Follitropin receptors in rat testis. Characterization with enzymatically 125I-labeled human follitropin.

The interaction between enzymatically radioiodinated human follitropin and the follitropin receptors in testis homogenate was investigated in immature and adult rats. The 125I-labeled human follitropin exhibited high binding activity with specific binding of up to 17% in the presence of an excess of testis homogenate. Approx. 50% of the bound hormone could be eluted at pH 5, and the receptor purified tracer exhibited a 3.6-fold increase in binding activity when compared with the original tracer preparation. Quantitative analysis of equilibrium binding data was performed with corrections for the measured specific activity and maximum binding activity of the tracer hormone. The equilibrium association constants (Ka) determined 24 degrees C were not significantly different in immature and adult rat testis, and the mean value for Ka was 3.9 . 10(9) M-1. At 37 degrees C, the Ka value obtained using immature rat testis was 1.3 . 10(10) M-1. The association of 125I-labeled human follitropin with immature rat testis homogenate was time and temperature dependent. In the presence of an excess of unlabeled hormone, 30--60% of the preformed hormone . receptor complex was dissociated after 24 h incubation. A specific and sensitive radioligand-receptor assay for follitropin was developed using immature rat testis homogenate. The minimum detectable dose of purified human follitropin was 0.6 ng, and human urinary and pituitary follitropin, ovine follitropin and pregnant mare serum gonadotropin reacted in the assay with equivalent slopes. The potencies of highly purified pregnent mare serum gonadotropin and highly purified human follitropin were similar in the radioligand-receptor assay, consistent with the follitropin bioactivity of the equine gonadotropin.     

Prolactin receptors in the hypoprolactinemic male rat (IPL nude rat): measurement in testis and induction in liver

In order to evaluate the importance of PRL in the regulation of its own receptors, characteristics of specific binding for PRL were studied in membrane preparations from liver and testis of a new hypoprolactinemic male rat, the IPL nude male rat, and this was compared to those found for normal male rats. Under basal conditions, hepatic specific binding of PRL in IPL nude rats, as in normal rats was not detectable. Following castration, it became detectable in both groups, and was 6.99 +/- 0.78% and 6.34 +/- 0.87% for IPL nude and normal rats respectively. Under such conditions, the apparent affinity constant (Ka) and the binding capacity (Nmax) obtained were also similar for both groups (Ka) = 1.36 +/- 0.14.10(9) M-1, Nmax = 102 +/- 14 fmol/mg protein in IPL and Ka = 1.34 +/- 0.28.10(9) M-1, Nmax = 97 +/- 11 fmol/mg protein in normal rats) although a decrease in serum levels of PRL was observed in both groups. This decrease was greater for IPL nude rats. As already reported, estradiol injection following castration was able to further increase the percentage of PRL hepatic specific binding (4 times). Furthermore, our results demonstrated that the affinity constant was significantly increased by estradiol injection in both groups. On the other hand, for testicular PRL binding characteristics, a statistically significant difference was found between IPL nude and normal rats. The PRL specific binding percentage was 7.01 +/- 0.85% for the IPL nude rat and 10.07 +/- 0.64% for the normal rat. By Scatchard analysis, the Ka of testicular membranes for labelled oPRL was similar in both groups, while the capacity differed (Nmax = 9.82 +/- 1.25 fmol/mg protein for IPL nude rat and Nmax = 26.06 +/- 4.39 fmol/mg protein for normal rats). These data established the fact that IPL nude male rats presented characteristics of hepatic PRL receptors similar to those of normal rats, while their testicular oPRL binding significantly differed. These findings therefore suggest that in genetic hypoprolactinemic rats (IPL nude rats), PRL might be more involved in the regulation of testicular PRL receptors than in that of hepatic receptors.     

Further characterization of estrogen binding to rat testis cytosol

Binding of estradiol (E2), estriol (E3), RU16117, and moxestrol to testis cytosol from adult male rats was investigated. High-affinity binding sites were identified in the 8-9S region of sucrose density gradients; a second, high-capacity binding component in the 4S region was probably due to contamination with serum. Thermodynamic properties of the testicular estrogen binding site were quite similar to those of the uterine receptor. E2 had the highest affinity for testicular cytosol binding sites (Ka: E2 much greater than moxestrol greater than E3 greater than RU16117). Comparison of association rate (E2 greater than E3 greater than moxestrol = RU16117) and dissociation rate constants (E3 = RU16117 greater than E2 much greater than moxestrol) as well as studies in vivo revealed moxestrol as a long-acting and RU16117 as a short-acting compound. This difference may be useful for evaluation of the mediation of estrogen effects in the rat testis.     

The characteristic binding of catechol estrogens to estrogen receptors in 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors

The binding of catechol estrogens (2-hydroxyestrone, 4-hydroxyestrone, 2-hydroxyestradiol, and 4-hydroxyestradiol) to estrogen receptors in 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumor cytosols was investigated. Cytosol estrogen receptors exhibited high affinities (Ka = 1.12-1.88 X 10(8) M-1) for all catechol estrogens as well as estradiol. The receptor level of catechol estrogens (46.1-97.5 fmol/mg protein) was 1.6-3.0 times higher than that of estradiol; especially the binding of 4-hydroxyestrone to estrogen receptors was the highest of all catechol estrogens and estradiol. In judging the receptor level of more than 20 fmol/mg protein to be positive, the binding of catechol estrogens to estrogen receptors was approximately correlated with that of estradiol. The positive receptor level of catechol estrogens was found in a half of tumor cytosols which showed the negative receptor level of estradiol. These results suggested that characteristic estrogen receptors indicating high affinities for catechol estrogens might be present in rat mammary tumor cytosols.     

Expression and localization of estrogen receptor alpha in the C2C12 murine skeletal muscle cell line

The classical model of 17beta-estradiol action has been traditionally described to be mediated by the estrogen receptor (ER) localized exclusively in the nucleus. However, there is increasing functional evidence for extra nuclear localization of ER. We present biochemical, immunological and molecular data supporting mitochondrial-microsomal localization of ER alpha in the C2C12 skeletal muscle cell line. We first established [(3)H]17beta estradiol binding characteristics in whole cells in culture. Specific and saturable [(3)H]17beta estradiol binding sites of high affinity were then detected in mitochondrial fractions (K(d) = 0.43 nM; B(max) = 572 fmol/mg protein). Immunocytological studies revealed that estrogen receptors mainly localize at the mitochondrial and perinuclear level. These results were also confirmed using fluorescent 17beta estradiol-BSA conjugates. The immunoreactivity did not translocate into the nucleus by 17beta-estradiol treatment. Western and Ligand blot approaches corroborated the non-classical localization. Expression and subcellular distribution of ER alpha proteins were confirmed in C2C12 cells transfected with ER alpha siRNA and by RT-PCR employing specific primers. The non-classical distribution of native pools of ER alpha in skeletal muscle cells suggests an alternative mode of ER localization/function.     

High affinity estrogen binding by rabbit liver

Macromolecular binding components for [3H]estradiol-17beta are present to cytosol prepared from rabbit liver. When cytosol from sexually mature male liver was incubated with [3H]estradiol and analyzed for binding on low ionic strength sucrose gradients, two peaks of binding activity were detected. One peak had a sedimentation coefficient of 4--5 S and the other had a sedimentation coefficient of 8--9 S. The two components differed from each other regarding steroid specificity and various physiocochemical parameters. [3H]estradiol binding to the 4--5 S component was not inhibited by estrogens, 5alpha-dihydrotestosterone, progesterone or cortisol. Binding to this component did not appear to be saturable and label was rapidly stripped from it by charcoal. Estradiol binding to the 8--9 S component was estrogen specific, saturable and of high affinity. The specific binder dissociates on high ionic strength sucrose gradients and sediments as a 4--5 S moiety. The specific binding protein has a Kd of 3.05 . 10(-10) M and a dissociation half-time of 33 h and there are 35.2 fmol of binding sites/mg cytosol protein. Estrogen binders are also present in liver cytosol from sexually mature female and sexually immature male rabbits. During prolonged incubation of [3H]estradiol with mature male liver cytosol at 0--5 degrees C polar metabolites of estradiol are produced.     

Presence of two types of estrogen binding sites in mouse testis

The characteristics of cytosol estrogen binding sites in BALB/c mouse testis were investigated. The cytosol prepared from the whole testis contained two classes of the specific estrogen binding sites by Scatchard and Rosenthal plot analyses. The first binding site (first binder) had high affinity for 17 beta-estradiol (E2; Kd = 4.9 X 10(-9) M) and binding specificity as observed in the typical estrogen receptor. The second binding site (second binder) had lower affinity for E2 (Kd = 4.8 X 10(-8) M) and the binding was inhibited less vividly by diethylstilbestrol (DES) and antiestrogens in comparison with that for the first binder. Postlabeled sucrose density gradient analysis in a low salt medium revealed that the major radioactive peak of the first binder appeared at 7S region, while that of the second binder sedimented at 4S region. The 7S component showed an appreciable binding to the nuclei, while the 4S component did not show a significant binding ability to the nuclei. Much higher concentrations of the first and the second binders were found in Leydig cells preparations. These results demonstrate the presence of two types of the specific estrogen binding sites in the mouse testis especially in Leydig cells.     

Appearance of the rat testicular receptor for calcitriol (1,25-dihydroxyvitamin D3) during development

In the present study we have examined the developmental changes in the concentration of receptors for calcitriol in high-salt cytosol from the rat testis. Receptors for calcitriol were undetectable (less than 0.4 fmol/mg protein) until day 24, after which there was a rapid increase to reach adult levels (6-8 fmol/mg protein) between day 50-60. The lack of receptors in high-salt cytosol from the immature rat testis is not due to degrading enzymes, since cytosols prepared from the combination of equal volumes of testis homogenates from immature and adult rats had binding levels exactly half of that found in "adult controls". Furthermore, the increase in specific binding of [3H]calcitriol during development is due to an increase in the number of receptor sites, and is not due to a change in the apparent affinity of the receptors (Kd approximately equal to 1 X 10(-11) M at 0 degrees C). These results may explain why we previously were unable to demonstrate calcitriol receptors in cultured Sertoli cells and peritubular cells isolated from 19-day old rats. Furthermore, they indicate that calcitriol may be of minor importance for testicular function in the immature rat. The role of calcitriol in the pubertal and adult testis remains to be established.     

Nuclear triiodothyronine receptors in the testis of the growing rat are regulated by the thyroid status]

The effects of thyroid status on the binding capacity, association constant (Ka) and receptor occupancy during postnatal rat testis development were evaluated. Hypothyroidism (induced by oral administration of 0.05% methimazole from the day of birth) increased the total T3 binding capacity in the testis, retarding the normal developmental decrease in T3 receptor number (mean maximal binding capacities estimated by Scatchard analysis for 21-day-old eu- and hypothyroid rats were 117 and 173 fmol/mg DNA, respectively). The rat thyroid status also affected the percentage of T3 receptor occupancy but not the affinity of binding (as measured by Ka). The postnatal developmental changes in T3 binding capacity induced by hypothyroidism were completely reversed by T3 replacement. These results suggest that T3 nuclear receptors in the developing rat testis are modulated by thyroid hormones.     

Characterization of a [3H]methyltrienolone (R1881) binding protein in rat liver cytosol

The binding of radiolabelled methyltrienolone 17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-trien-3-one (R1881) to adult male rat liver cytosol has been characterized in the presence of Na-molybdate to stabilize steroid-hormone receptors, and triamcinolone acetonide to block progestin receptors. Using sucrose density gradient analysis, male liver cytosol contains a [3H] R1881 macromolecular complex which sediments in the 8-9S region. 8S binding of R1881 to male rat serum, female liver cytosol or cytosol from a tfm rat cannot be demonstrated. Further metabolism of [3H] R1881 following 20h incubation with male rat liver cytosol was excluded: In the 8S region 97% of [3H] R1881 was recovered by thin layer chromatography. Characteristics of this [3H] R1881-8S binding protein include high affinity (Kd = 2.3 +/- 41 nM) and low binding capacity (18.8 +/- 3.3 fmol/mg cytosol protein), precipitability in 0-33% ammonium sulfate, and translocation to isolated nuclei following in vivo R1881 treatment. Whereas, the cytosol R1881-receptor is competed for by dihydrotestosterone, testosterone, and estradiol, [3H] estradiol binding in the 8S region is not competitive with androgens but does compete with diethylstilbestrol. The nuclear androgen binding site has a Kd = 2.8 nM for [3H] R1881, and is androgen specific (testosterone greater than 5 alpha-dihydrotestosterone greater than estradiol greater than progesterone greater than cyproterone acetate greater than diethylstilbestrol greater than dexamethasone greater than triamcinolone). Since a number of liver proteins including the drug and steroid metabolizing enzymes are, in part, influenced by the sex-hormone milieu, the presence of a specific androgen receptor in male rat liver may provide valuable insight into the regulation of these proteins.     

Testicular toxicity and mutagenicity of steroidal and non-steroidal estrogens in the male mouse.

The mutagenicity and toxicity of diethylstilbestrol (DES), 17 beta-estradiol and zeranol on the male mouse germ cells were investigated with meiotic micronucleus assays in vivo and in vitro, sperm-head abnormality test and morphometry. Further, the developmental effects of DES on testicular morphology were explored. Micronucleus induction was observed at 10(-7) M concentration of DES and 17 beta-estradiol in vitro, but other treatments yielded negative results. The micronucleus assay in vivo revealed a small number of micronuclei in early haploid spermatids 17 days after a single subcutaneous injection of DES 50 mg/kg, whereas estradiol and zeranol gave negative results. The sperm-head abnormality rates were significantly elevated 5 weeks after treatments with high doses of DES, 17 beta-estradiol and zeranol, and testicular morphometry revealed transient changes in the volume densities of testicular tissue components. Prenatal and neonatal estrogen administration resulted in permanent alterations in seminiferous epithelium and dilatation of the rete testis, but did not affect micronucleus or sperm-head abnormality rates. The mutagenicity and toxicity of hormones in the mouse testis paralleled the hormonal activity of these compounds. Early estrogenization was the most sensitive toxicity test, followed by in vitro meiotic micronucleus induction, whereas the sperm-head abnormality assay and morphological analysis did not reveal subtle changes.     

Estrogen-binding protein from rat preputial gland: purification and characterization.

Cytosol from the rat preputial gland has been shown to contain a protein which binds both estrone and estradiol. The protein, after a 26-fold purification from the cytosol of female Sprague-Dawley rats, migrated as one band during electrophoresis in sodium dodecyl sulfate on acrylamide gel. The electrophoretic mobility indicated a molecular weight of 15,000. The association constant for estrone as determined by equilibrium dialysis was 1.2 X 10(7) M-1, while that for 17beta-estradiol was 3.3 X 10(6) M-1. Progesterone, cortisol, testosterone, or diethylstilbestrol did not bind to the purified protein, whereas 17alpha-estradiol or estriol bound only slightly. In the presence of retinoic acid, but not retinol, the binding of estrone was reduced. Optimum binding for estrone was at pH 6.5 to 8.5.     

Binding characteristics of estrogen receptor (ER) in Atlantic croaker (Micropogonias undulatus) testis: different affinity for estrogens and xenobiotics from that of hepatic ER.

An estrogen receptor (ER) was identified in cytosolic and nuclear fractions of the testis in a marine teleost, Atlantic croaker (Micropogonias undulatus). A single class of high affinity, low capacity, and displaceable binding sites was identified by saturation analysis, with a Kd of 0.40 nM in cytosolic extracts and a Kd of 0.33 nM in nuclear extracts. Competition studies demonstrated that the receptor was highly specific for estrogens (diethylstilbestrol > estradiol > estriol = estrone) and also bound several antiestrogens. Testosterone and 5alpha-dihydrotestosterone had much lower affinities for the receptor, whereas no displacement of specific binding occurred with 11-ketotestosterone or any of the C21 maturation-inducing steroids. A variety of xenoestrogens, including o,p'-dichlorodiphenyltrichloroethane (DDT), chlordecone (Kepone), nonylphenol, hydroxylated polychlorinated biphenyls (PCBs), and the mycotoxin zearalenone, bound to the receptor with relatively low binding affinities, 10(-3) to 10(-5) that of estradiol. A comparison of the binding affinities of various ligands for the testicular ER and the hepatic ER in this species revealed that the testicular ER was saturated at a lower [3H]estradiol concentration (1 nM vs. 4 nM). The binding affinities of several compounds, including testosterone and nafoxidine, exhibited marked differences for the two ERs; and most of the estrogens and xenoestrogens tested had higher binding affinities for the testicular receptor. Minor amounts of estradiol (0.12 ng/g tissue/h) were produced by testicular tissue fragments incubated in vitro, and estradiol was detected in male Atlantic croaker plasma. The identification of a testicular ER and evidence that estradiol is produced by the testes in croaker suggest that estrogens participate in the hormonal control of testicular function in teleosts.     

Estrogen receptors in the adrenal cortex of the possum (Trichosurus vulpecula)

1. Specific [3H]estradiol binding activity with characteristics of estrogen receptors was found in the cytosols and nuclear extracts of the adrenal cortex proper and special zone of the brushtail possum (Trichosurus vulpecula). 2. The specific estradiol receptor had a sedimentation coefficient on sucrose gradients of approximately 9S and a molecular weight on gel filtration of more than 200,000. The adrenal cortex cytosol binds [3H]estradiol with high affinity (Ka 5.5 X 10(9) M-1), and limited capacity (Bmax 62.7 fmol/mg cytosol prot). In competition experiments with different steroids the receptor showed a high affinity for four estrogens and a very low affinity to androgens, progesterone and cortisol. 3. There was no difference in the affinity and maximum binding capacity of the cytosols from cortex proper in male and female animals, but the binding capacity of the special zone of females was half that of cortex proper. Estradiol receptors were found in the kidney, liver, lung, testis and muscle but only in the adrenal and prostate was the binding capacity relatively high compared with the uterus. 4. The specific binding capacity of [3H]estradiol to cytosols of adrenal cortex at different stages of the estrus cycle and pregnancy was unrelated to that of the uterus. In the adrenal the receptor concentration was lowest at estrus, when uterine concentration was high, while in late pregnancy the binding of adrenal cortex and uterus cytosols was almost the same. 5. The possible physiological significance of the presence of a specific estrogen receptor in male and female possums is discussed.     

Organometallic estrogens: synthesis, interaction with lamb uterine estrogen receptor, and detection by infrared spectroscopy

As an integral part of the development of a new technique using organometallic markers for the detection of hormone receptors by FT-IR spectroscopy, a series of estradiol derivatives labeled with Cr(CO)3 or Cr(CO)2CS fragments on the A ring has been synthesized. The stereochemistry of one of these steroids, alpha-[3-(dimethyl-tert-butylsiloxy)-17 beta-estradiol]dicarbonyl(thiocarbonyl)chromium(0), has been established by X-ray diffraction. The organochromium-labeled steroids are stable in aqueous methanol solution, and their relative binding affinities to estrogen receptor have been determined; these values vary from 0.4 to 28%. The complex exhibiting the strongest affinity, [3-O-(3-hydroxypropyl)-17 beta-estradiol]-chromium tricarbonyl complex, has been prepared in a tritiated form with a high specific activity (4.1 Ci/mmol). This tritiated hormone binds reversibly to the estradiol receptor in lamb uterine cytosol with an affinity (Kd = 0.85 nM) and number of binding sites (n = 770 fmol/mg of protein) close to the values observed for estradiol itself. The level of nonspecific binding is low, and the hormone is not bound significantly to other nontarget tissues. The observation that the binding affinity of the steroid depends on which side of the steroidal A ring the organometallic label is bound demonstrates the nonequivalence of the two sides of the A ring with respect to the receptor site. The FT-IR spectra of the organochromium markers in the v(CO) region can be used for the detection of the estradiol receptor in lamb uterine cytosol.     

Testicular FSH receptor numbers and affinity in bulls of various ages

Bulls (N = 42) ranging in age from 1 day to 5.5 years were used to determine whether a change in the concentration of FSH receptors in the bovine testis occurred as bulls matured. 125I-labelled human FSH was used as the ligand to evaluate binding to bovine testicular membranes. Membrane fractions were collected by centrifugation of testicular homogenates at 120 g and recentrifugation of the 120 g supernatant at 1250 g. Relative binding activity of membrane sedimented at 1250 g was determined after incubation of membranes with 125I-labelled FSH for 16--18h at 25 degrees C, followed by centrifugation (1250 g) to separate bound from free hormone. Specifically bound FSH when expressed as fmol/mg protein was negatively correlated with age (r = -0.73). The association constant (Ka) determined by Scatchard analysis was the same for bulls at all ages with a mean (+/- s.e.m.) Ka = 1.5 +/- 0.3 x 10(9) M-1. Concentration of FSH receptors on a per mg protein basis declined rapidly from birth to 2.5 years of age and remained low up to 5.5 years of age. On a whole testis basis the total number of receptors increased as the bulls matured. After 2.5 years of age total testicular binding did not change.     

Life-long effect of a single neonatal treatment with estradiol or progesterone on rat uterine estrogen receptor binding capacity.

Rats treated with a single dose of 17 beta-estradiol or progesterone within 24 h of birth were subjected to ovariectomy at 8 weeks of age and were nine days later examined for the binding capacity of the uterine estradiol receptors by saturation and competition tests (with diethylstilbestrol used as competitor). The Bmax value of the neonatally estradiol-treated rats (6.78 x 10(-10) M) was significantly decreased relative to the control (1.99 x 10(-9) M). The competition analysis affirmed these results. Neonatal progesterone treatment also accounted for a significant decrease (1.25 x 10(-9) M) in receptor concentration relative to the control (1.66 x 10(-9) M). Considering the competition analysis the decrease was less than in the case of estradiol and not even significant by saturation analysis. The uterine mass did not differ between the experimental and control rats, but part of those treated with estradiol developed ovarian cysts. It follows that not only synthetic steroids (DES, allylestrenol), but also an excessive presence of the physiological steroid hormone during the critical period of receptor maturation can account for a decrease in uterine receptor concentration in adulthood.     

A protease acting on the estrogen receptor may modify its action in the adult rabbit epididymis

We have previously shown that the cytosolic estrogen receptor in adult rabbit epididymides sediments as an congruent to 3 S species on sucrose gradients containing 0.01 M KCl while that from immature rabbit epididymides sediments at congruent to 9 S. This age-dependent decrease in sedimentation coefficient is attributable to the appearance of a leupeptin-sensitive protease as the animals mature. We now show that if adult epididymides are homogenized in buffer containing leupeptin, the 9 S receptor can be demonstrated, indicating inhibition of receptor degradation. In vitro nuclear uptake studies conducted in the absence of leupeptin indicated that the proteolyzed receptor was not an efficient nuclear binder. When leupeptin was present, nuclear uptake increased 6-fold and it was accompanied by depletion of receptor from the cytosol. Binding of the receptor to nuclei was specific since it could be inhibited by unlabeled estrogens but not by unlabeled 5 alpha-dihydrotestosterone or progesterone. In vitro mixing experiments indicated that the proteolytic activity was associated with the crude nuclear fraction since, in the absence of leupeptin, they had reduced ability to bind estrogen receptor present in immature epididymal cytosol. Specific in vivo binding of [3H]estradiol by adult and immature rabbit epididymides could be demonstrated. The time course of in vivo binding of [3H]estradiol by adult rabbit epididymal nuclei indicated maximum binding (70 fmol/g tissue) at 30 min following injection. By 60 min, the amount of binding had decreased to about 25 fmol. The accessory sex organs, which do not contain the protease, also exhibited maximum binding (150 fmol/g tissue) at 30 min. However, at the 60 min period binding was still about 140 fmol. Processing the tissues in buffers containing leupeptin had no effect on the results obtained. These results are interpreted to indicate that the presence of the protease decreases nuclear binding of the estrogen receptor and shortens nuclear occupancy. This combination of factors may be responsible for the decrease in estrogen action in the adult rabbit epididymis.     

Effect of estradiol-17β on glucose and proline uptake in plasma membrane vesicles from the R3230AC rat mammary carcinoma

Purified plasma membrane vesicles isolated from R3230AC rat mammary tumors displayed carrier-mediated and stereospecific uptake. Uptake was shown to be proportional to protein concentration, sensitive to increasing osmolarity, and inhibited only by substrates entering by the same carrier. Carrier-mediated glucose uptake was inhibited rapidly by estradiol-17β and phloretin in a dose-dependent manner, whereas proline uptake was not affected by estradiol-17β. The data suggest that the inhibition of glucose by estradiol and phloretin, originally observed in whole cells, occurs by an interaction of the steroid with a component on the plasma membrane. In contrast, the lack of effects of estradiol on proline transport into vesicles implies that intracellular components may have mediated the estrogen-induced effects observed in whole cells.     

Chronic estrogen treatment causes an alteration in uterine estrogen receptor dynamics of rats

Estrogen receptor content and dynamics in the uteri obtained from chronically estrogenized rats were analyzed. 12 day treatment with a subcutaneous implantation of a diethylstilbestrol pellet resulted in maximal stimulation of uteri with regard to wet tissue weight, DNA content, as well as progesterone receptor content without significant alteration of the estrogen receptor level. Estrogen receptor dynamics in just ovariectomized or ovariectomized and diethylstilbestrol-stimulated rats elicited by a single injection of estradiol were next examined using the exchange methods. The cytosol receptor content rapidly declined, with a small and temporary accumulation of the nuclear receptor in the uterus from rats continuously exposed to diethylstilbestrol during the preceding 12 days. A relatively rapid cytosol receptor replenishment was also observed in rats pretreated with diethylstilbestrol. This was accompanied by a rapid decrease in the nuclear receptor level to 70% of the preinjection value at 5 h after estradiol administration. These data are in contrast to findings on uteri of ovariectomized and nonestrogen-treated rats, in which a single injection of estradiol resulted in a prolonged nuclear receptor retention and a delayed cytosol receptor replenishment. Adrenalectomy did not result in a significant change of receptor dynamic patterns, suggesting that adrenal steroids do not play a role in the alteration of receptor dynamics elicited by continuous stimulation with diethylstilbestrol. These observations suggest that a continuous exposure of rat uteri to the estrogen causes an altered regulation of estrogen receptor dynamics by the homologous steroid compared to those in chronically estrogen-deprived rats.