Study of the specificity of the estradiol-binding system in the uteri of guinea pigs]

The authors analyzed the affinity of the steroids belonging to the estran series to the estradiol-binding system of the guinea pig uteri. The presence of free hydroxyl groups in positions 3 (phenol) and 17beta and their interorientation proved to determine the interaction with the recptor system of the guinea pig uterus. The data obtained indicated that the biological activity of the steroids was determined by the peculiarities of their structural interaction with the receptor systems of the uteri.     

Dynamic Studies on Estrogen Response in Fetal Guinea Pig Uterus: Effect of Estradiol Administration on Estradiol Receptor,Progesterone Receptor and Uterine Growth

Abstract

The uterus of the guinea pig fetus has been shown to respond to estradiol treatment by an increase in uterine wet weight and a stimulation of the progesterone receptor protein. A study of the kinetics of these two parameters of estrogen response in the fetal uterus was undertaken in order to correlate these responses with changes in the estrogen receptor. Administration of estradiol to pregnant guinea pigs (1 mg/kg/body weight) leads to a rapid stimulation of the progesterone receptor by 6h after treatment which reaches maximal values by 15.5h, which are increased 7-fold in estradiol-primed guinea pigs above values in untreated animals. The estradiol receptor undergoes rapid translocation from the cytosol into the nucleus by 1h after hormone treatment and is retained in the nucleus for at least 6h. At the same time, there is a 50% decrease in the total occupied and available estradiol receptor concentration at 6h after treatment. Estradiol treatment also provokes an increase in wet weight of the fetal uterus which is significantly greater after 3 consecutive days of treatment (171% ± 24 (S.D.) above wet weights of untreated uteri which were considered as 100%) than after only 1 day (121% ± 25 (S.D.)). These estrogen responses were found to be of long duration since uterine wet weights and progesterone receptor concentrations remained well above control values even 5 days after a single treatment with estradiol. In conclusion, the fetal uterus responds to estradiol treatment by a slow increase in wet weight and a rapid stimulation of the progesterone receptor protein with a concomitant loss in estradiol receptor concentration.     

Receptor and Biological Response to Estriol in the Fetal Uterus of Guinea Pig

Abstract

The binding of ³H-estriol was examined in the fetal uterus of guinea pig. The physico-chemical characteristics of the binding of ³H-estriol to macromolecules are similar to the typical receptor protein for estrogens. Different estrogens (estriol, estradiol, estrone and diethylstilbestrol) compete with this binding but progesterone and testosterone have no effect. The binding affinity has a Kd of 5.5 ± 1.6 ± 10^?10M. By ultra-centrifugation in sucrose gradient, two specific components with sedimentation coefficients of 8 and 45 are found. Competition studies suggest that the same specific binding sites may be present for estriol (E₃) and for estradiol. The s.c. administration of E₃ to the pregnant guinea pig (1 mg/day per kg body weight for 3 days) provokes two biological responses in the fetal uterus: a uterotrophic effect and a significant increase in the progesterone receptor. The increase in the fetal uterine weight is 50–70% in relation to the non-treated animals and the progesterone receptor concentration is 10–14 times higher than in the control animals. These effects are similar (or slightly higher) than in animals primed with equimolecular quantities of estradiol. In contrast, single daily injections of E₃ to newborn guinea pig, results only in weak uterotrophic activity.

It is concluded that estriol is capable of causing a biological response in the uterus during intra-uterine life.     

Characteristics of the nuclear translocation of progesterone receptor in fetal guinea pig uterus "in vivo", "in vitro" and in organ culture

The translocation of progesterone receptor from the cytosol into the nucleus was studied under "in vivo" and "in vitro" conditions in the uteri of guinea pig fetuses exposed to progesterone or a synthetic progestin, R5020. Progesterone treatment of estrogen-primed fetuses leads to a rapid (before 1h) transfer of cytosol progesterone receptor into the nucleus which is, however, short-lived (less than 3h). A rapid decrease in the retention of the estrogen receptor in the nucleus also occurs. In the "in vitro" incubations of whole fetal uteri, translocation of progesterone receptor is temperature-dependent and specific for progesterone and R5020; estradiol and cortisol have no effect. Putative progesterone receptors can also be induced in explants of fetal guinea pig uteri in organ culture which translocate from the cytosol into the nucleus under the same "in vitro" conditions as in whole uteri. Fetal uterine progesterone receptor, either stimulated "in vivo" by estrogen-priming or induced in organ culture, translocates from the cytosol into the nucleus and this process seems to be accompanied by a decrease in retention of the estrogen receptor in the nucleus which appears to be the mechanism by which progesterone antagonises estrogen action in fetal guinea pig uterus.     

Autoradiographic studies on the fetal and newborn uteri of the guinea pig after injection of 3H-progesterone into non-treated and into estradiol-primed animals

Summary The localization and retention of radioactivity in the uteri of fetal and newborn guinea pigs was studied after subcutaneous injection of ³H-progesterone into intact and estradiol-primed animals. In the fetal uterus of the animals treated with estradiol, a significant increase in the accumulation of silver grains was observed, mainly localized in the stroma and myometrium, but very little is detected in the uterine gland. On the other hand, in the uteri of newborns, the effect of estradiol on radioactivity retention was significantly less intense and the radioactivity is mainly localized in the epithelium and the uterine gland. These data correlate well with the quantitative evaluation of the concentration of progesterone-specific-binding sites in the non-treated and in the estradiol-primed fetal and newborn guinea pigs.     

Effect of porcine relaxin and estradiol-17β on the incorporation of tritiated thymidine by fibroblasts isolated from guinea pig uteri

Fibroblasts isolated from the uteri of 5 guinea pigs were cultured in vitro to determine the effect of relaxin and estradiol. Both relaxin and estradiol increased thymidine uptake by fibroblasts. Thymidine incorporation with 1.5 μg/ml relaxin and no estradiol was 107% of control while 600 pg/ml estradiol and no relaxin gave 112% of control. Thymidine incorporation in the presence of 1.5 μg/ml relaxin and 600 pg/ml estradiol was 128% of control. This indicated a significant interaction between relaxin and estradiol.     

The metabolism of estradiol-17β by the pregnant guinea pig uterus in vitro

The in vitro metabolism of [³H estradiol-17β-by the uterus was studied in non-pregnant, prenant (day 30-term) and post-parturant guinea pigs. Following incubation of tissue sections for one hour is Krebs-Ringer phosphate buffer, five major metabolites could be extracted from the medium or tissue depending upon age of gestation: estrone-3-glucuronide, estrone-3-sulfate, estradiol-3-glucuronide and estradiol-3-sulfate. Both sulfated estrogens were detected at each age of gestation studied, whereas the glucuronides, mainly of estrone, were not detected until approximately day 50. Thereafter, as term (day 65–70) was approached, their percentage contribution to total radioactivity increased at the expense of estradiol and the sulfates. Following parturition, total metabolites of estradiol rapidly decreased, particularly the glucuronides. No conjugates were detected in uteri from nonpregnant guinea pigs. In addition, no conjugates were found in the pre-partum mouse, rat and hamster or in human endometrium obtained immediately after birth. The data suggest that, in the guinea pig, a biochemical factor in the termination of normal pregnancy is the control of tissue levels of active estrogen (estradiol) by conjugation with glucuronic acid.     

Biological responses and histological studies of estriol and estriol sulfate in fetal and newborn uteri of guinea pig

The biological responses of estriol (E₃) and of estriol-3-sulfate (Ea₃-S) in the fetal and newborn uteri of guinea pig were studied. After a treatment of E₃ (1 mg/kg/day) or E₃-S (1.4 mg/kg/day) to pregnant guinea pigs (49–60 days of gestation) for 6 days, both estrogens provoke a significant uterotrophic effect in the fetal uterus which increases in weight 1.8–2.5 times in relation to the non-treated animals. The stimulation of progesterone receptor (PR) is also very intense, 7–12 times in relation to the control animals.In another series of experiments newborn guinea pigs (2-day old) were treated with 100μg/animal of E₃ or 140 μg/animal of E₃-S for a short (2 days) or a long (12 days) period. Concerning the uterotrophic effect, the weight of the uterus increases 1.8–2.5 times (in relation to the non-treated animals) after a 2-day treatment and the effect continues to increase up to 4–5 times in the 12-day treated animals. In opposition to the fetal uterus, the effect on PR provoked by E₃ or E₃-S is very limited (only an increase of 1 time in relation to the non-treated animals); the effect is even less intense after the 12-day treatment. Histological studies show an intense hypertrophic effect particularly in the epithelium of the endometrium in the fetal and newborn uteri for both E₃ and E₃-S. In the newborns the effect is also intense on the epithelium of the uterine gland.It is concluded that: (1) Estriol and estriol sulfate are very active and with similar intensity on the uterine weight before and after birth; (2) The stimulatory effect on PR decreases very significantly after birth and after a long treatment; (3) E₃S can act as a potent hormonal precursor.     

Limited proteolysis of cytoplasmic and nuclear uterine estradiol receptors yields identical estradiol-binding fragments.

Limited tryptic hydrolysis of the estradiol cytoplasmic receptor from calf uterus has been demonstrated to yield in a high-salt buffer a stable estradiol-binding molecule with the following characteristics: sedimentation coefficient 4.0 +/- 0.1 S; Stokes radius 3.5 +/- 0.05 nm; molecular weight 60000 (for an assumed v value of 0.73 ml g-1) and frictional ratio 1.36. Nuclear KCl extracts, prepared from uteri preincubated at 37 degrees C with labeled estradiol, were analysed by Sephadex G-200 chromatography and sucrose density gradient centrifugation. The following molecular parameters were found for the estradiol-receptor complex: sedimentation coefficient 4.4 +/- 0.1 S; Stokes radius 4.12 +/- 0.02 nm; molecular weight 77000 and frictional ratio 1.47 (v = 0.73 ml g-1). Limited tryptic proteolysis of this extract gave an estradiol-binding fragment with molecular characteristics identical to the trypsin-modified cytoplasmic receptor. In addition, mild tryptic digestion of whole labeled nuclei allowed us to solubilize almost quantitatively the nuclear [3H]estradiol in a macromolecular bound form. The molecule thus obtained showed molecular parameters very similar to the 60000-dalton trypsin fragments obtained from high-salt cytoplasmic and nuclear extracts. These molecules were undistinguishable by gel electrophoresis analysis at six different acrylamide concentrations. These results in conjunction with those derived from dissociation kinetics experiments and ligand specificity studies indicate the cytosolic protein is a functional part of the nuclear receptor. Based upon these and other studies we suggest that proteolytic cleavage of the estradiol-receptor complex, which results in the removal of the estradiol-binding sites from the nuclear recognition sites of the molecule, could play a role in the inactivation of the estradiol receptor in vivo.     

Modulation of the progesterone receptor in the fetal uterus of the progesterone-primed guinea pig in vivo and in organ culture

Guinea pig fetuses were treated with progesterone for 7 days before placing fetal uteri in organ culture to see if progesterone pre-treatment of fetuses in utero would permanently inhibit the spontaneous rise in progesterone receptor which occurs in organ culture. The data show that: the basal level of progesterone receptor in fetal uteri was not affected by the progesterone treatment and progesterone receptor concentrations in vitro were also not inhibited. When guinea pig fetuses were treated sequentially with progesterone and estradiol, estradiol failed to provoke an uterotrophic effect but it retained its ability to stimulate progesterone receptor concentrations.     

Estradiol receptor: phosphorylation on tyrosine in uterus and interaction with anti-phosphotyrosine antibody.

Estradiol receptor from rat uteri incubated with [32P] orthophosphate has been purified by diethylstilbestrol--Sepharose followed by heparin--Sepharose chromatography. The purified receptor, analyzed by centrifugation through sucrose gradients after incubation with monoclonal antibodies against purified estradiol receptor, appears to be labeled with 32P. The receptor preparation has been further purified by immunoaffinity chromatography and submitted to SDS--poly-acrylamide gel electrophoresis. A heavily 32P-labeled 68 kd protein and a very lightly 32P-labeled 48 kd protein, probably a proteolytic product of the 68 kd protein, were detected. Phosphoamino acid analysis of the receptor eluted from the immunoaffinity column shows that its 32P-labeling occurs exclusively on tyrosine. This is the first report on phosphorylation on tyrosine of a steroid receptor in tissue. It is consistent with our previous finding that a uterus estradiol receptor-kinase, which confers hormone binding ability to the estradiol receptor, in vitro phosphorylates this receptor exclusively on tyrosine. Calf uterus receptor binds with high specificity and affinity to monoclonal anti-phosphotyrosine antibodies covalently bound to Sepharose (Kd = 0.28 nM). Dephosphorylation of the receptor by nuclei containing the calf uterus nuclear phosphatase abolishes the interaction with antibodies. These results suggest that also in calf uterus, estradiol receptor is phosphorylated on tyrosine. Anti-phosphotyrosine antibodies bound to Sepharose have been used to partially purify the estradiol receptor from calf uterus.     

Estradiol binding in the cytosol and nuclear pellet of electrolytically treated rat uteri

Intact mature rat uteri positioned in saline buffer at 0–2°C between platinum electrodes operating at a voltage in the range 1–4 VDC, with periodic reversal of polarity, are conditioned so that the cytosols obtained from these uteri lose 40% of specific and 32% of non-specific estradiol binding capacity and 90% of the dissolved protein. By contrast, estradiol bound in the nuclear pellet following incubation of the uterus in solution of the hormone shows a 50% increase in quantity if the uterus was electrolytically pretreated. The results are regarded as reflecting the removal from intact tissue by electrolytic treatment of a substance that inhibits and controls the translocation of estradiol receptor from the cytoplasm to the nucleus.     

Changes in the levels of protein and steroid hormones in the plasma and steroid hormone receptors in the uterus of normal cycling guinea pigs

This study deals with the estrous cycle of guinea pigs in relation to sexual behavior, uterine weight, levels of gonadotropins, steroid hormones, and steroid hormone receptors in the uterus. The guinea pigs in this study showed cyclic changes in various reproductive functions broadly similar to other laboratory species studied. The increase in the uterine weight coincided with high concentration of steroid hormones (estradiol and progesterone) secreted during proestrus and estrus. The elevated levels of steroid hormone receptor concentrations in the uterus during these periods also confirm the role of these hormones. The rise in progesterone level from day 14 of the cycle was associated with lordosis and its related behavior. It was noted that the "estrus behavior" is the most accurate external marker for ovulation and sexual receptivity to males. It was also observed that there is an association between follicle-stimulating hormone and luteinizing hormone during the preovulatory period that was not demonstrated in previous studies.     

Radioautographic study of the effect of estradiol-17 , estrone, estriol, progesterone, testosterone and corticosterone on the in vitro uptake of 2,4,6,7- 3 H estradiol-17 by uterine eosinophils of the rat

The in vitro uptake of 2,4,6,7-tritiated estradiol-17beta in uterine eosinophils of the rat was inhibited by the presence of nonradioactive estradiol-17beta, estrone, and estriol, but not by progesterone, testosterone, or corticosterone. This action is attributed to competition between tritiated estradiol and the various estrogenic compounds for the same binding site. Compounds without any estrogenic activity do not compete. The proposal is made that the eosinophil binding system and the 8S-5S binding system are involved in different mechanisms of estrogen action. The parallelism between the doses of estradiol and estriol needed to promote certain estrogenic early effects in the uterus, and the affinity of these steroids for the eosinophil uptake sites, suggests that uterine eosinophils might be responsible for some of these early effects, such as water imbibition, histamine releasing activity, and estrogen priming effect.     

Effects of Salt Extraction on the Quantitation of Nuclear Estrogen Receptors: Interference by Secondary Estrogen Binding Sites

Abstract

The effects of salt-extraction on type I and type II estrogen binding sites were examined in uterine nuclei. Injection (10 ug) of estradiol or estriol in adult ovariectomized rats induced maximum numbers (80–100%, ~ 1 pmole/uterus) of 0.4 M KCL resistant type I estrogen complexes at 1 hour. Only estradiol, which sustained these levels for long periods of time (4–24 hours) stimulated true uterine growth.

Likewise, a single injection of estradiol, but not estriol, also elevated nuclear type II sites throughout the entire uterine growth period (1–48 hours). However extraction of these nuclei from estradiol injected rats with 0.4 M KCL increased the numbers of type II sites from ~ 1 pmole/uterus (non-extracted nuclei) to ~ 8 pmoles/uterus (salt resistant plus salt-extractable fractions). Sixty percent of these sites were resistant to salt-extraction. Continuous exposure to either estradiol or estriol by beeswax implants stimulated nuclear type II sites which were highly resistant (80%) to KCL-extraction, and additional sites were not exposed by high salt. Thus chronic treatment with both estrogens “locked in” nuclear type II sites such that they were resistant to KCL-extraction. This resistance of type II sites to salt-extraction correlated with the ability of estradiol and estriol implants to stimulate true uterine growth. The procedures presented here for nuclear preparation and assay have reduced non-specific binding considerably in the uterine system, and may eliminate the need to perform exchange assays on salt-extracted nuclei in other systems.     

Estrogen responsiveness of fetal guinea pig uterine cells in culture

Cells from the uterus of the guinea pig fetus have been grown as a monolayer culture in serum-containing medium. Cells from the first subculture showed high concentrations of progesterone receptor (PR; 9.3-13.8 pmol/mg DNA) even after 9 days in medium containing charcoal-treated serum and estradiol did not induce any further increase. The antiestrogens, tamoxifen and monohydroxytamoxifen, both had an inhibitory effect which could be overcome by estradiol. The progestins, progesterone and R5020, as well as the antiprogestin, RU38486, also decreased the PR concentration. Estrogen receptor (ER) levels did not vary with the compounds tested but were found to be low compared to concentrations found in the fetal guinea pig uterus at 55-65 days of gestation. None of the compounds tested had any effect on the growth of the fetal uterine cells so that the modulation of PR concentrations was dissociated from the regulation of cell growth. It is concluded that estrogens are necessary but not sufficient factors in the control of PR levels in fetal uterine cells. The establishment of a culture system for separate types of fetal uterine cells will permit us to study in vitro the factors involved in the growth effects of estrogens and the control of PR synthesis.     

Biology and receptor interactions of estriol and estriol derivatives in vitro and in vivo

The biological effects of estriol (E3) have been studied in three estrogen targets, namely, the rat uterus in vivo and in vitro, in primary human endometrial cell cultures and in MCF-7 human breast cancer cells in culture. Studies on the temporal relationships between estrogen receptor binding and biological responses in the uterus using estriol and several more long-acting estriol derivatives, namely, 17α-ethynyl estriol, estriol-3-cyclopentyl ether, and 17α-ethynyl estriol-3-cyclopentyl ether, indicate that estriol is a short-acting compound with a brief duration of action. Estriol is a poor stimulator of uterine growth and plasminogen activator activity in vivo. Chemical modifications of the estriol molecule produce long-acting derivatives that result in a prolonged input of hormone receptor complexes into the nucleus and a prolonged and marked stimulation of uterine growth. In human endometrial cells in primary tissue culture, E₃ has 12% the affinity of estradiol (E₂) for cytosol estrogen receptor and it is quite effective yet slightly less potent than estradiol in stimulation of progesterone receptor synthesis. Low concentrations of E₃(10⁻¹⁰ M) stimulate growth of MCF-7 cells in vitro and dose-response curves show E₃ to be only slightly less effective than E₂. In these endometrial and breast cancer cell systems in vitro, there is no metabolism of E₃ while E₂ is metabolized to estrone.Hence, estriol is an effective estrogen in vitro. In vivo, it is short-acting, but it can be made a full estrogen agonist when given at a sufficiently high concentration or in a chemically modified form which prolongs its activity by enabling effective concentrations of the compound to be maintained in the blood and in target tissues.     

Influences of estradiol and of catechol and non-catechol estrogens on the output of prostaglandins in uteri from spayed rats

The effects of 17-beta estradiol and of some catechol and non-catechol-estrogens on the synthesis and output of prostaglandins (PGs) E and F by uteri from ovariectomized rats, were explored. Uteri from castrated animals released twice as much PGE than PGF. When uterine tissue was obtained from spayed rats injected prior to sacrifice with a low dose of 17-beta estradiol (0.5 + 1.0 microgram, on two consecutive days), the output of PGE diminished significantly. With a higher dose of the hormone (0.5 + 50.0 micrograms) the depressive influence on the synthesis and release of PGE was even more marked, whereas the output of PGF rose significantly. Low or high doses of estrone or of estriol failed to affect the release of either one of the PGs determined. On the other hand, 2-0H-estradiol at a low dose had no action but at a higher one inhibited the release of PGE without influencing PGF. Neither low nor high doses of 2-0H estriol or of 2-0H estrone affected the synthesis and release of uterine PGs. It was also observed that all the compounds tested evoked a significant uterotrophic action. It appears plausible that some catechol metabolites of 17-beta estradiol, but not other catechol-estrogens, could be involved in the mechanism of action of 17-beta estradiol modulating the production of PGs by the rat uterus.     

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 10mg/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 hypertrophie and hyperplastic effect particularly in the uterine epithelium.In the newborn uterus (6-day old), tamoxifen (s.c. injection of 0.6μg/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.