One of the important functions of the mammalian ovary is the production of steroid hormones that have a multitude of physiologic and behavioral effects upon the animal. Unlike the testes or adrenals, the ovaries alter both qualitatively and quantitatively the output of steroids in a cyclic fashion which accounts for the mainifestations of the estrous or menstrual cycles. The control of the synthesis of these steroids by pituitary gonadotropins has been under investigation for some time but many aspects remain unresolved. The rate limiting step in steroid production is well recognized as the conversion of cholesterol to pregnenolone within the mitochondria. However, other steps are known to be under gonadotropic control; among these are the induction of several enzymes. The goal of our research is the delineation of the role played by, and the control of, 17Alpha-hydroxylase and C17,20-lyase in ovarian steroid production. These enzyme activities, thought by many to be actions of a single microsomal cytochrome P450, are involved in the conversion of progestins (pregnenolone and progesterone) to androgens. The androgens then serve as substrates for synthesis of estrogens by the aromatase of the ovary. We will use the measurement of tritiated water, produced by the conversion of 17Alpha3H-progesterone to 17-hydroxyprogesterone as an index of hydroxylase activity. The amount of C14H3C00H (acetic acid) produced from 21(C14)-progesterone will be used to evaluate C17,20-lyase activity. The ovarian content of cytochrome P450 and cytochrome C reductase will be measured spectrophotometrically and correlated with the activity of the enzymes under a variety of conditions. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis will be used to visualize changes in microsomal proteins and to correlate these changes with those of enzyme activity and cytochrome content. The enzymes will be altered by treatment of intact and hypophysectomized prepubertal rats with human chorionic gonadotropin or pregnant mare's serum gonadotropin alone or in combination with inhibitors of ribonucleic acid (actinomycin D), protein (cycloheximide, puromycin) or steroid (aminoglutethimide) synthesis. Also the inhibitory effect of prolacin upon the enzyme system stimulated by gonadotropin will be examined. The results will contribute to our understanding of the mechanisms by which steroid hormone synthesis, particularly androgen and estrogen synthesis, is controlled.
