KINETIC-STUDIES ON ANDROSTENEDIONE PRODUCTION IN OVARIAN MICROSOMES FROM IMMATURE RATS

Androstenedione formation from progesterone by P-450(17-alpha,lyase) was investigated in ovarian microsomes of immature rats treated with pregnant mare serum gonadotropin. Successive monooxygenase reactions in the formation of androstenedione without the intermediate leaving P-450(17-alpha,lyase) were demonstrated by a double-substrate double-label experiment using [C-14]progesterone and 17-alpha-[H-3]hydroxyprogesterone as substrates and also by specific reduction in the concentration of intermediate 17-alpha-hydroxyprogesterone in the reaction medium by reaction of liposomal P-450C21. A detailed kinetic study on the reactions of P-450(17-alpha,lyase) in microsomes was conducted in the steady state. Kinetic parameters indicated the C17,C20-lyase reaction for 17-alpha-hydroxyprogesterone (K(m) = 80 nM) to be strongly inhibited by progesterone (K(i) = 8 nM). In the presence of a high concentration of progesterone, as in the case of in vivo rat ovary, most androstenedione is concluded to be formed directly from progesterone by successive monooxygenase reactions catalyzed by P-450(17-alpha,lyase). 20-alpha-Dihydroprogesterone competitively inhibited the C17,C20-lyase reaction for 17-alpha-hydroxyprogesterone with K(i) = 23 nM, but had only slight effect on progesterone metabolism to androstenedione. 20-alpha-Dihydroprogesterone, thus, cannot be a regulator for androstenedione formation in rat ovary.