Introduction

Studies in this laboratory on estrogen-binding protein started as a result of the publication of the elegant work of Jensen and his colleagues in the early 60′s [5]. Note-boom and Gorski [6,7] showed that the binding of estrogen in the uterus was principally in the nucleus and cytosol, was stereospecific, and probably associated with a protein. Toft and Gorski [13, 14]showed that a cytosol protein that bound estrogen could be resolved on sucrose gradients. This was followed up by Toft, Shyamala and Gorski [15]who showed that the binding of estrogen could be carried out in cell-free systems. A possible role for the binding protein was demonstrated by the finding that estrogen appeared to cause the binding protein to migrate into the nucleus [4, 11, 12]. The thinking in this laboratory about the estrogen receptor is presented in reviews [4; Current Topics in Developmental Biology, 1969, ed. Monroy & Moscona]. Suggested effects of the receptor on gene expression have been previously reported. [3].

The regulation of estrogen binding protein concentration appears to involve four periods in which different conditions prevail. The first period occurs during development and would start with the development of uterine primordia. At the present time, only the postnatal period in the rat has been studied [2].

Following development, the next period in the rat runs from about 10 days to sexual maturity in the rat. This is a period of relatively little change in receptor numbers per cell, and synthesis and turnover are in equilibrium.

The third stage occurs when the uterus is exposed to estrogen (Fig. 1). The hormone is bound to the large binding protein (8S) in the cytoplasm and then appears to move into the nucleus [3]. As a result of this movement, the binding protein is depleted in the cytoplasm and is then gradually replenished. This replenishment process has turned out to be very complex and possibly reflects complexities in the structure of the binding protein. This is most dramatic when estrogen is injected, but also occurs during the natural rise and fall of estrogen levels during the estrous cycle.

The fourth and final period is that which occurs following continued estrogen exposure, such as in pregnancy. We have not looked at this stage to any great extent, and we will therefore exclude this period from the following discussion.

Ontogeny of the estrogen binding protein Period 1: The development of uterine binding

The ability of the rat uterus to respond to estrogen increases during the first 10 days after birth [5]. If the estrogen binding protein has some relationship to tissue response, one might expect the binding protein concentration to increase in a manner corresponding to the change in uterine response. Fig 2 shows that the concentration of estrogen binding sites per unit of DNA or per cell increase about 4-fold during the first 10 days of postnatal development [2]. That this is a change in concentration and not physical state of the binding protein is shown in Fig. 3. The binding protein has the same relative affinities at 4, 10, and 22 days, as estimated by Scatchard plots. The similarity in size is indicated by the sedimentation velocity of 8S in both 10 and 22 day old rats. The change in concentration of estrogen binding sites was shown not to be dependent on the rats’ ovaries [2]. Other sites of control might be the pituitary or other endocrine glands, but no experimental evidence is yet available. We also have no information on binding site concentrations in prenatal uteri or in other species; either pre- or postnatal.

Period 2: Equilibrium

Once the rat reaches 10 days of age, estrogen binding site concentration per unit of DNA or per cell does not change, but rather it appears to reach an equilibrium between sythesis and degradation. The rate of synthesis is difficult to determine; however, an estimate of degradation rate can be obtained and, therefore, an indirect estimate of syn-

A. Sucrose density gradient profiles of uterine cytoplasmic fractions from 10 and 22 day old rats. 3H-estradiol (5 × 10−4 μg) was added to 0.2 ml of cytosol prepared from one rat uterus and layered on 5-20% sucrose gradients. Gradients were centrifuged at 35,000 rpm for 17.5 hr on a Model L. Spinco ultracentrifuge using a SW-39 rotor. B. Scatchard plots of EBP binding determined by the glass binding method. Kd for all three groups was approximately 3.0 × 10−9M and the number of binding sites 0.9 μμ moles/one 22 day uterus, 0.6 μμ moles/two 10 day uteri, and 0.23 μμ moles/three 5 day uteri [2]. thesis at equilibrium [6]. The protein synthesis inhibitor, cycloheximide, can be used to block synthesis of the binding protein while degradation continues. Fig. 4 shows the sucrose gradient patterns of estrogen added to uterine cytosol from rats treated for various time periods with cycloheximide. Treatment for 8 hr resulted in only a 5% drop in binding capacity. A summary of several turnover studies using three different assays for specific estrogen binding is shown in Fig. 5. These data have been used in Fig. 6 to calculate the rate of synthesis and the 1/2 life of estrogen binding sites on a per cell basis. We have also shown an analysis of rates of synthesis and turnover calculated from the data shown in Fig. 2 and based on the formulations of Berlin and Schimke [1]. It can be seen that estimates of synthesis and turnover by the two methods are very similar and add to our confidence in using them. These calculations suggest that the developmental period involves the establishment of a new rate of synthesis at about the time of birth, which then reaches a new equilibrium at 10 days after birth.

The rates of synthesis calculated above are quite low and could be handled by one polysome unit (one messenger RNA with proper number of ribosomes), making peptide bonds at approximately 1/7 the rate of polysome that synthesize hemoglobin.

Period 3: Depletion-replenishment cycle after estrogen

The model of estrogen...