The influence on diaminobenzidine staining of four variables: prefixation in aldehyde, temperature and pH of incubation, and H2O2 concentration, was investigated in catalase-, as well as in peroxydase-containing material. Catalase from five different sources and five types of peroxidase were examined. It is concluded: (a) when cells are incubated without prior fixation, in a DAB medium at room temperature and pH 7.3 with 0.003% H2O2, peroxidases produce a visible cytochemical stain, while catalases do not; (b) the cytochemical reaction elicited by catalases is stimulated by prior aldehyde fixation in specified conditions, and incubation at 45 degrees C and pH 9.7 with 0.06% H2O2; (c) under the latter circumstances several peroxidases also stain. Ultrastructural preservation is satisfactory in tissues incubated prior to fixation.

Release of apoptogenic factors into the cytosol including cytochrome c is triggering the execution phase of apoptosis through activation of cytoplasmic effector caspases. How loss of function of the electron transport chain can be reconciled with an adequate energy supply necessary for executing the apoptotic program was studied in granulosa cell (GC) sheets cultured up to 72 h without gonadotrophic support. Cytochrome c was localized ultrastructurally by oxidation of diaminobenzidine tetrahydrochloride both in living and fixed cells. In uncultured GC sheets all cells show staining over their entire mitochondrial population. In 72 h cultured sheets in the absence of FSH pre-apoptotic GC's display two subsets of mitochondria: normal sized stained mitochondria and small orthodox mitochondria without demonstrable cytochrome function. Apoptotic cells contain several mitochondria with preservation of respiratory function besides unstained orthodox mitochondria. The cytochrome c containing mitochondria typically display dilated intracristal spaces, a mitochondrial conformation related to increased ATP production. Cytochrome c release was confirmed by Western blotting. In 72 h cultures supplemented with FSH, GC's displayed staining over their entire mitochondrial population. In cultures lacking FSH, but partially protected from apoptosis through caspase inhibition, the cytochrome c release was not inhibited. Thus in the present studied model dysfunction of only a subset of mitochondria is instrumental to initiate the apoptotic program while a functional electron transport chain is maintained until the degradation phase in a subset of respiring mitochondria. Cell Death and Differentiation (2000) 7, 331 ± 337.

Summary ― Follicular atresia in the ovaries of Japanese quail was studied by cytochemistry and electron microscopy. Three

The biosynthesis, transport and degradation of catalase have been studied in the guinea pig liver parenchymal cell using 2-allyl-2-isopropylacetamide (AIA) as an inhibitor of de novo formation of catalase. Total catalase activity was assayed biochemically; cytoplasmic catalase was measured microspectrophotometrically after quantitative diaminobenzidine staining of the liver. By morphometry, number and size of peroxisomes in catalase stained sections were determined. From our data we conclude that (1) the final step in the catalase formation takes place inside peroxisomes, (2) catalase is transported from the peroxisomes into the cytoplasm, (3) in the cytoplasm catalase is degraded. These conclusions in part confirm the topological model on the intracellular catalase biosynthesis pathway of Lazarow and de Duve (1973) except for the presence of cytoplasmic catalase which is released from the peroxisomes as proposed earlier by Jones and Masters (1975).