The Biosynthesis of Cholesterol Side-Chain Cleavage Cytochrome P-450 in the Rabbit Corpus Luteum Depends Upon Estrogen*

To gain a better understanding of the luteotropic action of estrogen, we have investigated the effect of estrogen on the synthesis of the enzyme, cholesterol side-chain cleavage cytochrome P-450 (P-450scc) in the rabbit corpus luteum. Using an established protocol, rabbits were treated with estradiol, and the estradiol was then withdrawn on day 9 of pseudopregnancy, which caused an 88% fall in serum progesterone within 48 h. In other rabbits, estradiol was replaced at 48 h which stimulated a 6.6-fold increase in serum progesterone concentration within the next 24 h. Luteal tissues were incubated with [35S]methionine and homogenized, and a mitochondrial fraction lysate was obtained. Equal trichloroacetic acid-precipitable radioactivity was taken for immunoprecipitation using a well-characterized polyclonal antiserum against bovine adrenal P-450scc. The immunoisolated proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and radioactivity was visualized by autofluorography. The results indicate that the rate of synthesis of P-450scc in 48 h-estradiol withdrawn animals was markedly reduced, and by 72 h of withdrawal was barely detectable. When estradiol was reintroduced, the synthesis of P-450scc was increased. Despite the prominent changes in P-450scc synthesis, immunoblotting revealed only a minimal (approximately 30%) decrease in relative P-450scc content by 72 h after estradiol withdrawal. Analyses of DNA and protein contents of luteal tissues revealed an increase in DNA per mg luteal tissue, a decline in total tissue protein/DNA ratio, but no change in mitochondrial fraction protein/DNA ratio after estrogen withdrawal. The results indicate that de novo synthesis of P-450scc in the corpus luteum is sensitive to estrogen; however, the estrogen-sensitive rate-limiting step(s) for steroidogenesis are at other sites in the steroid biosynthetic pathway.