Estrogens and the Hypothalamus: Nuclear Receptor and RNA Polymerase Activation

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Abstract
To further the understanding of estrogen action in the central nervous system, we have developed a procedure for quantitation of nuclear estrogen receptor (RnE) in adult rat brain. Crude chromatin is separated from soluble and membranous fractions by centrifugation of brain homogenate through 1.2 M sucrose pads. Incubation of the pellet with [3H] estradiol at elevated temperature and precipitation of receptor-steroid complexes with protamine sulfate allows measurement of RnE. The yield of DNA by this procedure is 70–80% and the amount of RnE measured is linear with respect to the amount of DNA added. Using this procedure, we have measured hypothalamic RnE in the ovariectomized adult rat as a function of time after injection of 5 ug estradiol and found a peak RnE accumulation of about 30 fmol/hypothalamus at 1 h. By 12 h, the levels of RnE return to control (uninjected) values. We have also measured the time course of estradiol induced activation of hypothalamic endogenous nuclear RNA polymerases I and of II, suggested to be initial steps in estrogen action. RNA polymerase I activity was unaffected by estrogen treatment, while RNA polymerase II activity showed a transient activation with a time course approximating that of RnE accumulation. For early times after estrogen treatment (10–90 min), the amount of RnE present in the hypothalamus is correlated with the extent of RNA polymerase II activation. The absence of sustained activation of RNA polymerases I or II in the hypothalamus is consistent with the lack of long term retention of RnE in nuclei of this, tissue and the lack of estrogen induced hypertrophy and hyperplasia.