The Characterization of a Malachite Green Stainable, Glutaraldehyde Extractable Phospholipid in Rabbit Spermatozoa

Biochemical analyses of glutaraldehyde supernatants from ejaculated, saline-washed rabbit spermatozoa indicate that considerable quantities of choline and phosphorus are removed from the spermatozoa during fixation. Thin-layer chromatography of glutaraldehyde supernatants from brain, skeletal muscle, heart muscle, and spermatozoa has demonstrated a substance to be present which contains choline, phosphorus, and aldehyde groups. The compound chars slowly and stains with malachite green. Furthermore, the compound is absent from glutaraldehyde supernatants of tissues fixed in the presence of malachite green. Histochemical studies demonstrate that the postacrosomal segment of ejaculated rabbit spermatozoa contains appreciable quantities of phosphorus, choline, and malachite green stainable material (MGA-material). Mass spectral analyses have given additional evidence that MGA-material from rabbit spermatozoa may be a plasmalogen (1-alkenyl-2-acylphospholipid). Inhibition of malachite green staining of rabbit spermatozoa is accomplished when spermatozoa are pretreated with mercuric chloride or heat. Spot tests indicate that malachite green possesses a selective affinity for certain phospholipids with a staining specificity for the fatty acid components. It is concluded that the postacrosomal segment of ejaculated rabbit spermatozoa is an area of at least one choline containing phospholipid and that glutaraldehyde, in conjunction with malachite green, stains and retains this substance within rabbit spermatozoa during preparation for fine structure studies.