The objective of the Proposed Research is to define the molecular events that mediate the induction of aromatase activity in ovarian granulosa cells, adipose tissue stromal cells, and placental cells; the principal cell types in women that synthesize estrogens from C19-steroid precursors. The putative stimulatory factors for aromatase activity in these cell types are: follicle-stimulating hormone, androgens, and dibutyryl cyclic AMP in ovarian granulosa cells; dexamethasone, adrenocorticotropin and dibutyryl cyclic AMP in adipose stromal cells; and human chorionic gonadotropin and dibutyryl cyclic AMP in human placental cells. Aromatase is an enzyme complex comprised of a specific form of cytochrome P-450 and of NADPH-cytochrome P-450 reductase. Aromatase cytochrome P-450 and NADPH-cytochrome P-450 reductase will be purified from human placental microsomes; polyclonal antibodies to these proteins will be raised in rabbits, and monoclonal antibodies will be obtained from mouse spleen cell-myeloma cell hybridomas. These antibodies will be used to study the effects the putative stimulatory factors on the rates of synthesis of aromatase cytochrome of P-450 and NADPH-cytochrome P-450 reductase in estrogen-producing cells, as well as the effects of stimulatory factors on the cellular levels of translatable mRNA species that encode for these proteins. The molecular weights of the aromatase enzyme components, immunoisolated from in vitro translation assays, will be compared to those of the native proteins to determine whether either of these enzyme proteins is synthesized in a precursor form. In addition, the cellular levels of aromatase cytochrome P-450 and NADPH-cytochrome P-450 reductase, as well as the translatability of the mRNA's that encode for these proteins, will be studied in granulosa cells derived from ovarian follicles at different stages of maturity. Poly(A)-containing RNA from human placenta will be used to prepare a complementary DNA (cDNA) specific for aromatase cytochrome P-450 mRNA. This cDNA probe will be used in hybridization studies to quantify the effects of putative stimulatory factors on the levels of this species of mRNA in estrogen-producing cells. These studies will provide new insight into the cellular mechanism(s) by which estrogen synthesis is regulated.
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