Identification of outer mitochondrial membrane cytochrome b₅ as a modulator for androgen synthesis in Leydig cells

Outer mitochondrial membrane cytochrome b₅ is an isoform of microsomal membrane cytochrome b₅. In rat testes the outer mitochondrial membrane cytochrome b₅ is present in both mitochondria and microsomes, whereas microsomal membrane cytochrome b₅ is undetectable. Outer mitochondrial membrane cytochrome b₅ present in the testis was localized in Leydig cells with cytochrome P-450_17α, which catalyzes androgenesis therein. We therefore analyzed the functions of outer mitochondrial membrane cytochrome b₅ in rat testis microsomes by using a proteoliposome system. In a low but physiological concentration of NADPH-cytochrome P-450 reductase and excess amount of progesterone, outer mitochondrial membrane cytochrome b₅ stimulated the cytochrome P-450_17α-catalyzed reactions, 17α-hydroxylation and C17-C20 bond cleavage. The effects were different from those by micro. somal membrane cytochrome b₅ as follows: preferential elevation of the 17α-hydroxylase activity by outer mitochondrial membrane cytochrome b₅ in an amount-dependent manner versus that of the lyase activity by microsomal membrane cytochrome b₅ at the low concentration, and the inhibition of both activities at the high concentration. At a low concentration of progesterone reflecting a physiological cholesterol supply, outer mitochondrial membrane cytochrome b₅ elevated primarily the production of 17α-hydroxyprogesterone and then facilitated the conversion of the released intermediate to androstenedione. Thus, we demonstrated that outer mitochondrial membrane cytochrome b₅ and not microsomal membrane cytochrome b₅ functions as an activator for androgenesis in rat Leydig cells.