Properties of boar sperm plasma membranes (PM): proteins released by washing and differential solubility in salts, detergents, and sensitivity to surface radiolabelling

In this study a variety of properties of boar sperm plasma membrane proteins were examined. A qualitative and quantitative analysis of proteins washed from boar sperm revealed that large numbers and a variety of polypeptides (Ps) are easily removed from sperm upon washing. Initially (by the second wash), Ps are released from the plasma membrane (PM) of epididymal sperm and primarily correspond to those in epididymal fluid, but eventually (fifth wash) Ps are released that are not seen in epididymal fluid nor as components of the PM. These Ps appear to originate from the sperm cytosol and signal the damaging effects of extensive washing on sperm. Upon washing, ejaculated sperm release Ps characteristic of both epididymal fluid and accessory sex glands. Epididymal Ps are almost completely released by the fourth wash; accessory gland proteins appear to be more tenaciously bound and continue to be released with further washing. Most basic accessory gland Ps bind strongly enough to resist the series of washes necessary for the preparation of PM vesicles. About one-half of ejaculated sperm lose motility after five washes, but evidence of massive release of internal Ps, such as seen in epididymal sperm, is not noted. In the epididymis and after ejaculation, sperm are coated with numerous Ps which are released upon washing; many are released nonspecifically and rapidly, others are more firmly bound. These analyses extend the surface map of boar spermatozoa to include a description of loosely bound proteins and their origin. These results also indicate that the qualitative and quantitative changes in surface membrane protein composition, occurring after simple washing, are significant and may confound the interpretation of surface composition changes in studies which rely solely on immunological or radiolabelling procedures. In order to determine the nature of the binding of major polypeptides (Ps) to the lipid bilayer of boar sperm plasma membranes (PMs), the solubility of Ps in solutions of different ionic strength and in detergents was examined. Several major polypeptides (identified in previously published surface maps) were extracted by hypotonic and hypertonic salt solutions, suggesting that electrostatic interactions play a major role in their binding to the bilayer. Other major proteins were extracted only by detergents, suggesting that these proteins are embedded deeply into the bilayer. These extraction procedures also provided a new strategy for isolating specific Ps in large quantities. Radiolabelling procedures identified about 80 surface-exposed Ps, some of which are major constituents of the PM and others which are quantitatively minor components. Labelling of PM vesicles reveals about sixfold more Ps than does labelling of whole sperm. Increased labelling appears to be the result of surface accessibility of PM constituents after removal of loosely bound Ps from epididymal fluid and seminal plasma during the washings which accompany the preparation of PM vesicles from whole sperm. These results prescribe caution when interpreting changes in surface organization and membrane structure which are dependent solely on the use of radiolabels.