The regulation of estrogen secretion plays a crucial role in many physiological processes, particularly reproduction, fetal development, sexual behavior, and in several pathological processes, such as estrogen-dependent carcinomas and heart disease in aging males. Despite the large number of studies describing the regulation of estrogen synthetase (aromatase) in human steroidogenic tissues by gonadotropins steroids and cAMP, very few studies address the mechanism of aromatase activity regulation. The eventual goal of this project is to determine the mechanisms by which this cytochrome P-450 enzyme system is regulated by physiological factors in cell cultures derived from estrogen-secreting human tissues. To accomplish this objective access is required to the tools necessary to properly carry out definitive studies of this important system, i.e., purified aromatase protein components and monospecific antibodies against these components. Since the NADPH-cytochrome P-450 reductase component of aromatase is already purified to greater than 99% homogeneity on this project, and monospecific (polyclonal and monoclonal) antibodies against this protein are available, our efforts will concentrate on the purification to homogeneity of the aromatase P-450 component, already partially purified with rabbit antiserum against it on hand. Both protein components will be characterized and the reconstitution of aromatase studied to determine Km values and how the sensitivity to carbon monoxide inhibition depends on the relative ratios of the two protein components. Monoclonal antibodies against the aromatase P-450 component will be procurred using a unique detection scheme, that utilized the transfer of 3H from a non-volatile to a volatile compound in the absence of homogeneous antigen. We also propose to synthesize mixed sequence synthetic oligonucleotide probes (15- to 25-mer) based on a partial amino acid sequence of the protein components. Because of ethical limitations on human experimentation, we intend to fully develop human cell cultures from term placenta, granulosa and theca cells from pre- and peri-ovulatory ovarian follicles, and endometrial and adipose tissue stromal cells for eventual use in determining the mechanism of aromatase regulation. Studies using antibodies against placental aromatase to determine cross-reactivity against non-placental aromatase will be conducted.
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