In vitro assays of aromatase and their role in studies of estrogen formation in target tissues

Abstract

In recent years, there has been increasing recognition of the importance of steroid aromatization in organs besides the steroidogenic ones. Estrogens formed from C-19 precursors in peripheral tissues can clearly contribute to the levels of estrogens in the circulation, while aromatization in target cells may be an important determinant of the concentration of estrogens to which a particular population of target cells is exposed. Assays sufficiently sensitive and simple enough to permit quantification and characterization of aromatase activity in peripheral and target tissues are now available. One of these, based on the quantification of 3H2O formed from [1-beta-3H]androstenedione has, in our hands, a sensitivity of 15 fmol, is highly reproducible, and is relatively simple. It has been validated for stoichiometry between amounts of 3H2O and [6,7-3H]estradiol formed from [1-3H]androstenedione and [6,7-3H]androstenedione, respectively. Using this assay, we have been able to quantify aromatase activity in discrete brain nuclear regions dissected from Vibratome sections of fetal rat brains and thereby to identify in discrete areas sex differences and temporal changes during development that are obscured when larger tissue specimens are used. However, aromatase activity in brain nuclei as well as other target organs, including breast tissue, is likely to be concentrated in specialized cell populations. This heterogeneity limits the interpretation that can be placed on data obtained from standard "test tube" assays on homogenized tissue. We have used quantitative cytochemical assays for enzymes and cytochrome P-450 to identify regional specialization in the membrana granulosa of preovulatory follicles and localize cells that may be involved in steroidogenesis, including aromatization. Our findings underscore the need for new quantitative cytochemical and immunocytochemical assays to localize and to measure the amount and activity of aromatase in situ within identified populations of target cells.