Identification of gp17 glycoprotein and characterization of prostatic acid phosphatase (PAP) and carboxypeptidase E (CPE) fragments in a human seminal plasma fraction interacting with concanavalin A.

The decapacitating fraction of human seminal plasma, which strongly interacts with concanavalin A, is constituted by high mannose-type N-linked glycoproteins, most of them of less than 44 kDa. Each component with apparent molecular mass of 30, 18, and 17 kDa respectively, as judged by SDS-PAGE, was submitted to "in gel" digestion with trypsin followed by HPLC separation of the peptides and sequencing. They were characterized at microscale as gp17, an aspartyl protease that possibly contributes to liquefaction of the seminal plasma coagulum, two fragments of human acid phosphatase (17 and 30 kDa, respectively), and a 17-kDa fragment of carboxypeptidase E. Neither the fragments of prostatic acid phosphatase nor that of carboxypeptidase E had been described before in the human seminal fluid. Very weak bands, of apparent molecular masses 44 and 52 kDa, are consistent with presence of small amounts of parent compounds, prostatic acid phosphatase and carboxypeptidase E.

Inhibition of acrosine-like protease activity by a lectin affinity chromatographic bovine seminal plasma fraction containing the PDC-109 and aSFP proteins.

These studies showed that the fractionation of bovine seminal plasma based on lectin agarose affinity chromatography, employing lectins specific to asparagine linked oligosaccharides, and a lectin specific for fucosylated glycans, lead to products with an inhibitory effect on the acrosine-like protease activity. This effect decreases when glycocompounds containing fucosylated Lewis(x) structures are removed, suggesting that these compounds might have some role in the modulation of this activity in the bull. In the fraction devoid of high mannose, hybrid and non-bisecting lactosaminic oligosaccharide-containing glycocompounds, PDC-109 and aSFP proteins were detected and characterized at microscale.

Fucosylated glycoconjugates of the human spermatozoon. Comparison of the domains of these glycoconjugates with the alpha-fucosyl binding sites, and with lactosaminic glycoconjugates and beta-D-galactosyl binding site domains.

Fucosylated glycoconjugates play an important role in fertilization as the recognition signal of the zona pellucida. In this work, using "critical" concentrations of either, FITC Lotus tetragonolobus lectin or FITC alpha-L-fucosyl-BSA neoglycoprotein as molecular probes, population densities of fucosylated glycoconjugates and of their "complementary" molecules (carrying fucosyl receptors), were found all over the sperm surface with higher population densities in post acrosomal sheath, neck and midpiece. These results were compared with previously reported data on the population densities of lactosaminic compounds and their "complementary" molecules, obtained on same samples of spermatozoa. Statistical data demonstrate that fucosylated glycoconjugates share the same domains with biantennary N-acetyllactosaminic oligosaccharides carrying outer galactose and bisected N-acetylglucosamine residues. These domains highly differ with those of the lactosaminic glycoproteins carrying tri and tetraantennary N-acetyllactosaminic oligosaccharides. These studies also show that the domains of fucosylated glycoconjugates and their "complementary" molecules (carrying fucosyl receptors) locate in different zones of the spermatozoon than those of the compounds carrying beta-galactosyl receptors. Besides, the results suggest structural differences between fucosylated glycoconjugates of the acrosome, equatorial zone and post acrosomal sheath. This may be relevant to the different biological behavior of these compounds and zones, in fertilization.

Cytochemical detection of receptors specific for N-linked oligosaccharides of glycoproteins in the membrane of the human spermatozoon and their distribution in the different zones of that membrane.

The horseradish peroxidase (HRP), a glycoprotein rich in mannose and N-acetylglucosamine residues has been used as a ligand to detect receptors for N-glycosidic linked oligosaccharides of glycoproteins in the human spermatozoon. Specific binding of HRP occurred to the membrane and the binding sites were visualized with 3,3'-diaminobenzidine-H2O2 (DAB-H2O2) reagent, and by fluorescence when the FITC-peroxidase was used. This specific binding was suppressed by alpha-D-methyl-mannoside and human chorionic gonadotropin, decreased by follicle stimulating and luteinizing hormones and slightly diminished by N-acetylglucosamine. The distribution of the N-linked oligosaccharide specific receptors for glycoproteins in the different zones of the membrane of the spermatozoon was determined by counting the spermatozoa labeled in those zones. The pattern of the distribution is similar to that found for N-linked oligosaccharides containing glycoproteins of the same membrane. The similarity of these distributions together with the general model for cell-to-cell recognition suggest that the sperm-egg interaction mechanism could consist of dual interactions by double binding receptors.

Carbohydrates of the surface of the normal human spermatozoon.

Autoproteolysis of human spermatozoa produces oligopeptides with oligosaccharide chains of the N-glycosidic-linked type that are released from the "surface exposed" parts of glycoproteins. The products eluted in the previous washing of the spermatozoa have the same composition and solubility characteristics as the oligopeptides from the digestion. This suggests that autoproteolysis is a constant process that normally occurs on the spermatozoa membrane. The cytochemical characterization and localization of the N-glycosidic-linked oligosaccharide receptors on the human spermatozoa membrane after digestion, in the presence or absence of seminal plasma, indicates that only part of the oligosaccharides are cleaved. Their distribution on the different zones of the spermatozoon changed as the probability of detecting these receptors in the intermediate segment increased after proteolysis; this indicates that in this zone the receptors are cryptic ones that become exposed by the action of the proteolytic enzymes. In the presence of seminal plasma most receptors on the acrosome are eliminated.

Cytochemical characterization and localization of oligomannosidic oligosaccharide receptors on the normal human spermatozoa using fluorescent lectins: comparison of different fixation procedures.

Similar locations of the Con A and wheat germ lectin receptors were obtained by using fluorescent lectins, in nonfixed spermatozoa and in spermatozoa fixed with formaldehyde and methanol, showing that in samples with the same previous treatment, worked out by the same operator, in which enough determinations have been performed to eliminate individual variations, the different procedures of fixation produced similar results. The locatizations obtained with fluorescent lectins confirm previous results, produced with the peroxidase technique, indicating that the lectins interact with oligomannosidic oligosaccharide receptors situated mainly in the equatorial segment of the acrosome and postnuclear cap. They also indicate the presence of similar receptors that were not detected previously on the neck and intermediate segment. The larger size of the lectin-peroxidase-diaminebenzidine reagent compared to that of the fluorescent lectins suggests that the new receptors are semicriptic and were not detected by steric effects in the first case, but were able to interact with lower volume, fluorescent probes. It is suggested that these oligomannosidic chains could be recognition signals for the elimination of incompetent sperm during their passage through the female reproductive track. Also these oligosaccharides and its possible metabolic variations could be involved in the interaction between the acrosome-reacted spermatozoa with the zone pellucida.

Immunological response of guinea pigs sensitized with homologous antigens.

The chemical composition of some "immunologically" pure antigens isolated from guinea pig testis and spermatozoa was correlated with their antigenic behavior. Their immunological responses were compared to select the best materials for a further isolation of chemically pure antigens. The glycoprotein extracted from the spermatozoa (T Gly) has the highest immunological potency and seems to be a T and B, depending antigen, able to induce high humoral and cell responses producing germinal cell damage, testicular lesions, and aspermatogenesis.

The antigenic mucin-like glycoproteins of human seminal plasma.

Immunodiffusion, immunoelectrophoresis, SDS-polyacrylamide gel electrophoresis, polyacrylamide gel electrophoresis, ultracentrifugation analysis, and gel permeation chromatography in chaotropic and detergent mediums showed that the human seminal plasma obtained and stored in the usual way is mainly composed by heterodispersed glycoproteins of low molecular weight. The glycoprotein components of the human seminal plasma which do not interact specifically with concanavalin A (con A) were separated by column chromatography on Con A-Sepharose according to nonspecific ion exchange and hydrophobic interactions with the protein. The carbohydrate composition of the glycoproteins and the existence of the N-acetylgalactosaminylserine (or threonine) linkage, as well as their aggregation properties show that they are mucin-type glycoproteins. They resulted "immunologically pure" although the carbohydrate chains show the structural heterodispersion usually found in mucins. The glycoproteins have a common structural pattern for the terminal nonreducing, determinant-bearing sequences of their oligosaccharide chains which, together with the low molecular weights and the known fact that mucus secretions of normal cells contain only one or two types of glycoproteins, suggest that they are fragments produced by endogenous proteolytic degradation of a native mucin.

Immunohistochemical localization of concanavalin A and wheat germ lectin receptors in the normal human spermatozoa.

A semiquantitative immunohistochemical technique for the detection of N-acetyl alpha-D-neuraminic acid, N-acetyl beta-D-glucosamine and its beta-(1 leads to 4)-linked internal chains, alpha-D-glucopyranosyl and alpha-D-mannopyranosyl and its alpha-(1 leads to 2)-linked internal chains and sterically related, nonreducing, end-chain residues of oligosaccharide chains of glycoproteins or glycolipids on the surface of membranes was developed using Con A and wheat germ lectins. When this method was applied to the localization of carbohydrate receptors on the membrane of the normal human spermatozoa, it was found that the Con A and wheat germ lectin receptors were mainly located in the equatorial and post nuclear cap with few receptors located in the acrosome and neck. None of them were found in the intermediate segment plus tail. Con A receptors were alpha-D-mannopyranosyl end-chain residues and wheat germ lectin receptors were N-acetyl beta-D-glucosamine (1 leads to 4)-linked internal chains. These groups occur together in the oligomannosidic type of N-glycosidic-linked oligosaccharide chains of glycoproteins and so the use of both lectins on desialycated membranes or on those which contain nonclustered N-acetyl neuraminic acid residues may be of help to localize this type of glycoprotein oligosaccharide chains. Con A receptors were not removed after proteases digestion, suggesting the possibility that they are part of intrinsic spermatozoal antigens.

Immunoserological, immunohistochemical and immunobiological studies on an antigenic glycoprotein isolated from human seminal plasma.

The seminal plasma and a fraction of the spermatozoal membrane solubilised by sarkosyl contain similar antigenic determinants. The glycoprotein isolated from the seminal plasma also contains determinants which are immunologically identical to those of the sarkosyl soluble fraction. Three of these determinants were identified as: two oligosaccharide chains with terminal N-acetyl neuraminic acid and with subterminal alpha-L-fucopyranose and beta-D-galactopyranose residues respectively, and an oligosaccharide chain with beta-D-galactopyranose as non-reducing end-chain. The receptors on the spermatozoa to anti-glycoprotein and anti-seminal plasma antibodies are localized. The specificity of the receptors localised in the neck region depends entirely on the presence of N-acetyl neuraminic acid as end-chain group while the specificity of the receptors to the anti-seminal plasma antibodies in the intermediate segment plus tail and to the anti-glycoprotein antibodies in the post-nuclear cap depends also on the subterminal units. At least two other receptors in the equatorial zone are ended by residues different from the N-acetyl neuraminic acid. One of them, which is also found in the acrosome, does not have specificity for anti-glycoprotein antibodies, while the other, which is not in the acrosome, has anti-glycoprotein antibodies specificity and may have a terminal beta-D-galactopyranose residue.

Affinity chromatography on concanavalin A-Sepharose of antigenic fractions of human seminal plasma.

Elution diagrams obtained on affinity chromatography show that the antigenic fractions of human seminal plasma studied, namely (a) the non-dialyzable components of human seminal plasma and (b) its trichloroacetic acid-soluble fraction, (c) the trichloroacetic acid-soluble fraction of whole human seminal plasma and (d) the pronase digested human seminal plasma, are complex mixtures of glycoproteins with minor amounts of polysaccharides. Some of these glycoproteins contain significant percentages of carbohydrates while others contain only trace amounts. Most of the glycoproteins carry non-reducing end-chain groups comprising alpha-D-glucopyranosyl, alpha-D-mannopyranosyl or sterically related residues.

Antigens from human seminal plasma. Part III: studies on two new antigens from the tricholoracetic acid soluble fraction.

Two new antigens were isolated from the trichloroacetic acid soluble fraction of human seminal plasma: a polysaccharide (G-3) and a glycopeptide (G-4). Both were homogeneous, and hand sedimentation constants at infinite dilution of 2.9 X 10(-13) and 2.0 X 10(-13) resectively. Chemical and physicochemical studies suggest that both are branched molecules composed mainly of alpha-D-glucopyranosidic residues. The peptide component of G-R contains a high proporiton of the basic amino acids arginine and lysine. Since both antigens are polydispered, the low percentage of sialic acids or of some amino acids determined might have antigenic significance.

Isolation and characterization of two antigens from human seminal plasma.

A simple method is described for the isolation of two glycopeptides from human seminal plasma. One of them cross-reacts with human spermatozoa, a fact which will help the study of antigenicity of sperm cells. The glycopeptides obtained (G1 and G2) are homogenous, as judged by disc electrophoresis, ultracentrifugation, immunodiffusion and immunoelectrophoresis. The weight average molecular weight of both glycopeptides was about 10 000 for both fractions. Differences in the amino-acid and carbohydrate composition of both glycopeptides were observed. The content of histidine was higher in one of them (G1). Only slight differences in the content of sialic acid of both glycopeptides were observed.