Glycosidases in porcine follicular fluid and their effect on zona pellucida-AWN 1 spermadhesin interaction.

Oligosaccharide moieties on the surface of the oocyte belong to the key molecules that direct the course of fertilization and are subjected to changes during oocyte maturation in the follicle. In our study, we focused on the activities of five glycosidases in the fluids from porcine secondary and preovulatory follicles (α-l-fucosidase, α-d-galactosidase, ß-d-galactosidase, ß-D-N-acetylhexosaminidase, and α-d-mannosidase). All of them were detected active at neutral and acidic pH. However, changes in their activities associated with follicle development were observed only in the case of α-d-mannosidase, which was increased (P < 0.001), and ß-d-galactosidase, which was decreased (P < 0.001) at neutral pH, and of α-d-galactosidase and ß-N-acetylhexosaminidase, which were decreased (P < 0.0001) at the acidic pH. The comparison of glycosidases from follicular fluid and from blood plasma using red native electrophoresis revealed that most of the glycosidases are present in more than one isoenzyme form; some of them were detected mainly in the follicular fluid. Finally, we tested the effect of glycosidases on the interaction between zona pellucida and AWN 1 spermadhesin (putative sperm receptor of zona pellucida) and demonstrated that the effect of both ß-d-galactosidase and to a lesser degree α-d-mannosidase led to a decrease in this interaction. We can hypothesize that these two glycosidases modulate the amount of zona pellucida oligosaccharide moieties and/or their structures for an optimal sperm binding in pigs.

Purification and enzymatic characterization of tobacco leaf ß-N-acetylhexosaminidase.

The kinetic properties of ß-N-acetylhexosaminidase purified from tobacco (Nicotiana tabacum L.) leaves have been investigated. In addition to chromogenic pNP derivates, N,N'-diacetylchitobiose and N,N',N″-triacetylchitotriose were also used as substrates of ß-N-acetylhexosaminidase. The highest reaction rate and the affinity for the substrate were observed for pNP-GlcNAc; however, an excess of this substrate inhibits the reaction. The reaction rate with pNP-GalNAc as the substrate was found to be about 85% of that obtained with pNP-GlcNAc. The hydrolysis of acetylated chitooligomers by ß-N-acetylhexosaminidase followed by separation and quantification using capillary electrophoresis was slower compared to pNP-GlcNAc. The pH optimum of ß-N-acetylhexosaminidase for individual substrates was found at 4.3-5.0 and the temperature optimum was 50-55 °C. Gel permeation chromatography and red native electrophoresis determined the relative molecular weight as 280 000 and the isoelectric point as 5.3. The inhibition of ß-N-acetylhexosaminidase by monosaccharides GlcN, GalN, GlcNAc, GalNAc in combination with substrates pNP-GlcNAc and pNP-GalNAc was studied and the type of inhibition and the inhibition constants were determined.

The antimicrobial action of histones in the reproductive tract of cow.

An infection of any part of female reproductive tract can severely interfere with fertility and reproduction. The fluids and epithelium from the lumen of the female reproductive tract (uterus, oviduct and ovarian follicle) are a known source of antimicrobial action in several species. In this study, we compared the antimicrobial properties of fluids from the reproductive tract of a cow. After removal of small molecules, we demonstrated that there is an antimicrobial activity connected with a fraction of compounds with a molecular mass range between 3500 and 30,000. The most probable candidates responsible for the observed antimicrobial effect were subsequently identified by mass spectroscopy as histones H2A type 2-C, H2B type 1-K, H3.3, and H4. The antimicrobial role of histone H2B was further confirmed by using an antibody against this histone.

Native red electrophoresis--a new method suitable for separation of native proteins.

A new type of native electrophoresis was developed to separate and characterize proteins. In this modification of the native blue electrophoresis, the dye Ponceau Red S is used instead of Coomassie Brilliant Blue to impose uniform negative charge on proteins to enable their electrophoretic separation according to their relative molecular masses. As Ponceau Red S binds less tightly to proteins, in comparison with Coomassie Blue, it can be easily removed after the electrophoretic separation and a further investigation of protein properties is made possible (e.g. an enzyme detection or electroblotting). The tested proteins also kept their native properties (enzyme activity or aggregation state).

Native polyacrylamide electrophoresis in the presence of Ponceau Red to study oligomeric states of protein complexes.

Native polyacrylamide electrophoresis in the presence of two reversible protein anionic stains (Ponceau S and Ponceau 2R) was used to study the oligomeric states of soluble proteins. A mild binding of the used protein stains to nondissociated protein oligomers imposed a charge shift on the proteins resulting into separation of protein species according to their size under physiological conditions. Adsorbed stains could be easily removed after electrophoresis by washing of polyacrylamide gel with buffer and protein complexes could be visualized either by the detection of their enzyme activity or by using a nonspecific protein stain. The specific detection of enzyme activity of glycosidases, lactate dehydrogenase, or phosphatases was shown as an example.

3-aminobenzanthrone, a human metabolite of the carcinogenic environmental pollutant 3-nitrobenzanthrone, induces biotransformation enzymes in rat kidney and lung.

3-aminobenzanthrone (3-ABA) is the metabolite of the carcinogenic air pollutant 3-nitrobenzanthrone (3-NBA). 3-ABA was investigated for its ability to induce cytochrome P450 1A1 (CYP1A1) and NAD(P)H:quinone oxidoreductase (NQO1) in kidney and lung of rats, and for the influence of such induction on DNA adduct formation by 3-ABA and 3-NBA. NQO1 is the enzyme that reduces 3-NBA to N-hydroxy-3-aminobenzanthrone (N-OH-3-ABA) and CYP1A enzymes oxidize 3-ABA to the same intermediate. When activated by cytosolic and and/or microsomal fractions isolated from rat lung, the target organ for 3-NBA carcinogenicity, and kidney, both compounds generated the same DNA-adduct pattern, consisting of five adducts. When pulmonary cytosols isolated from rats that had been treated i.p. with 40 mg/kg bw of 3-ABA were incubated with 3-NBA, DNA adduct formation was up to 1.7-fold higher than in incubations with cytosols from control animals. This increase corresponded to an increase in protein level and enzymatic activity of NQO1. In contrast, no induction of NQO1 expression by 3-ABA treatment was found in the kidney. Incubations of 3-ABA with renal and pulmonary microsomes of 3-ABA-treated rats led to an increase of up to a 4.5-fold in DNA-adduct formation relative to controls. The stimulation of DNA-adduct formation correlated with a higher protein expression and activity of CYP1A1 induced by 3-ABA. These results show that by inducing lung and kidney CYP1A1 and NQO1, 3-ABA increases its own enzymatic activation as well as that of the environmental pollutant, 3-NBA, thereby enhancing the genotoxic and carcinogenic potential of both compounds.

Redox cycling in the metabolism of the environmental pollutant and suspected human carcinogen o-anisidine by rat and rabbit hepatic microsomes.

We investigated the ability of hepatic microsomes from rat and rabbit to metabolize 2-methoxyaniline (o-anisidine), an industrial and environmental pollutant and a bladder carcinogen for rodents. Using HPLC combined with electrospray tandem mass spectrometry, we determined that o-anisidine is oxidized by microsomes of both species to N-(2-methoxyphenyl)hydroxylamine, o-aminophenol, and one additional metabolite, the exact structure of which has not been identified as yet. N-(2-Methoxyphenyl)hydroxylamine is either further oxidized to 2-methoxynitrosobenzene (o-nitrosoanisole) or reduced to parental o-anisidine, which can be oxidized again to produce o-aminophenol. To define the role of microsomal cytochromes P450 (P450) in o-anisidine metabolism, we investigated the modulation of this metabolism by specific inducers and selective inhibitors of these enzymes. The results of the studies suggest that o-anisidine is a promiscuous substrate of P450s of rat and rabbit liver; because P450s of 1A, 2B, 2E, and 3A subfamilies metabolize o-anisidine in hepatic microsomes of both studied species. Using purified enzymes of rat and rabbit (P450s 1A1, 1A2, 2B2, 2B4, 2E1, 2C3, 3A1, and 3A6), reconstituted with NADPH:P450 reductase, the ability of P450s 1A1, 1A2, 2B2, 2B4, 2E1, and 3A6 to metabolize o-anisidine was confirmed. In the reconstituted P450 system, rabbit P450 2E1 was the most efficient enzyme metabolizing o-anisidine. The data demonstrate the participation of different rat and rabbit P450s in o-anisidine metabolism and indicate that both experimental animal species might serve as suitable models to mimic the fate of o-anisidine in human.

Saccharide-mediated interactions of boar sperm surface proteins with components of the porcine oviduct.

The role of boar seminal plasma proteins attached to the sperm plasma membrane during ejaculation has been studied in saccharide-mediated events in the female reproductive tract. Heparin-binding (Hep(+)) proteins (DQH sperm surface protein, and AQN and AWN spermadhesins) and their aggregated forms (fractions II and III) interacted more strongly with both oviductal epithelium cells and fluid than non-heparin-binding (Hep(-)) proteins (PSP I and PSP II spermadhesins) and their heterodimer (fraction IV), and interactions correlate with affinity of these proteins to yeast mannan. Indirect immunofluorescence (IMF) showed that the AQN 1 spermadhesin and fraction II bind to the apical glycocalyx of the ampulla, as well as the isthmic and uterine tubal junction regions of the oviductal sections. IMF demonstrated the recognition of AQN 1 and fraction II and mannosyl components of oviductal epithelium. We suggest that Hep(+) proteins (especially AQN 1, fraction II) on sperm could enable sperm binding to oviductal epithelium and thus participate in formation of the sperm oviductal reservoir. Interactions of Hep(+) proteins to oviductal epithelium were inhibited by mannan, hyaluronic acid and sialylated O-glycoproteins. No or slight inhibition was observed with sulphated polysaccharides (heparin, chondroitin sulphate) and simple monosaccharides. Besides that, attachment of boar seminal plasma proteins to oviductal epithelium cells was affected by oviductal fluid, the natural environment in the oviduct. Moreover, the ability of hyaluronic acid to inhibit the interaction of sperm surface proteins to the oviduct might play a role in sperm release from the oviductal reservoir and in the capacitation process.

Mannan-binding proteins from boar seminal plasma.

The interaction of boar seminal plasma proteins and sperm with yeast mannan was investigated by the enzyme-linked binding assay (ELBA) and specific detection of proteins after SDS electrophoresis and blotting using biotinylated derivative of the polysaccharide. Heparin-binding proteins (especially AQN 1 and DQH proteins) and their aggregated forms showed affinity to yeast mannan. Besides that, these proteins were shown to bind to oviductal epithelium. The mannan-binding activity of boar proteins and sperm was inhibited most efficiently by ovomucoid, ovalbumin and N-glycans released from ovalbumin, but not with d-glucose, d-mannose and their phosphates. On the other hand, yeast mannan inhibited both the interaction of boar seminal plasma and sperm with heparin and the binding of these proteins to porcine oviductal epithelium. Yeast mannan immobilized to divinyl sulfone-activated Sepharose was used for the isolation of mannan-binding proteins. Proteins adsorbed to the immobilized polysaccharide were analyzed by RP-HPLC, SDS electrophoresis and N-terminal amino acid sequencing. AQN and AWN spermadhesins and DQH protein (names are derived from the N-terminal amino acid sequence) were identified as components of the isolated fraction. The results suggest an involvement of mannan-binding proteins in the formation of the sperm oviductal reservoir in pig. The ability of these proteins to interact both the complex d-mannose-containing saccharide structures and the heparin may also play an important role in sperm release from the oviductal reservoir or the capacitation process.

Isolation of non-heparin-binding and heparin-binding proteins of boar prostate.

Proteins of boar prostate secretion were separated by affinity chromatography on heparin-polyacrylamide to non-heparin-binding (H) and heparin-binding (H+) protein fractions. H- and H+ fractions were then subjected to RP HPLC. Elution profiles of H-and H+ fractions of prostate secretion were compared with those of seminal plasma and the amounts of corresponding proteins were compared. Besides, the isolated proteins were characterized by SDS-PAGE. In the H- fraction of prostate secretion, PSP I and PSP II spermadhesins and in the H+ fraction AQN 2 and AWN 1 spermadhesins were found in substantially lower amounts than in seminal plasma. On the contrary, beta-microseminoprotein was identified in abundant amounts both in H- and H+ fractions of boar prostate secretion. AQN 2 and AWN 1 spermadhesins were proved by their antibodies. Some seminal plasma proteins originating mainly in seminal vesicles could also be secreted by the prostatic gland. beta-Microseminoprotein was found to be produced mainly by the prostate.