In Vitro Assays of Aromatase and Their Role in Studies of Estrogen Formation in Target Tissues1

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 estro gens 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 quan tification and characterization of aromatase activity in periph eral and target tissues are now available. One of these, based on the quantification of 3H2O formed from [1-/?-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 tem poral 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 popula tions. 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 fol licles and localize cells that may be involved in steroidogenesis, including aromatization. Our findings underscore the need for new quantitative cytochemical and immunocytochemical as says to localize and to measure the amount and activity of aromatase in situ within identified populations of target cells. The primary role of steroid aromatase in steroidogenic glands in determining levels of circulating estrogens is self-evident. But steroid aromatase is now recognized to be distributed widely in tissues outside steroidogenic glands. The cumulative effect of these so-called peripheral sites on estrogen produc tion rates and on circulating hormone levels can be gauged from in vivo studies such as those discussed by others at this conference (9). Clearly, extraglandular tissues and organs with a capacity to aromatize C-19 precursors can have a measur able effect on the amount of estrogen to which target organs are exposed. The importance of this in normal females during the reproductive years may be debated. There is, however, little doubt that extraglandular aromatization as a source of circulating estrogens assumes significance when the contri bution made by the ovaries is diminished or eliminated through either the normal process of aging or use of drugs that arrest cyclic ovarian function and that this contribution may be in creased by obesity (4, 8). The significance of aromatization in peripheral tissues, how ever, may not be limited to the contribution made to circulating estrogen levels. At least some of the peripheral tissues or organs in which conversion of C-19 precursors to estrogens has been demonstrated, notably the brain and mammary gland, are also important targets for the action of estrogen. Fat cells must also be considered in this category, since they not only aromatize steroids (2, 13, 17) but also possess estrogen re ceptors, concentrate estrogens in their nuclei, and form pro gesterone receptors in response to stimulation by estrogens (6, 7, 25). Aromatization in target organs therefore must be considered as part of the general phenomenon of formation of steroid metabolites within target cells, metabolites with actions different from those of the circulating steroids from which they are derived. This phenomenon, first identified in relation to 5areduction (1, 28), may be viewed as a mechanism for diversi fication and amplification of hormone action. This mechanism should allow for the generation at discrete sites of concentra tions of active metabolites different from those in the general circulation and for controlling these local concentrations by modulating the activity of the enzymes responsible for the formation of the metabolites. The brain is one target organ in which the significance of in situ estrogen formation has been investigated in some detail over the last decade. There is evidence now that estrogen formation in this organ can play a crucial role in both neuronal differentiation during development and neuronal function in the differentiated organism (11). It may be useful, therefore, to examine some of the principles and concepts that are evolving from studies of brain and their relevance to the significance of aromatization in breast tissue. The level of aromatization in the hypothalamus and amyg dala, as in the breast, is low. Hypothalami of adult rats were found to convert in vitro only about 0.02% of a C-19 substrate to estrogen during a 1-hr incubation. The corresponding figures for hypothalami of neonatal rats are greater but still less than 1% of the substrate (26). Yet, as discussed at this symposium (11), there is good evidence to support the thesis that, at least in this species, aromatization in the CMS2 plays an essential 1Presented at the Conference "Aromatase: New Perspectives for Breast Cancer," December 6 to 9, 1981, Key Biscayne, Fla. This work was supported by NIH Grant HD09542. tion. 2 The abbreviations used are: CNS, central nervous system; PC, postconcep-