C. Sumida

Pharmacodynamic and biological effects of anti-estrogens in different models

The biological response to anti-estrogens is very variable and depends on the animal species considered, the target organ, the parameter studied, and the experimental conditions. Anti-estrogens can bind specifically, (1) to the estrogen receptor, (2) to the typical anti-estrogen specific binding site, and (3) to low density lipoproteins in the plasma. Using a monoclonal antibody against the estrogen receptor, different immunological characteristics of the anti-estrogen-receptor complex can be observed. This difference could explain some of the different biological effects. Studies using different human mammary cancer cell lines (hormone-dependent) show that anti-estrogens are active in decreasing cell proliferation. Also, anti-estrogens can block proteins specifically produced by these cells. Some of these proteins could act as growth or inhibitory factors. Estrogen sulfates are the main precursors of estradiol in breast tissues and this conversion is significantly decreased by anti-estrogens. It is accepted that the main pathway of action of anti-estrogens is through the estrogen receptor, but recent information suggests the possibility that this is not the only step in the mechanism of action of anti-estrogens.

Specific [3H]-estradiol binding in the fetal uterus and testis of guinea pig. Quantitative evolution of [3H]-estradiol receptors in the different fetal tissues (kidney, lung, uterus and testis) during fetal development

Abstract Specific estradiol receptors have been found in fetal uterus in the cytosol fraction and in the nuclear extracts obtained by successive extractions with (A) 0.1 M Tris-HCl-0.0015 M EDTA, (B) 0.3 M NaCl-0.01 M Tris-HCl, and (C) 1 M NaCl-0.01 M Tris-HCl. In uterine cytosol, an estradiol-specific component with a sedimentation coefficient of 8.5-9 S in sucrose density gradient was detected. The K d of the binding of estradiol in fetal uterine cytosol is 4 × 10-10 M. Similar [3H] -estradiol receptors are present in fetal testis. A systematic study of the evolution of specific [3H]-estradiol binding during fetal development has shown the following results. The specific binding of [3H]-estradiol per mg protein in fetal kidney cytosol increased from no detectable binding at 34–35 days of gestation to 35 fmol/mg protein at 59 days with a subsequent decrease to 22 at 24 h after birth. Specific binding in the three nuclear extracts studied (0.1 M Tris, 0.3 M NaCI and 1 M NaCl) also increased from 11 fmol/mg protein at 34–35 days of gestation to 147 fmol/mg protein at 59 days and decreased to 50 at 24 h after birth. In fetal lung cytosol, specific [3H] -estradiol binding increased dramatically from 24 fmol/mg protein at 34–35 days to 434 at 59 days, decreasing to 345 fmol/mg protein at 24 h after birth. Nuclear binding also increased from 27 fmol/mg protein at 34–35 days to 329 at 59 days and decreased to 114 at 24 h after birth. In the fetal uterus, specific binding in the cytosol fraction is 900 fmol/mg protein at 34–35 days of gestation and increases to 500 fmol/mg protein at the end of gestation (55–60 days). In contrast, fetal heart showed very little or no specific binding of [3H]-estradiol.

Mineralocorticosteroid receptors in the foetal compartment

Abstract Mineralocorticosteroid receptors have been found in the foetal kidney of the guinea pig (at 25–40 days of gestation) in experiments carried out both in vivo and in vitro. More than 50% of the total d-aldosterone receptors were found in the nucleus. 20% of the total nuclear radio-activity was extracted by the 0.1 M Tris-HCl solution and 50% by the 1 M NaCl -0.01 M Tris solution, suggesting that at this period of the foetal evolution the aldosterone receptors are localized principally in the chromatin fraction. The formation of these [ 3 H]-aldosteronc-macro-molecule complexes is very rapid, maximum values being found at 4 min of incubation at 37°C. The nuclear receptors, but not those of the cytosol fraction, are temperature-dependent, d-Aldosterone and deoxycorticosterone compete with the [ 3 H]-aldosterone complex. On the other hand, d-aldosterone has no effect on the [ 3 H]-aldosterone-macromolecule complex if reincubation is carried out with the nuclear extracts (at 4°C) after extraction. Also, the 0.1 M Tris and 0.01 M Tris-1 M NaCl nuclear extracts do not form [ 3 H]-aldosterone complexes after incubation (separately or combined) at 4°C or 37°C, suggesting that the configuration necessary for the formation of the [ 3 H]-aldosterone complexes is altered by the extraction procedure. Incubation of the purified nucleus produces more [ 3 H]-aldosterone-macromolecule complexes (per mg of protein) than incubation with the total cell or with the crude nuclear fraction. These data suggest that unbound aldosterone can cross the nuclear membrane and form the aldosterone complexes without the participation of a cytosol intermediate.

Stimulation of progesterone receptors by phorbol ester and cyclic AMP in fetal uterine cells in culture

The role of growth factor signal transducers in the induction of the progesterone receptor by epidermal growth factor (EGF) and the potential sites of EGF antagonism by an antiestrogen were studied in fetal uterine cells in culture. The effects of EGF and estradiol were not additive, suggesting that EGF and estradiol are acting through common mechanisms where antiestrogens could possibly intervene. Fetal uterine cells in culture were found to contain specific, high affinity binding sites for [125I]EGF. Estradiol treatment of the cells led to a higher number of binding sites, but the site of action of 4-hydroxytamoxifen is not the EGF receptor because this antiestrogen had no effect on EGF binding. Activation of protein kinase C by a phorbol ester (12-O-tetradecanoylphorbol 13-acetate) increased progesterone receptor levels to a similar extent as EGF or estradiol. Increasing the intracellular cAMP concentrations by either adding dibutyryl cyclic AMP or activating adenylate cyclase with forskolin also raised progesterone receptor concentrations. Neither the phorbol ester nor dibutyryl cAMP had any effect on cell proliferation. 4-Hydroxytamoxifen completely abolished the effects of the phorbol ester and cAMP. In conclusion, the levels of an estrogen-induced steroid hormone receptor can be regulated by molecules involved in the signal transduction pathway of peptide factors. Moreover, in fetal uterine cells, a potent antiestrogen appears to act as a multiple antagonist but only on an estrogen-inducible response.

Steroid hormone receptors in fetal guinea-pig kidney

Cytosol and nuclear subcellular fractions isolated from fetal kidney of guinea pigs (35–55 days of gestation) form steroid macromolecule complexes both “in vivo” and “in vitro” with [3H]-aldosterone or [3H]-estradiol but not with [3H]-progesterone. d-Aldosterone competes in the formation of the cytosol and nuclear [3H]-aldosterone complexes but estradiol has no effect. Estradiol competes in the formation of the [3H]-estradiol complexes of both the cytosol and the nuclear extracts but d-aldosterone has no effect, suggesting the presence of two different receptors for these hormones in the fetal kidney during this period of development. In the [3H]-estradiol experiments 30 min after administration of this hormone to the fetus or 15 min after incubation at 37°C with the cell suspensions of the fetal kidney, the bulk of the [3H]-complexes (60–70%) is located in the nucleus; of this, 60% is found in the chromatin fraction. 62–95 % of the radioactivity in the complexes consists of non-metabolized estradiol. In addition to estradiol, estrone and estriol were found to compete for binding sites albeit less intensively, whereas testosterone and cortisol had no effect. Ultracentrifugation in a sucrose density gradient (in 0.5 M NaCl) of the 0.3 M NaCl and 1 M NaCl nuclear extracts yielded a component with a sedimentation coefficient of 3.7 S in which estradiol had a significant competitive effect. Experiments carried out with the [3H]-estradiol cytosol-complex obtained at 4°C and then reincubated with the purified nuclei at 4° or 37°C show that the quantity of [3H]-estradiol found in the different nuclear extracts was 2.5–3 times greater at 37°C than at 4°C. By the application of the Scatchard method, the presence of two binding sites was established, the first with a dissociation constant of 2.5 × 10−10 M and the second with a Kd of 7.7 × 10−9 M. When [3H]-estrone was administered subcutaneously “in situ” to the fetus, it was noted that most of the radioactive material in the 3H-macromolecule complexes of the cytosol and nuclear extracts of the kidney is [3H]-estradiol. It is concluded that during this period of fetal development (35–55 days of gestation): estradiol receptors are present in the fetal kidney; in the interconversion of estrone ai estradiol in the same fetal tissue the reaction proceeds predominantly in the formation of estradiol.

Estrogen concentrations and effect of estradiol on progesterone receptors in the fetal and new-born guinea-pigs

Abstract Estradiol concentration in plasma of fetal guinea-pigs, determined by radioimmunoassay, varies from 9 to 40 pg/ml and the concentration of estrone between 80 to 105 pg/ml. The concentrations of both estrogens do not change significantly during fetal evolution. The total number of specific estradiol binding sites in fetal guinea-pig uterus reaches 18 to 20pmol/mg DNA at the end of gestation, and the concentrations of estradiol plus estrone is ∼1500 pg per g of this tissue which correlates well with the number of specific binding sites occupied by the endogenous estrogens (2–4 pmol/mg DNA). In the fetal uterus, estradiol receptors appear at about mid-gestation (from 34 days) but progesterone receptors are detectable only after 50–52 days of gestation. After estradiol treatment of the pregnant guinea pig (1 mg/kg/day for 3 days), the following changes take place in the fetal uterus: (1) Induction of progesterone receptor protein at an earlier period (37–42 days) when this receptor is not detectable in control animals. (2) Five to ten-fold increase in progesterone receptors at the end of gestation. (3) An increase of [3H]-progesterone uptake by fetal uterus (localized by autoradiography). (4) The stimulation of progesterone receptors is of long duration. Five days after treatment the values are similar to those obtained 24 h after the administration of the last dose of estradiol. (5) Similar effects were noted also in fetal ovary, but not in lungs, kidney, brain and heart or placenta. (6) The induction of progesterone receptors by estradiol is significantly lower in the uterus of new-born guinea pigs (2–7 days old). It is concluded that estradiol expresses its biological effect in the fetal compartment, increasing the levels of progesterone receptors.

Anti-estrogens in fetal and newborn target tissues

The antagonistic effects of progesterone and of the anti-estrogens, tamoxifen and nafoxidine, to estrogen responses were studied in the target tissues of fetal and newborn guinea pigs. In the fetal uterus, progesterone inhibits the stimulatory effect provoked by estradiol on uterine growth, on progesterone receptor and on the acetylation of nuclear histones. Progesterone also blocks the synthesis of new progesterone receptor protein in organ culture. Tamoxifen or nafoxidine (1 or 10 mg/kg/day injected to the mother for 3 days) provoke a uterotrophic effect similar to that of estradiol (1 mg/kg/day injected to the mother for 3 days) but these anti-estrogens have a limited effect on the progesterone receptor. Tamoxifen given together with estradiol antagonizes the effect of the estrogen on the acetylation of histones but the anti-estrogens do not block the effect of estradiol on uterine growth. Histological studies show that both estradiol and tamoxifen provoke a dramatic hypertrophic and hyperplastic effect particularly in the uterine epithelium. In the newborn uterus (6-day old), tamoxifen (s.c. injection of 0.6 micrograms/g body weight) and estradiol (injection of 30 ng/g body weight) provoke a similar uterotrophic effect and both have a limited effect on the progesterone receptor. In the fetal thymus estradiol provokes a selective decrease in the larger and actively proliferating lymphoid cells of the cortical zone. Tamoxifen has a similar effect but to a much lesser extent than estradiol. On the other hand, tamoxifen antagonizes the effect of estradiol on this fetal tissue. It is concluded that during fetal life progesterone antagonizes the effect of estradiol but tamoxifen can act as an agonist or an antagonist of estrogen action which is a function of the type of response or organ considered.

Biological responses of tamoxifen in the fetal and newborn vagina and uterus of the guinea-pig and in the R-27 mammary cancer cell line

The biological and morphological responses of tamoxifen were studied in two models: the uterus and vagina of fetal and newborn guinea-pigs: R-27 cells--a mammary cancer cell line (tamoxifen resistant) derived from the MCF-7 cancer cell line. Tamoxifen (TAM) alone or in combination with estradiol (E2) was administered to pregnant (50-52 days of gestation) or to newborn (2-day-old) guinea-pigs for a long period (12 days). TAM alone produced a great trophic effect on the uterus and vagina which was markedly enhanced when TAM was administered together with E2. Histological studies showed that TAM provokes morphological changes in both the endometria and the myometria and this effect was also greater when TAM was administered together with E2. In the fetal uterus and vagina, the ultrastructural studies showed that TAM induces morphological alterations in different cytoplasmic organelles. This effect was much more intense in newborns where TAM provoked a significant vacuolization of the epithelial cells. Concerning progesterone receptor (PR) in the fetal or newborn tissues (uterus or vagina) TAM provoked a less intense effect than those provoked by E2, but TAM did not block the effect provoked by E2. It was observed that [3H]TAM binds specifically to the estrogen receptor (ER) of fetal guinea pig uterus and this complex is partially recognized by a monoclonal antibody which recognizes the activated form of this receptor, supporting the suggestion that the biological action of TAM is mediated by the ER. The biological and ultrastructural effects provoked by TAM (1 X 10(-6) M), estriol (E3)(5 X 10(-8) M) and the combination of TAM + E3 were studied in the R-27 mammary cancer cell line in culture. E3 stimulated the PR content by 7-10 times. However, TAM did not provoke a significant decrease in the concentration of PR, and in the mixture of TAM + E3 the concentration of PR was of the same order as that in E3 treatment. Ultrastructural observations indicate an intense concentration of ribosomes in the pericytoplasmic area after exposure to E3 and with exposure to TAM an increase in vacuoles and a significant enlargement of the size of the mitochondria were observed. It is concluded that TAM in the target tissues of fetal and newborn guinea pigs acts as a real estrogen and in the R-27 mammary cancer cell line TAM does not block the effect provoked by E3, however it does provoke intense ultrastructural modifications.

The complexity of anti-estrogen responses

The actions and biological responses of anti-estrogens are a function of: the experimental conditions, the parameters, the organ and the animal species considered. Target tissues for estrogens in the guinea-pig during the perinatal period are interesting models to explore the action of anti-estrogens. The summary of the data indicates: (1) In the fetal uterus of guinea-pig in in vivo experiments (after injection to the maternal compartment) tamoxifen acts as a real agonist concerning growth, as a partial agonist concerning the stimulation of the progesterone receptor. (2) In in vitro experiments (in organ culture of fetal uterus or in isolated cells) anti-estrogens (tamoxifen or 4-hydroxy-tamoxifen) act as antagonists and also inhibit the effects provoked by estrogens. (3) In the uterus and vagina of newborn guinea-pigs, tamoxifen and its derivatives: 4-hydroxytamoxifen and N-desmethyltamoxifen act as real agonists concerning the uterotrophic and vaginotrophic effects, and also stimulate the amount of DNA per organ, but concerning the progesterone receptor in the uterus, in the short treatment anti-estrogens act as partial agonists but they have no effect in the long treatment. In the vagina in the short treatment anti-estrogens provoke no significant effects, but in the long treatment they are full agonists. In neither of the two biological responses studied (growth and progesterone receptor) does tamoxifen and its derivatives block the action of estradiol. (4) The use of a monoclonal antibody to the estrogen receptor revealed quantitative differences in the activation of the estrogen receptor when bound to estradiol or tamoxifen. This observation was in agreement with the lesser extent of binding to DNA-cellulose of the tamoxifen-estrogen receptor complex as compared with the estradiol-estrogen receptor complex. This fact suggests an impaired activation of the estrogen receptor induced by tamoxifen which might be related to the different biological responses provoked by estrogens and anti-estrogens.

Ontogeny of Steroid Receptors in the Reproductive System

Publisher Summary Steroid hormones play an important role during the crucial periods of gestation—namely, conception, nidation, embryonic development, and fetal maturation. Progesterone and the estrogens are two of the steroid hormones that play a basic role in pregnancy. During the course of normal pregnancy, the production rates and plasma concentrations of these steroid hormones can vary significantly, sometimes increasing 100-300 times at the end of gestation, as in the case of estrogens and progesterone in pregnant women. Also during gestation, the production of various steroid hormones is the contribution of the secretion of three compartments: maternal, placental, and fetal. This production is different in the three compartments, both qualitatively and quantitatively, and also depends on the period of pregnancy as well as the species considered. The placenta makes most of the progesterone and estrogens produced during pregnancy. This chapter discusses various aspects of hormone action such as the presence of steroid hormone receptors in the fetal and placental compartments and the correlation of these receptors with biological effects during fetal development and in newborns. The presence of steroid receptors in different fetal tissues confirms that the mechanism of action can be similar to that postulated in target organs after birth. Good correlation exists among receptor binding, nuclear translocation, and biological responses elicited by the hormone in the fetus and the newborn.