The Estrogen Receptor of Rabbit Corpus Luteum: Binding, Dissociation, and Stability Characteristics

Abstract

Specific binding of estrogen to luteal cytosol and nuclei from pseudopregnant rabbits is a saturable process in vitro. High affinity, low capacity hormone retention is entirely attributable to components of cytosol; serum proteins play no part. The luteal estrogen receptor is unstable in conventional sucrose density gradient analysis and partially dissociates, even under conditions of low ionic strength, from a 6.7S to a 4S form, but stabilization can be achieved by maintaining cytosol temperature at 4 C and centrifuging through gradients containing tritiated estrogen and/or heparin. The heparin-stabilized receptor retains a strong binding specificity towards estrogens and displays little or no affinity for progesterone or 5a-dihydrotestosterone. Exposure of cytosol to an elevated temperature (37 C) completely destroys binding capacity. The 6.7S stabilized receptor dissociates to a 4S form under conditions of high ionic strength. At the same time, estrogen uptake is increased by nonspecific factors. Natural and synthetic estrogens compete for nuclear estradiol-binding sites, the order of preference being 17β-estradiol ≥ diethylstilbestrol > estriol ≥ estrone. The antiestrogen, nafoxidine hydrochloride, is a poor competitor. Testosterone and progesterone do not compete for nuclear estrogenbinding sites, but 5α-dihydrotestosterone and, more significantly, 20α-hydroxypregn-4-en-3-one in high concentrations do interfere with the nuclear uptake of estrogen. Nonspecific binding increases and specific binding decreases as the temperature of the exchange assay is raised. These results suggest that the estrogen recognition mechanisms of rabbit luteal cytosol are much more sensitive to temperature and mechanical manipulation than has previously been reported. This may explain the inconclusive nature of previous attempts to demonstrate an estrogen-induced stimulation of rabbit luteal progestogenesis in vitro. Moreover, the regulation of estrogen receptor uptake by rabbit luteal nuclei may be influenced by high concentrations of nonestrogenic steroids.