A major methodological problem in the intracellular localization of cholesterol is the nearly complete extraction of sterols during routine dehydration and embedding procedures for electron microscopy. Cholesterol digitonide (a sterol complex with digitonin), however, is qualitatively insoluble in these solvents. Mouse liver has been prepared as follows: (a) Flickinger's aldehyde fixative, 20 hr; (b) Flickinger's fixative containing 0.2% digitonin, 24 hr; (c) cacodylate wash, 24 hr; (d) 1 % OsO, 2 hr; (e) acetone dehydration; and (f) Epon 812 infiltration under vacuum, 28 hr. After the last step, an analysis of the tissue for sterol content under optimal analytical conditions demonstrates a retention of 99% of the unesterified cholesterol present in unfixed mouse liver. Liver prepared in an identical manner except for omission of digitonin is essentially devoid of sterols. Cholesterol isolated chromatographically from liver processed as outlined above has been identified unequivocally by mass spectrometry. Liver from step (f) also has been polymerized, thin-sectioned, and examined in the electron microscope. A remarkable quality of finestructural preservation is obtained. The major alteration encountered is the presence of small cylindrical "spicules," often occurring as tightly packed concentric lamellae, at membrane surfaces.

Routine electron microscopy in combination with subcellular localization of acid phosphatase has been employed to study the formation and fate of residual cytoplasmic bodies extruded into the tubular lumen shortly before spermiation. Prior to extrusion the spermatid cytoplasm contains lipid droplets, mitochondria, ribosomes, endoplasmic reticulum, the caudally migrated Golgi apparatus, and numerous multivesicular and multigranular bodies. These membrane-limited bodies and the Golgi zone stain heavily for acid phosphatase. Following extrusion the residual bodies undergo a series of alterations: (1) disruption of multigranular bodies with release of free granules; ( 2 ) sequestration of granules, ribosomes, and reticulum inside double-membrane-limited vacuoles derived from Golgi lamellae; (3) appearance of numerous, single-membrane-bound, cytoplasmic vacuoles; (4) fragmentation; (5) peripheral migration toward the tubular wall; and ( 6 ) phagocytosis of these migrating fragments by the Sertoli cells. The demonstration of acid phosphatase activity within free granules, the sequestering Golgi lamellae, and both classes of vacuoles suggests that initial residual body degradation occurs through lysosomal cytoplasmic autophagy.

Triparanol and 20,25-diazacholesterol inhibit cholesterol biosynthesis and result in the accumulation of desmosterol. AY-9944, another inhibitor, produces an accumulation of 7-dehydrocholesterol. Adult male C3H mice receive one of these drugs intraperitoneally. Livers, adrenal glands, and testes from each drug group are excised, and portions of each are analyzed by a modified Liebermann-Burchard reaction for quantitation of sterols. Adrenals and testes are examined also by electron microscopy. Fine-structural localization of acid phosphatase has been studied in triparanol-treated adrenal glands. Biochemical analysis reveals that 14-64% of the sterols occurs as desmosterol or 7-dehydrocholesterol. Fine-structural alterations in the adrenal glands and testes from each drug group are essentially identical. The predominant cytological feature is the occurrence of increased numbers of pleomorphic, unit-membrane-limited, electron-opaque, cytoplasmic inclusions. Hence, the cellular modifications following triparanol administration are not unique, as has been suggested. They represent a generalized phenomenon, probably related to inhibition of cholesterol biosynthesis, which is an effect common to each drug. Lead phosphate reaction product (indicating acid phosphatase activity) is demonstrable within these membrane-limited cytoplasmic bodies, identifying them as morphological lysosomes. The utilization of a lysosomal mechanism in sterol-synthesizing cells, which are accumulating cholestercl intermediates, is discussed.