Identification of immunoassayable estrogen receptor lacking hormone binding ability in tamoxifen-treated rat uterus

Using two different monoclonal antibodies to human estrogen receptor (ER), the enzymeimmunoassay was performed. The values of ER contents in human breast cancer and untreated rat uteri obtained by this procedure were correlated well with those by [3H] estradiol binding assay. When estradiol was injected to immature rats, the enzymeimmunoassay showed the uterine receptor dynamic pattern similar to those analyzed by exchange assays. In contrast, tamoxifen administration induced the immunoassayable but nonsteroid binding form of ER. This ER-like antigen was the heat-labile molecule with the sedimentation constant of 7 S while ER in untreated rat uterine cytosol sedimented at 9 S. These results suggest the presence of unique molecular state of ER induced by tamoxifen.     

Evidence for involvement of tyrosine in estradiol binding by rat uterus estrogen receptor

The possibility of tyrosine involvement in steroid binding by rat uterus estrogen receptor (rER) was investigated. Chemical modification of rER with reagents such as tetranitromethane (TNM) and N-acetylimidazole (NAcI) inhibited estradiol binding. Steroid binding was inhibited to a greater extent at pH 8 than at pH 6, indicating the participation of tyrosine (TNM has increasing affinity for cysteine ove tyrosine at pH 6). Inhibition patterns remained similar for incubations at 0-4 degrees C or 37 degrees C. NAcI inhibited rER steroid binding at 37 degrees C, but not at 0-4 degrees C. Hydroxylamine incubated in the presence of rER and NAcI appeared to reverse this inhibition. Thus, these findings indicate that the phenyl ring and possibly the phenolic hydroxyl of tyrosine are involved in steroid binding of the receptor.     

Alterations in estrogen receptor isoforms in the mammary gland and uterus of the rat during differentiation

1. Estrogen receptors in lactating mammary glands and uteri of rats which were 10 and 19 days postpartum exhibited molecular heterogeneity based on their surface charge properties. 2. The polymorphism of estrogen receptors detected by high-performance ion exchange chromatography may be monitored in-line with a radioisotope detector. 3. Estrogen receptors from the mammary gland and uterus of rats at 10 days of lactation exhibited primarily two receptor isoforms eluting at 200-250 mM and 250-300 mM phosphate, whereas three ionic isoforms (eluting at 50-150, 200-250 and 325-375 mM phosphate) were found in the mammary glands of rats at 19 days of lactation. Similar changes in the profiles of estrogen receptor isoforms were observed in uterine cytosol preparations at each stage of postpartum differentiation. 4. The elution pattern of receptor-associated radioactivity was not altered by the addition of diisopropylphosphate, a potent inhibitor of trypsin-like proteases, either before, during or immediately after homogenization. This indicates that the differences observed in the receptor elution profile of 10 and 19 day postpartum lactating mammary glands were not due to artifactual proteolysis. 5. In summary, our data indicate that the differentiation stage of lactating mammary glands may dictate the final profile of receptor isoforms detected.     

Non-stoichiometric nuclear-cytoplasmic redistribution of estrogen receptor in adult rat uterus, following estradiol injection

In immature and ovariectomized rats acutely injected with estradiol (E2), accumulation of estradiol receptor complexes (E2R) from the uterine cytosol to the nucleus has been shown to be quantitative by numerous investigators. In the present study, translocation of E2R from the cytosol to the nuclear fraction in adult and ovariectomized estrogen prestimulated rats was analyzed. Twenty micrograms of E2, dissolved in saline containing 10% ethanol and 1 g% bovine serum albumin (B.S.A.) were injected intraperitoneally to the animals and 2 h later E2R in the cytosol and crude nuclear fractions were assayed by exchange techniques. Unlike a 91% recovery of the depleted cytosol E2R in the nuclear fraction of ovariectomized rats, only 39.2 and 27.5% were recovered in the adult and ovariectomized estrogen prestimulated rat uterus respectively. Moreover, depending on the temperature and duration of nuclear suspension incubation, from 18 up to 80% of the recovered nuclear E2R were solubilized in the incubation medium and nuclear post-incubation washes and could be measured by hydroxylapatite treatment (HAP). Saturation assays showed a plateau from 12 nM E2 3H onwards up to 80 nM. The Kd values computed for the receptors in the nucleus and HAP in all the three groups were of the order of 2 X 10(-9) M. In conclusion, after E2 administration to adult or ovariectomized estrogen prestimulated rats, a stoichiometric recovery of the depleted cytosol E2R in the nuclear fraction was not observed, even when leakage of nuclear receptor into the medium in course of exchange was taken into account. Chronic estrogenization appeared to modify the dynamics of uterine receptor.     

Expression of peroxisome proliferator-activated receptor isoforms in the rat uterus during early pregnancy

Peroxisome proliferator-activated receptors (PPARs) play an important role in different compartments of the female reproductive system in rodents and humans. However, expressional profiles and physiological functions of PPARs in the endometrium prior to the placentation are not well understood. In this study, we determined expressional profiles of the PPARs during early pregnancy. Immunocytochemistry revealed that both PPARα and PPARβ/δ were strongly detected in the endometrial stroma on days 4.5–6.5 of pregnancy, which is just a starting time of implantation. Delayed implantation animal model showed that the expressions of PPARα and PPARβ/δ occurred after the initiation of implantation in the endometrial stroma. Moreover, an in vitro decidualization model further revealed that the expression of PPARα increased in the cultured rat endometrial stromal cells at 24 h after the decidualization treatment, but the expression of PPARβ/δ was delayed and increased at 48 h after the treatment. PPARγ was expressed in the endometrial stroma and its expression decreased significantly at 2.5 days post-coitum and maintained a low level of expression during the period of implantation. These results indicate that PPARα is expressed and induced by the initiation of implantation, prior to the expression of PPARβ/δ in decidualized endometrium. Increasing expression of PPARγ during fertilization and its decline during the period of implantation further suggest that PPARs may play important roles during early pregnancy.     

Preferential nuclear binding of estrogen in the formalin-fixed rat uterus

Rat uterus fixed overnight in buffered formalin retains the ability to specifically bind estradiol. However, the estrogen binding property of fixed tissue appears preferentially localized in the nuclear fraction regardless of hormonal status. Furthermore, the quantity of the nuclear estrogen receptor in fresh or fixed uterus is virtually identical in the presence or absence of estrogenic hormone. Yet, while both tissue preparations exhibit equivalent increases in the total nuclear receptor occupancy after hormone exposure, only the fresh uterus contains a major cytosolic estrogen binder which decreases in availability upon the estrogen-induced elevation of the nuclear bound steroid. However, the cytosolic estrogen receptor exhibits a significant loss in its ligand binding property after formalin exposure. Thus, the preferential localization of estrogen binding in the nuclear fraction of fixed whole tissue may just reflect that only the tightly bound nuclear estrogen receptor's functional and/or structural integrity survives long-term formation fixation. Our observation of estrogen binding in preserved tissue may also be a clinically useful tool in therapy analysis.     

Immunohistochemical localization of estrogen receptor in rat brain, pituitary and uterus with monoclonal antibodies

Localization of estrogen receptor (ER) in rat brain, pituitary and uterus is shown by the avidin-biotin complex technique using a monoclonal antibody, JS34/32. Immunostaining is observed in nuclei of certain neurons in the preoptic-septal region, hypothalamus and amygdala, and in cells of the anterior pituitary and uterus after estradiol stimulation. Staining is specific since preadsorbed JS34/32 antibody with purified cytoplasmic ER as well as a control monoclonal antibody do not show positive immunoreaction. In the brain, neither cytoplasmic nor nuclear staining is seen in the absence of estradiol stimulation, nor with the progesterone and dihydrotestosterone treatments. The distribution of ER-containing neurons in specific areas of the brain overlaps with the distribution of estrogen target neurons demonstrated by autoradiography. The results demonstrate the usefulness of the monoclonal antibody for the detection of ER in target tissues.     

Progesterone regulation of estrogen receptor in the rat uterus: A primary inhibitory influence on the nuclear fraction

The purpose of this study was to determine the short-term effects of progesterone action on estrogen receptor (Re) levels in the rat uterus. Ovariectomized, adrenalectomized rats were maintained on subcutaneous Silastic implants containing crystalline estradiol. Progesterone treatment with serum estradiol maintenance caused a rapid decrease (within 4 h) of total Re, attributable to loss of nuclear Re without a significant change in cytosol Re levels. Removal of estradiol implants resulted in an increase in total Re and cytosol Re at all time periods studied without a significant decrease in nuclear Re until 8 h. Combined estradiol withdrawal and progesterone treatment resulted in lower total Re levels and a more rapid decrease in nuclear Re than with estradiol withdrawal alone. These results demonstrate that progesterone rapidly and selectively decreases nuclear Re levels in rat uterus and suggest that this process is not dependent on cytosol Re or serum estradiol levels.     

Regulation of estrogen receptor protein and messenger ribonucleic acid by estradiol and progesterone in rat uterus

The objective of this study was to examine the mechanisms of estrogen receptor (ER) processing and replenishment in the uterus of ovariectomized rats after estradiol and progesterone treatment. Uterine ER binding activity, ER protein and ER mRNA were measured by receptor binding exchange assay, Western blot and slot blot, respectively. The regulation of ER levels in rat uterus by estradiol and progesterone was very dramatic. Changes in ER protein were faithfully reflected by changes in binding activity. Estradiol caused receptor “processing” within 4 h of administration followed by recovery or “replenishment” of ER levels to the initial level by 20 h. The term “processing” has previously been used to describe the loss of ER binding activity in the early phase of estradiol-action, but it was never clear whether the ligand binding site was inactivated by processing or if the receptor molecule actually disappeared. This study shows that receptor “processing” constitutes disappearance of receptor protein and the later “replenishment” phase represents new ER protein rather than recycling of “processed” receptor. Progesterone-action, on the other hand, influenced only the “replenishment” phase by blocking recovery of ER protein. ER mRNA was suppressed by estradiol at 8 h, after the receptor was “processed” and “replenishment” already initiated. Progesterone, on the other hand, did not alter the steady state level of the message. Other mechanisms, such as regulation of translation rate of existing mRNA and changes in the rate of degradation of ER proteins are more likely involved in acute regulation of ER by these ovarian steroid hormones.     

Effects of SERM (selective estrogen receptor modulator) treatment on growth and proliferation in the rat uterus

Background  

Selective estrogen receptor modulators (SERMs) have been developed in order to create means to control estrogenic effects on different tissues. A major drawback in treatment of estrogen receptor (ER) positive breast cancer with the antagonist tamoxifen (TAM) is its agonistic effect in the endometrium. Raloxifene (RAL) is the next generation of SERMs where the agonistic effect on the endometrium has been reduced.     

Aprotinin inhibits the hormone binding of the estrogen receptor from calf uterus

Micromolar concentrations of the proteinase inhibitor, aprotinin, produced a dose-dependent inhibition in the binding capacity of the estrogen receptor from calf uterus. Aprotinin inhibition was greater at 28 degrees C than at 4 degrees C and only occurred when conditions allowed the receptor transformation. When aprotinin was tested in the presence of transformation inhibitors, its effect was no longer seen. The binding capacity of the highly purified estrogen-binding subunit was similarly inhibited.