A novel oestrogen-binding species in rat granulosa cells

Abstract

ABSTRACT The dissociation constants (Kd) and steroid specificities of oestrogen-binding species in rat granulosa cell cytosol and nuclei have been studied. Preliminary work, where diethylstilboestrol was employed as competitor in binding assays, identified the oestrogen receptor in whole ovarian tissue nuclei (Kd 0·35 ±0.09 nmol/l) and cytosol (Kd 0·39 ± 0·03 nmol/l). Isolation of granulosa cells revealed that the majority of this receptor (75–96%) was present in these cells. Specificity studies on the binding of [3H]oestradiol in granulosa cell cytosol indicated the presence of an additional class of oestrogen-binding sites which were, however, not present in nuclei. Saturation analysis over an extended range of [3H]oestradiol concentrations and using unlabelled oestradiol as competitor revealed a binding species of Kd 45·8± 6·9 nmol/l (capacity 16·7 pmol/mg cytosol protein) for oestradiol in addition to the cytosol oestrogen receptor of Kd 0·58 ± nmol/l (capacity 2·8 pmol/mg cytosol protein). The low affinity of this novel species implies that the dextran-coated charcoal techniques used in previous studies on ovarian oestrogen-binding species would cause dissociation of ligand and not allow it to be measured. The second oestrogen-binding species displayed affinity for oestradiol-17β, oestriol, oestrone, testosterone, 5α-dihydrotestosterone, methyltrienolone, progesterone and the antioestrogens tamoxifen, nafoxidine and clomiphene citrate. The species, however, did not bind diethylstilboestrol, a characteristic shared with other low affinity cytosol oestrogen-binding species which have been reported in dog prostate, chick oviduct and male rat liver but not shared with uterine type II oestrogen receptors. It can be further distinguished from the oestrogen receptor by differential ammonium sulphate precipitation and the stability of its ligand binding at temperatures above 55 °C where the oestrogen receptor–ligand interaction is rapidly lost. Concentrations of nuclear oestrogen receptor in granulosa cells (2200 sites/cell) were similar to those found in other target tissues but a high proportion of this receptor (70%) was 'unoccupied' or available for binding at 4 °C and the majority (75%) was resistant to extraction with 0·4 m-KCl. As the second oestrogen-binding species could not be detected in granulosa cell nuclei it is unlikely to be involved directly in eliciting genomic responses to hormonal stimulation. It is more probable that it regulates the level of the free intracellular steroid to which the oestrogen receptor of the granulosa cell (the predominant site of oestrogen biosynthesis) is exposed. J. Endocr. (1984) 102, 83–91