Characterization of human seminal plasma proteins homologous to boar AQN spermadhesins.

Spermadhesins, proteins secreted by the boar sexual accessory glands, are believed to play an important role in sperm capacitation and primary contact of sperm and egg. We have previously found human seminal plasma proteins immunobiochemically related to boar AQN and AWN spermadhesins. In this study, we characterized further the AQN spermadhesin-related proteins, here designated as hSA (human spermadhesin-like) proteins. On Western blot, we immunodetected 14, 16 and 18 kDa forms of hSA proteins (hSA-14, hSA-16 and hSA-18, respectively) cross-reacting with rabbit antibody against AQN spermadhesins. Each relative molecular-mass form of hSA comprised three isoelectric isoforms (6.0, 6.8 and 8.4) as shown by 2D-PAGE. Glycoprotein analysis revealed that all hSA-16 and hSA-18 isoforms were N-glycosylated, and those of hSA-14 were non-glycosylated. Two isoforms of hSA-14 (pI 6.0 and 8.4) had affinity to heparin. Size-exclusion chromatography of human seminal plasma indicated that hSA proteins formed high molecular-mass complexes either with other hSA proteins or with seminal plasma lactoferrin and/or its fragments. Similarity of biochemical properties (relative molecular masses, isoelectric points and existence of non- and N-glycosylated forms) of hSA proteins and those of boar AQN spermadhesins, together with a previously described N-terminal amino acid sequence of one hSA protein identical to AQN spermadhesins, imply that hSA proteins are structurally related to boar AQN spermadhesins. However, localization of hSA proteins on the sperm tail and neck suggests that their biological role differs from that of boar AQN spermadhesins located on the sperm head.

Mutual interactions of boar seminal plasma proteins studied by immunological and chromatographic methods.

PROBLEM: Boar seminal plasma contains various types of proteins. Most of them belong to spermadhesins and some of them contain fibronectin type II domain. Almost all of these proteins are present under physiological conditions in aggregated forms differing in their relative molecular mass, composition, and binding properties. The study of mutual specific interactions between proteins of boar seminal plasma that could be involved in the formation of aggregated forms and most probably of sperm coating layers is the subject of our present communication. METHODS OF STUDY: Aggregated forms of boar seminal plasma proteins separated by size exclusion chromatography were analysed by Reversed-phase High-performance Liquid Chromatography (RP HPLC), SDS-PAGE and immunochemical methods (ELISA and immunoblotting). Mutual interactions between proteins were investigated by size exclusion chromatography of a mixture of separated monomer proteins and affinity chromatography of boar seminal plasma on immobilized spermadhesins. RESULTS: Composition of proteins that are adsorbed from boar seminal plasma to immobilized spermadhesins corresponds to that of aggregated forms found in seminal plasma. Mutual interactions between monomer forms observed by size exclusion chromatography are in good agreement with results of affinity chromatography. An existence of interactions between spermadhesins of the AQN and AWN families and the DQH sperm surface protein, as well as between proteins involved in the formation of heterodimer porcine seminal plasma I (PSP I)/PSP II was proved. CONCLUSION: Mutual specific interactions between protein components of boar seminal plasma were shown. These interactions participate in the formation of aggregated forms of proteins in seminal plasma and probably also in the arrangement and remodelling of protein coating layers of sperm. Aggregation of seminal plasma proteins is probably an important phenomenon in the fertilization process.