Matthew Freund

Isolation and Characterization of Membrane Vesicles from Human and Boar Spermatozoa: Methods Using Nitrogen Cavitation and Ionophore Induced Vesiculation

A method for isolation of plasma membrane vesicles from human and boar spermatozoa using nitrogen cavitation is described. The purity of the preparations were assessed by electron microscopy, marker enzyme assay and the sedimentation characteristics of fused plasma membrane-acrosomal membrane vesicles in sucrose gradients. PAGE-SDS profiles of plasma membrane polypeptides from boar spermatozoa were significantly different from those of human spermatozoa. Differences in electrophoretic profiles of polypeptides from different regions of the spermatozooon were also observed.

Morphologic Characteristics of the Chemically Induced Acrosome Reaction in Human Spermatozoa

The morphologic changes accompanying the acrosome reaction in human spermatozoa, as it is induced by the antibiotic A23187 and calcium ions, are described. The reaction is shown to be similar to that observed in other species when the reaction occurs spontaneously or is induced by physiologic fluids. The reaction in human spermatozoa differs from the chemically induced reaction in other species in that plasma membrane microfilaments, prominent in the boar, and tubular-like elements prominent in boar, rabbit, and monkey sperm, are not observed. Motility remains high when human spermatozoa are treated with A23187 and calcium and it is possible that these agents may be useful in the study of certain causes of infertility.

The interaction of living boar sperm and sperm plasma membrane vesicles with the porcine zona pellucida

Abstract Enzymological, morphological, and immunological methods were used to characterize further the interaction of noncapacitated boar spermatozoa with the porcine zona pellucida. Transmission electron microscopy showed that sperm usually bind to the zona over the head region of the cell. Only the plasma membrane is involved in this binding. Bound sperm will undergo the acrosome reaction when treated with calcium and the ionophore A23187. The ability of intact sperm to bind to porcine eggs in vitro and the ability of sperm plasma membrane vesicles to absorb univalent antibody to the sperm binding site for the zona were used to determine the effects of various physical, chemical, and enzymological treatments on the sperm binding sites. These sites were resistant to a number of enzymes including proteases and polysaccharidases, but were inactivated by heat and trichloroacetic acid. Binding sites on the zona were inactivated by extracts from small quantities of sperm. Binding was also blocked by Fab antibody to whole zonae absorbed to other swine tissue and by similarly absorbed Fab antibody to sperm plasma membranes. These data provide further support for the presence of zona recognition sites on the plasma membrane of noncapacitated boar sperm. The binding sites on the sperm plasma membrane do not appear to be peripheral membrane proteins nor major constituents of a surface glycocalyx.

Use of a fluorescent dye to measure drug-induced changes in the membrane potential of boar spermatozoa.

Abstract The fluorescence emission intensity of the dye 3,3′ dipentyloxo-carbocyanine iodide equilibrated with washed boar spermatozoa and valinomycin or gramicidin varies with external potassium or sodium concentration in a manner indicating that dye fluorescence is related to the plasma membrane potential. The membrane potential in turn is shown to be dependent on energy metabolism. The drugs propranolol, lidocaine and diphendydramine, which possess local anesthetic-like properties, induce a rapid depolarization which can be reversed by valinomycin at low drug concentrations and a slower apparently energy dependent depolarization at higher drug concentrations that is not reversible. Low concentrations of these drugs decrease forward progression of sperm but have little effect on the percentage motile cells. Theophylline increases the frequency of contraction of drug-treated cells but not their forward progression, these findings are discussed in terms of the role of the sperm membrane in the control of motility.