The Binding of Estradiol-17β to the Bovine Endometrial Nuclear Membrane

Abstract

Abstract The interaction of estradiol-17β with mature bovine endometrial tissue, and with isolated nuclei has been studied. The hormone binds to an insoluble nuclear fraction. This fraction contains membrane and evidence is presented to show that estradiol is bound to the nuclear membrane. Incubation of isolated nuclei and microsome fractions with estradiol-17β shows that the hormone binds essentially instantaneously to microsomes and nuclei. Such binding is non-saturable up to estradiol-17β concentrations as great as 2.5 x 10-6 moles per mg of membrane protein and it is likely that this interaction is not biologically significant. A second form of binding is observed in nuclei which is of higher affinity and saturable, with 507 ± 47 sites per nucleus. This class of binding sites is found to be blocked in cattle that are maintained in artificially induced estrus by feeding with diethylstilbesterol. The high affinity binding site is present only in the mature endometrium and is absent in nuclei from the mature myometrium (the muscular tissue surrounding the endometrium in the uterus) and the immature uterus. Potent estrogenic agents compete effectively with estradiol-17β for binding to this site, whereas weak estrogenic steroids (such as progesterone and testosterone) are inefficient competitors. The sensitivity of the high affinity site to pH and hydrolytic enzymes has been studied and compared with the effect of such agents on the low affinity, nonspecific, membrane site. Such a comparison underscores the inherent differences between these two sites of interaction. The thermodynamic parameters of the high affinity binding site have been measured and compared to reported values for the specific cytoplasmic receptor binding site. The nuclear high affinity binding site possesses a favorable free energy of interaction as a result of entropic forces (ΔS0 = +36.4 cal deg-1 mole-1, ΔH0 = -0.66 Cal mole-1). The number of high affinity nuclear sites varies dramatically during the estrous cycle in a bicyclic fashion. Sites are available during estrus and diestrus, whereas they are blocked in later metestrus and proestrus.