Investigation into the regulation of estrogen biosynthesis in the human is one of the virtually unexplores frontiers of steroid hormone biochemistry that has important ramifications into such diverse areas as reproduction, sexual development and cancer. The experiments proposed in this study are designed to investigate mechanisms by which the activity and biosynthesis of estrogen synthetase (aromatase), the enzyme which converts androgens to estrogens, are regulated by natural processes in steroidogenic tissue. Aromatase from the microsomal fraction of human term placental tissue, a rich and easily available source of the human enzyme, will be purified and the components of the cytochrome P-450 mono-oxygenase system responsible for aromatization will be identified and characterized. The re-constitution of aromatase activity from its purified components will be studied as well as the effects of environment (e.g., pH, ionic strength, temperature, lipid composition), steroid (e.g., progestins, corticoids, androgens, estrogens) and non-steroid compounds (e.g., gonadotropins, protaglandins, cyclic AMP) on reconstituted aromatase and on placental microsomal aromatase. The rate-limitine component of aromatase in placental microsomes, trophoblast cell culture derived from human choriocarcinoma, and human ovarian tissued homogenate will be determined by incorporating each purified aromatase component individually into the membrane fraction of the system of interest and measuring the effect on the aromatization rate. Antibody to the purified aromatase components will be obtained and used, in conjunction with pulse-labeling with radioactive amino acids, to measure the rate of biosynthesis of these components in steroidogenic tissue (primary placental cell/organ culture, choriocarcinoma cell culture, primary ovarian granulosa cell culture) before and after stimulation of estrogen secretion by gonadotropins, prostaglandins and cyclic AMP.
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