The Cholesterol Side Chain Cleavage Enzymes in Immature Rat Ovary

Cholesterol side chain cleavage reaction has been studied in ovaries of immature female rats pretreated with pregnant mare serum gonadotrophin and human chorionic gonadotrophin. The cleavage enzymes were found to be in the mitochondria and to have the characteristics of a mixed function oxidase requiring NADPH and oxygen. Using [4‐ 14 C]cholesterol as substrate the main product obtained was progesterone while pregnenolone and 20α‐hydroxypregn‐4‐en‐3‐one, were formed in smaller amounts. This shows that ovarian mitochondria contains a Δ 5 –3β‐hydroxysteroid‐dehydrogenase, a Δ 5–6/4–5 isomerase and a 20α‐hydroxy steroid dehydrogenase. The proposed intermediates in cholesterol side chain cleavage (20α‐hydroxy cholesterol and 20α, 22ɛ‐dihydroxy cholesterol) were not detected. Mitochondrial production of progesterone in the presence of NADPH was further stimulated by succinate while fumarate was without effect. In the presence of NADPH, NADH was found to be inhibitory while NAD + gave rise to an increase in pregnenolone, the overall reaction being unaffected. Excess of NADPH causes an accumulation of pregnenolone and slightly decreases the overall reaction. The action of reduced glutathione (GSH) and ascorbic acid‐like reducing agents of physiological occurrence has been studied. It was found that GSH in small amounts stimulates and in larger amounts inhibits the side chain cleavage reaction. Ascorbic acid in physiological amounts produced an inhibitory effect. The action of adenosine 3′,5′‐cyclic‐monophosphoric acid (3′5′AMP) and other nucleotides (AMP, ADP, ATP) was tested. 3′5′AMP markedly stimulates the conversion of cholesterol to pregnenolone and decreases the synthesis of progesterone. The action of AMP shows some similarities with that of 3′5′AMP but the action of ADP and ATP was found to be different.