Characteristics of High Affinity Progesterone Binding to Rat Placental Cytosol*

Abstract

An exchange assay has been developed to study the progesterone receptor from rat placental cytosol prepared on the 12th day of pregnancy. Specific binding of progesterone was optimal in a buffered medium containing 10 mm Tris, 30% (vol/vol) glycerol, and 1 mm dithiothreitol, pH 7.8, at 4 C for 18–24 h. There was no detectable degradation in binding up to at least 43 h of incubation under these conditions. However, binding rapidly declined at elevated temperatures. Exposure of placental cytosol to N-ethymaleimide eliminated specific binding without affecting nonspecific binding, whereas exposure to HgCl₂ decreased specific binding by 91% but enhanced nonspecific binding 324%. Scatchard analysis of [³H]progesterone binding to receptor yielded a K_d of 3.4 ± 0.3 nm and 0.25 ± 0.04 pmol binding sites/mg placental cytosol protein or 11.3 ± 1.8 pmol/placenta (mean ± sem for eight experiments). Several structurally similar corticosteroids were weak competitive inhibitors of progesterone binding. The Ki was 46.0 ± 13.0 nm for llβ-hydroxyprogesterone, 189.9 ± 8.3 nm for deoxycorticosterone, and 194.0 ± 78.7 nm for corticosterone, and cortisol did not compete with progesterone for receptor (mean ± sem for three experiments). Estradiol administration enhanced the concentration of the placental cytosol receptor 200% over the control level on day 12 of pregnancy. Progesterone injection 24 h after last estradiol injection depleted receptor concentration 1 and 4 h later, but replenishment was observed 8 h post injection. Thus, a placental cytosol progesterone receptor exists in the rat which exhibits characteristics similar to those described for the progesterone receptor from rat uterus. Furthermore, these studies strengthen the notion that the action of progesterone on the maintenance of placental function may be mediated via mechanisms consistent with the general concept of steroid hormone action. (Endocrinology106: 1861, 1980)