Molan, Peter C.Al-Somai, Niaz Mohamed Rashed2026-08-172026-08-171994https://hdl.handle.net/10289/18546The detrimental effect of bovine seminal plasma on sperm is believed to be the action of its constituent proteins and peptides. The major proteins in seminal plasma are known to be aggregates of peptides of low molecular weight. Various researchers have reported that the anti-sperm activity in seminal plasma is spread over a wide range of molecular weights. High molecular weight cationic material from seminal plasma is believed to be more damaging to sperm than high molecular weight anionic material. The substance or substances responsible for the detrimental effect have not been fully characterised. The current study was undertaken to examine, more thoroughly, the detrimental effect of bovine seminal plasma on sperm, to isolate and characterise the component( s) of low molecular weight from the aggregate, and to study the interaction between these components with regard to the formation of aggregates and their effect on sperm motility. In order to achieve this goal, a method had to be developed to isolate the anti-sperm component(s), to give a better dissociation of the component(s) from the macromolecules and to disaggregate the macromolecules to their constituents. Preliminary investigation, using gel permeation chromatography, indicated that low molecular weight fractions from bovine seminal plasma were more detrimental to sperm motility than those of higher molecular weight. By dialysis in the presence of citrate or under acidic conditions followed by gel permeation chromatography under acidic conditions, the low molecular weight components were released from the aggregates and they were assessed for their detrimental effects on sperm. Although they were found to have more detrimental effect than the parent aggregate, they tended to reaggregate to higher molecular weights under neutral conditions. Methods evaluated as ways of achieving disaggregation were: dialysis in membranes with cut-off of various molecular weights; gel permeation chromatography under dissociating conditions; selective precipitation with isopropanol; immobilisation of the cationic proteins on a cation exchanger, with extensive washing with acetonitrile at pH 3 to dissociate the anionic component(s), then elution with 0.01 mol/l NaOH; separation of the hydrophobic from the hydrophilic component(s) by immobilisation on a hydrophobic interaction column (Phenyl Superose); and reverse phase chromatography on FPLC columns to separate proteins and peptides. Further sequential use of these methods on FPLC columns for final purification resulted in the isolation of the anionic and cationic peptides in a reasonably homogenous form. All the fractions obtained using the above methods were assessed for their effects on sperm washed by centrifugation in Ficoll solution to free them from seminal plasma. The washed sperm were suspended in a defined diluent to provide a good test system for examining the effects of seminal plasma and its fraction on sperm. From attempts to isolate the damaging component from seminal plasma, it appeared that relatively high molecular weight cationic or anionic proteins retained anti-sperm activity while they were in aggregated form, however, the extent of activity depended on the form of aggregation. The isolated cationic peptide was highly basic in nature, highly active against sperm and failed to form an aggregate by itself under neutral pH. The isolated anionic peptide was hydrophobic in nature, inactive on sperm and failed to form an aggregate by itself. A combination of the active cationic and the inactive anionic peptide succeeded, in a certain ratio, in the formation of a large aggregate at neutral pH. This was demonstrated by a reaggregation study with radiolabelled anionic peptide which showed incorporation in aggregates which eluted at the high molecular weight region indicating that the two peptides were required for formation of aggregates. The combination of the two peptides reduced the detrimental effect of the active cationic peptide. It is hypothesised that the aggregates in seminal plasma are an assembly of the active cationic peptide with the inactive anionic peptide. This aggregation is a way of attenuating the damaging effect while the release of these low molecular weight from the large aggregates accentuates the damaging effects. The well established antibacterial activity in seminal plasma is highest in the form of high molecular weight aggregates. Aggregation in the form of high molecular weight components may serve to protect the male reproductive tract against bacterial invasion and protect the sperm from the toxic effect of the low molecular weight components by being in the form of high molecular weight aggregates.enAll items in Research Commons are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.Thesis