To test the validity of filipin cytochemistry for localization of cholesterol in testicular cells, we compared the results obtained by this technique with those obtained by a two-step enzymatic method involving cholesterol esterase and cholesterol oxidase. In all the animals models tested (guinea pig, mink, and mallard duck) the disappearance of subsurface filaments along Sertoli cell junctional membranes was accompanied by a significant increase in the number of filipin-cholesterol complexes/microns 2 in these membranes. Enzyme histochemistry allowed localization of free cholesterol in the limiting membrane of multivesicular bodies, in membranes within lysosomes, in mitochondrial membranes, and in junctional membranes, with or without subsurface filaments. The method also permitted selective visualization of cholesterol esters in lipid droplets. We conclude that filipin mapping of cholesterol induces false-negative cytochemical results. The enzymatic method is superior to filipin because it allows localization of free cholesterol in junctional membranes and of cholesterol esters in lipid droplets. This compartmentalization of the compounds may represent the basis of a system that helps to maintain constant free cholesterol levels in the testis.
Cell culture of middle-ear epithelium of the guinea pig. Histochemical localization of mitochondrial enzymatic activities in cultured cells.