Molecular mechanisms of steroid hormone action

Abstract

The differentiated chick oviduct is a target tissue for progesterone. Administration of the hormone to estrogen-primed chicks causes a rapid induction of new messenger RNA, resulting in the appearance in cytoplasm of specific mRNA species. One of these species is the mRNA coding for the egg white protein avidin, which is produced only in response to progesterone. The cytoplasm contains receptors which bind progesterone specifically and transport it as a complex into oviduct nuclei. Only a small fraction of the receptors are active in vitro; this fraction is increased to 30% by brief warming of the receptor-hormone complexes before incubation with the nuclei. The binding reaction is slow, and the receptors cannot be released from nuclei by DNase treatment. The reaction occurs preferentially with oviduct nuclei, which contain at least twice as many acceptor sites (8000/nucleus) as other chick tissues (< 2000/nucleus). The binding constants are the same in all tissues ( K d ~ 10 −8 M), and are identical to the constant for receptor binding to isolated chromatin. We have isolated the progesterone receptor component which binds to the chromatin. Oviduct cytosol from laying hens was prepared and progesterone receptors were precipitated with ammonium sulfate (30% sat.). The re-dissolved pellet was eluted from a steroid-affinity column (Sepharose 4B-BSA-deoxycorticosterone) with 3M urea. The receptors were reconstituted by dialysis and labeled with [ 3H]-progesterone. The [ 3H]-progesterone-receptor complexes were then purified by sequential chromatography and elution with the indicated slats at pH 74 from DEAE-cellulose (0.2 M KCl), phosphocellulose (0.26 M KCl), and hydroxylapatite (0.15 M K x PO 4). The peak fraction was finally chromatographed on an agarose A-1.5 M column ( K av = 0.28). Yield was 1 %, and purity approached the theoretical maximum specific activity, 10 9 d.p.m./mg protein.