chromatographies and preparative thin layer chromatography. The structure of this ganglioside, determined by chemical and enzymatic methods was: (see article). Its fatty acids were mainly long chain saturated ones (20 : 0, 22 : 0, 24 : 0). Its long chain bases consisted of 27% C(16:1) sphingosine and 68% C(18:1) sphingosine.     

Identification of 17-18 kDa zona pellucida binding proteins from boar spermatozoa

Zona pellucida (ZP) binding proteins from boar spermatozoa were compared with antigens recognized by ACR.2 and ACR.3 monoclonal antibodies. The ZP binding proteins of 55, 53, 45 and 38 kDa are identical with various forms of boar acrosin immunologically detected by ACR.2 antibody. The ZP binding proteins of 17 and 18 kDa are recognized by ACR.3 antibody. The N-terminal amino acid sequence of the 17 kDa protein revealed that it is not derived from an acrosin molecule.     

Tyrosine protein kinase in boar spermatozoa: identification and partial characterization.

A protein-tyrosine kinase has been isolated from a detergent-soluble extract of boar spermatozoa, using poly(Glu, Tyr)4:1 as a substrate. The purification procedure involves sequential column chromatographies on phosphocellulose, polyamino acid affinity and Sephadex G-100 molecular sieving, and results in more than a 1200-fold enrichment. Analysis of the most purified preparation by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a major Coomassie blue-stained band of molecular mass 42 kDa. The Tyr-protein kinase does not seem to be autophosphorylable. The Km value for poly(Glu, Tyr)4:1 is relatively low, 2.3 microM, and the tyrosine-polymer phosphorylating activity is apparently inhibited by tyrphostin. The characteristics shown by this new tyrosine kinase--the first to be described in mature male germ cells--support the hypothesis that it belongs to the group of non-receptor-associated tyrosine kinases.     

Isolation and characterization of a sulfated glycoprotein from rabbit uterus.

Crude glycoproteins were extracted with 0.15 M NaCl from the pooled endometrial scrapings of rabbit uteri after treatment with estrogen. The crude glycoproteins were fractionated with ammonium sulfate, followed by DEAE-cellulose column chromatography, treatment with CM-Sephadex C-25 and gel filtration on Sephadex G-200. Subsequently, purification of an acidic glycoprotein was carried out by gel filtration on Sephadex G-200 and then Sepharose 4B. The results of electrophoresis and enzymatic digestion, together with analytical data and the infrared spectrum indicated that the acidic glycoprotein was a sulfated glycoprotein.     

The isolation and characterization of a concanavalin A receptor from boar spermatozoa surface

Boar spermatozoa were radioactively labeled by either lactoperoxidase-catalysed iodination or galactose oxidase oxidation followed by reduction with tritiated sodium borohydride. Plasma membrane glycoproteins were solubilized with the non-ionic detergent Nonidet P40 and separated by affinity chromatography on concanavalin A-Sepharose. A major water-soluble concanavalin A receptor of molecular weight greater than 160 000 was isolated by gel filtration and ion-exchange chromatography. Its amino acid and carbohydrate composition were determined. This glycoprotein is susceptible to digestion by trypsin or chymotrypsin.     

Isoelectric focusing of boar spermatozoa.

The isoelectric points of washed spermatozoa from intact boars and from boars after removal of the seminal vesicles were determined using isoelectric focusing on natural pH gradients. Normal boar spermatozoa focused at a higher pH than spermatozoa from boars without seminal vesicles. The isoelectric point of the latter was increased to a value approaching normal by preincubation in normal seminal plasma. This indicates that seminal plasma alters the membrane surface charge of boar spermatozoa on ejaculation.     

Isolation and biochemical characterization of the alpha- and beta-subunits of glycoprotein IIb of human platelet plasma membrane.

The alpha- and beta-subunits of glycoprotein IIb (GPIIb) of human platelet plasma membrane were isolated in fully reduced, partially reduced and alkylated, and fully alkylated forms, by size-exclusion chromatography after reduction of pure GPIIb. The sugar moiety of GPIIb alpha accounts for 16.4% of its total weight, whereas that of GPIIb beta accounts for only 10.2%. The molar percentages (per 100 mol of total amino acids) of neuraminic acid and galactose in the alpha-subunit more than double those in the beta-subunit, whereas galactosamine is present only in GPIIb alpha. From the amino acid and sugar compositions the acidic nature of both subunits was confirmed. The Mr values obtained, 114,000 for GPIIb alpha and 22,200 for GPIIb beta, are in very good agreement with those obtained by physical methods. We found by stepwise reduction of pure GPIIb with dithioerythritol that GPIIb alpha and GPIIb beta are joined by a single interchain disulphide bridge, while the remaining half-cystine residues participate in intrachain bonds, six in GPIIb alpha and one in GPIIb beta, the intersubunit disulphide bond being that reduced first. Neither of the two subunits is liberated from isolated plasma membranes when this GPIIb interchain bond is reduced in isolated membranes.     

F-actin in acrosome-reacted boar spermatozoa.

Biochemical and immunoelectron microscopic methods have been used to analyze the distribution of actin in boar spermatozoa and its state of aggregation before and after acrosome reaction. F-actin was detected on sperm head and tail by electron microscopy using an improved phalloidin probe: incubation with a fluorescein-phalloidin complex and an anti-fluorescein antibody, followed by labeling with protein A-gold complex. Gold particles, indicating the presence of F-actin, were localized on the sperm surface of the acrosome-reacted spermatozoa. Specific labeling was localized (1) between the outer acrosomal membrane and the plasma membrane in the equatorial region, (2) between the outer surface of the fibrous sheath and the plasma membrane in the postacrosomal region, (3) around the connecting piece and the neck region, and (4) on the external surface of the fibrous sheath in the principal piece of the tail. Furthermore, after NP-40 extraction, the SDS-PAGE revealed a difference in solubility between reacted and unreacted boar spermatozoa, reflecting actin polymerization. We conclude that most actin in the acrosome reacted boar spermatozoa is polymeric.     

Immunochemical and biochemical characterization of a glioma-associated extracellular matrix glycoprotein

A novel human glioma-associated extracellular matrix (ECM) glycoprotein has been identified by murine monoclonal antibody 81C6. The glycoprotein, designated GMEM, is expressed in the ECM of glioma and mesenchymal cell cultures, in the perivascular matrix of endothelial proliferations of human gliomas, and in the stroma of human glioma xenografts in athymic mice, where it has been used as a target antigen for monoclonal antibody tumor localization and radioimaging. We report here on the immunochemical and biochemical characterization of GMEM. Polyacrylamide gel analysis of immunoprecipitated [3H]-leucine- and [3H]-glucosamine-labeled ECM from the human glioma cell line U-251MG has shown that GMEM is a high-molecular-weight macromolecule (Mr approximately 1,000,000) composed of Mr approximately 230,000 disulfide-bonded glycoprotein subunits. Immunoprecipitation, immunoblot, and one-dimensional peptide map analysis have shown that GMEM is distinct from human fibroblast and plasma fibronectin. These results support previous immunohistology and absorption analysis findings, indicating that GMEM is distinct from fibronectin, laminin, and glycosaminoglycans secreted by U-251MG.     

Binding of a 15 kDa glycoprotein from spermatozoa of boars to surface of zona pellucida and cumulus oophorus cells.

A highly purified 15 kDa glycoprotein isolated from ejaculated spermatozoa was used to raise antisera in female rabbits. An indirect immunofluorescence technique was used to detect the antigen in the seminal vesicle tissue and on the acrosomes of ejaculated, native and capacitated, boar spermatozoa. No immunoreactivity was detected on cells of the seminiferous tubules (spermatogonia, spermatocytes, and spermatids), on spermatozoa in the ductus epididymis and in cells of the epididymal and testicular tissues. These observations support the view that the 15 kDa protein is produced in the seminal vesicle secretory epithelium, and is attached to the sperm plasma membrane during the exposure of spermatozoa to seminal vesicle compounds. The observations that the antigen remained on the acrosome of ejaculated spermatozoa after capacitation and blocked sperm-oocyte binding in vitro suggest that the antigen plays a role in sperm-egg interactions. The strong immunoreactivity exhibited by cumulus cells after incubation of antisera with the porcine egg surrounded by cumulus cells shows the possible importance of the 15 kDa glycoprotein for contact of spermatozoa with cells of the cumulus oophorus surrounding the egg.     

Isolation and characterization of a pulmonary glycoprotein from human amniotic fluid.

A glycoprotein of the molecular weight of 36 000 has been isolated from human amniotic fluid. The glycoprotein was found to contain sialic acid, galactose, mannose, fucose, glucosamine, hydroxyproline and relatively high amounts of glycine. End-group analyses resulted in a single NH2-terminal residue indicating that the glycoprotein was homogeneous. The data indicate that this unique collagen-like glycoprotein, which is immunologically identical to a major alveolar glycoprotein found in alveoli of patients with alveolar proteinosis, is also a major protein in the human amniotic fluid. The idea that the pulmonary constituents enter the amniotic fluid cavity during fetal lung development is also confirmed by this report.     

Bovine renal glomerular basement membrane. Isolation and characterization of a glycoprotein component.

Bovine glomerular basement membrane was extracted with 6 M guanidinium chloride and the soluble material fractionated on a Bio-Gel A-1.5m column in 1% Na dodecyl-SO4. A single component was obtained by reduction of a selected column fraction with 2-mercaptoethanol followed by chromatography on an analytical Bio-Gel A-1.5m column and shown to be homogenous by electrophoresis and ultracentrifugation. It consists of 90% protein and 8.6% carbohydrate by weight. The amino acid composition is characterized by the presence of low amounts of hydroxyproline and hydroxylysine, and substantial amounts of aspartic acid, glutamic acid, half-cystine, and glycine. It contains all the monosaccharide constituents present in the whole basement membrane indicating the presence of both heteropolysaccharide and disaccharide units; the presence of the latter unit was demonstrated unequivocally by ion exchange chromatography. The component contains 1 heteropolysaccharide unit and 4 dissaccharide units/molecule of Mr equals 70,000. The molecular weight of component VII was determined by several methods. Molecular weight values of 68,000 +/- 3,000 and 72,000 +/- 2,000 were determined in 6 M guanidinium chloride by the methods of sedimentation equilibrium and gel filtration chromatography, respectively, and values of 136,000 +/- 3,100 and 140,000 +/- 2,000 were determined in 1% Na dodecyl-SO4 by the methods of polyacrylamide gel electrophoresis and gel filtration chromatography, respectively. Circular dichroism spectra indicate that component VII assumes a random coil conformation in 6 M guanidinium chloride and a more disordered conformation in 1% Na dodecyl-SO4 than standard proteins used in calibration of polyacrylamide gels and gel filtration column. These results indicate that the minimal molecular weight of component VII is about 70,000 and that the anomalous behavior in Na dodecyl-SO4 is due in part to its conformation.