Fine structural localization of 3beta-hydroxysteroid dehydrogenase in rat corpus luteum

Synopsis A procedure for the ultracytochemical demonstration of 3-hydroxysteroid dehydrogenase (3-HSD) is described and the results for the localization of this enzyme in the lutein cells of the rat corpus luteum are presented.The procedure involved pre-fixation with aldehydes and incubation of small blocks. Short fixation (maximum 30 min) in 0.1% glutaraldehyde, 2% depolymerized paraformaldehyde or in 6.25% hydroxyadipaldehyde was found to be the best compromise for the preservation of both 3-HSD activity and fine structure. The method utilizes 3-hydroxy-5-androstan-17-one as substrate, tetranitro blue tetrazolium as a final electron acceptor, and phenazine methosulphate or menadione as an intermediate electron carrier to bypass the NADH₂-diaphorase. Control and inhibitor (cyano-ketone) experiments provide evidence for the specificity of the cytochemical reaction.Results showed that 3-HSD activity is localized on or in membranes of smooth endoplasmic reticulum, in outer compartments of mitochondria, and possibly within smooth endoplasmic reticulum cisternae and intracristal spaces of mitochondria.Localization of NADH₂-diaphorase activity in the same tissue was also examined. With the ferricyanide techniques, the reaction product was found to be associated mainly with the mitochondria.