Analysis of the progesterone receptor in the beagle uterus and oviduct

Abstract

Uterine and oviductal cytosols from immature and cycling adult beagles contain macromolecules which bind progestins specifically and with high affinity as determined by an exchange assay utilizing the synthetic progestin, R5020. The [ 3H]-R5020 bound to the progesterone receptor 2 to 3 times more tightly than progesterone and apparently did not bind to corticosteroid-binding globulin (CBG). Cytosols were incubated with 30 nM [ 3H]-R5020, in the presence or absence of a 250-fold excess of unlabeled R5020, for 4 to 6 h at 4°C, conditions appropriate for exchange of labeled R5020 for endogenously bound steroid. Dextran-coated charcoal was used to separate bound from free ligand. The progesterone receptor in the female beagle, characterized using this standard assay, was tissue and steroid specific. Uterine cytosols contained two [ 3H]-R5020 binding peaks (4S and 7–8S) on sucrose density gradients. The progesterone receptor bound [ 3H]-R5020 with an apparent dissociation constant ( K D ) of approximately 2.5 nM according to Scatchard plot analysis. The concentration of uterine and oviductal progesterone receptors was under the inductive influence of estradiol. Measurable specific [ 3H]-R5020 binding increased dramatically following administration of estradiol-17 β, but was absent in uteri and oviducts of untreated pups. These data have demonstrated the presence of a specific progesterone receptor in the beagle oviduct and uterus which displayed characteristics similar to those reported for progesterone receptors in other mammalian species.