Analysis of [ 3H]estradiol binding to nuclei prepared from epididymides of sexually immature intact rabbits

Abstract

The estrogen receptor that is present in the epididymis of sexually immature rabbits is capable of entering nuclei in the time- and temperature-dependent manner characteristic of such reactions. Nuclear translocation reaches a maximum after 1 h of incubation at 23°C. Even short incubations (15 min) at 37°C destroy the cytoplasmic receptor and as a consequence nuclear translocation at this temperature is precluded. We have noted that a small, but demonstrable, amount of receptor enters nuclei at 0°C. Nuclear translocation is absolutely dependent upon estradiol binding to the receptor as shown by the fact that inhibitors of estradiol binding to the receptor (unlabeled estradiol, estrone and estriol) prevent translocation. Compounds that do not prevent estradiol binding to the receptor (5α-dihydrotestosterone, progesterone and cortisol) have no effect on the translocation of the estradiol-receptor complex to nuclei. Nuclei incubated with [ 3H]estradiol in the absence of receptor did not take up the hormone. Nuclear binding of estradiol falls into two categories: (a) that which is readily extractable with 0.4 M KCl; and (b) that which is resistant to KCl extraction. The resistant fraction is further subdivided into a fraction from which label can be readily extracted with ethanol and an ethanol-resistant residual fraction. The KCl-soluble fraction represents about 30% of the total radioactivity associated with nuclei. Acceptor sites for the estradiol-receptor complex are not limited to target cell nuclei since the complex is apparently bound to nuclear constituents in two presumptive nontarget tissues (spleen and skeletal muscle). However, the number of acceptor sites in target cell nuclei is demonstrably greater than in nontarget cell nuclei.