Nature of the phosphotungstic acid-chromic acid (PACP) stain for plasma membranes of plants and mammalian sperm

Abstract

The selective staining of plasma membranes of plants and porcine spermatozoa given by a mixture consisting of 1% phosphotungstic acid in 10% chromic acid (PACP) applied following periodic acid destaining of glutaraldehyde-osmium tetroxide-fixed electron microscope sections is reduced or eliminated by prior extraction of the tissues with lipid solvents, including ethanol. The ethanol-soluble fraction of sperm contains constituents which restore the PACP-staining reaction when added to ethanol-extracted and lyophilized sperm. Analysis of the ethanol extracts by thin layer chromatography revealed two major components which reacted with both phosphotungstic acid (PTA) and alpha-naphthol detection reagents. These PTA-positive constituents were concentrated in plasma membranes of sperm; components with similar mobilities were found in fractions of plasma membranes from plants. Addition of the PTA-positive constituents from either sperm or plants to extracted and lyophilized sperm restored the PACP staining. The findings are interpreted to mean that one or more low molecular weight constituents (saccharides or glycolipids), rather than glycoproteins, concentrated in plaslma membranes are responsible for the unique PACP staining in both plants and porcine sperm.