20 alpha-Hydroxysteroid dehydrogenase activity in the rat corpus luteum; a quantitative cytochemical study

A quantitative cytochemical method for the demonstration of 20 alpha-hydroxysteroid dehydrogenase activity (20 alpha-HSD) in the regressing corpora lutea of the adult rat ovary is described. The method employs unfixed tissue sections and relies upon the oxidation of 20 alpha-hydroxy-4-pregnen-3-one (20 alpha-OH-P) with nitro blue tetrazolium as the hydrogen acceptor. The enzyme was dependent upon NADP+ for its activity and was inactive when 20 beta-hydroxy-4-pregnen-3-one (20 beta-OH-P) was used as a substrate. The apparent Km values for 20 alpha-OH-P and NADP+ were 3 x 10(-4) M and 2.5 x 10(-5) M respectively. Inhibition of 20 alpha-HSD activity by steroids was demonstrable at pH 8. Androstenedione was by far the most potent inhibitor, followed by progesterone (the product of the enzyme activity) 17 alpha-hydroxyprogesterone, Compound S and 20 beta-OH-P. At pH 6.8, a pH more favourable to the progesterone leads to 20 alpha-OH-P reaction, only progesterone and 17 alpha-hydroxyprogesterone were inhibitory. Testosterone was without demonstrable effect at either pH.