Periodate Treatment and “All-or-None” Dehydrogenase Staining Phenomena

Abstract

It has been shown that when suspensions of fresh rodent sperm are incubated in the presence of tetranitro-blue tetrazolium (TNBT) and stained for succinic dehydrogenase (SDH),this enzyme is localized in the mitochondria of the middle-piece. Rat and mouse spermatozoa treated in this manner and postfixed (suspension) in 4°C Millonig's osmium tetroxide for brief periods demonstrate SDH reactivity in mitochondrial profiles that indicate no SDH, almost complete activity, or most commonly, intermediate staining reactions in the internal membranes (Figure 1). In the last instance, the cristal membranes display a heterogeneous formazan (reduced TNBT) staining pattern not in agreement with the Seligman “all-or-none” hypothesis concerning mitochondrial oxidative enzymatic reactivity.In order to test this hypothesis and circumvent some of the attendant difficulties in these procedures (penetration of reaction mixture components, fixatives), all cytochemical reactions and postfixation were performed on suspensions of sperm from the cauda epididymides of mature rats. An attempt was made to divorce electron densities due to osmium postfixation by employing 1% aqueous sodium periodate (pH 3-4) as a mitochondrial membrane stabilizer through the embedding procedures. Previous use of this treatment allowed preservation of the membranes of these organelles in positive contrast as evidenced by uranyl acetate staining of sectioned material (Figures 2 & 3).