Regulation of uterine epidermal growth factor (EGF) receptors by estrogen in the mature rat and during the estrous cycle

Previous work has shown that the immature rat uterus contains epidermal growth factor (EGF) receptors and that tissue levels of this receptor are increased by the administration of exogenous estrogens. This study was undertaken to determine if estrogen administration also elevated EGF receptor levels in the mature animal and if the growth factor receptor levels varied in concert with endogenous estrogens throughout the estrous cycle. In the mature, castrate rat administration of estradiol, but not non-estrogenic steroids, causes a 2-3-fold elevation of uterine EGF receptors as judged by ligand binding. This increase is maximum in 18 h and is due to an increase in the number of binding sites. In cycling animals EGF receptor levels are low at metestrus, rise at diestrus, reach a maximum (approximately twice metestrus values) at proestrus, and then return at estrus to metestrus levels. These changes in EGF receptor levels parallel changes in plasma estrogens and occupied nuclear estrogen receptor reported by other workers. These results indicate that uterine EGF receptors are increased by exogenous estrogens in both mature and immature animals, and support a physiological role for estrogens in the regulation of this growth factor receptor.     

Immunohistochemical detection of estrogen receptors in the canine uterus and their relation to sex steroid hormone levels

Cyclic changes in estrogen receptor expression in the uterine tissue of 60 female dogs were evaluated, using an immunohistochemical technique on formalin-fixed paraffin-embedded sections. The expression of estrogen receptors in the uterine horns, body and cervix was quantified by means of an immunohistochemical score. A negative correlation was found between staining scores in the uterine horns and serum progesterone levels. Generally, staining scores in the uterine horns were highest during proestrus, declined during estrus and were lowest during early metestrus. During anestrus high staining scores for estrogen receptors were observed, indicating sensitivity for estrogens in a sexual quiescence stage. Compared with the uterine horns, high staining scores were found in the uterine body and cervix during estrus and metestrus. No positive staining for estrogen receptors was detected in 1 pregnant uterus. Fluctuations in estrogen receptors were more pronounced in endometrial stroma cells than in epithelial cells of the uterine horns. The importance of stromal cells in the sexual cyclicity of the canine uterus should not be underestimated when studying uterine endocrinology and pathology.     

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.     

Progesterone and estrogen receptor distribution in the endometrium of the mare

An immunoperoxidase staining technique was used to localize receptors for progesterone and estrogen in the uterus of the mare. Specific staining for receptors was limited to cell nuclei. During estrus, stromal cells tended to stain more intensely for both receptor types than myometrial cells or luminal and glandular epithelial cells. During diestrus, staining intensities in stromal and myometrial cells tended to decrease. Staining intensities of epithelial cells were not affected by the cycle stage. Early pregnancy did not markedly affect the staining intensities of pregnant mares compared with the nonpregnant mares on Day 14 of diestrus. In mares susceptible to endometritis from which samples were taken during diestrus, stromal and myometrial staining for estrogen receptors was more intense than in endometrium from genitally-normal mares.     

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 of nerve growth factor and its receptors NTRK1 and TNFRSF1B is regulated by estrogen and progesterone in the uteri of golden hamsters

Experiments were conducted using female golden hamsters to identify the presence of nerve growth factor (NGF) and its receptors NTRK1 and TNFRSF1B in the uteri of female animals and regulation on their expression by estrogen and progesterone. NGF and its receptor NTRK1 were immunolocalized to luminal epithelial cells, glandular cells, and stromal cells. TNFRSF1B was immunolocalized in luminal epithelial and glandular cells, with no staining found in stromal cells of the uterine horns of normal cyclic golden hamsters. Strong immunostaining of NGF and its receptors NTRK1 and TNFRSF1B was observed in uteri on the day of proestrus as compared to the other stages of the estrous cycle. Results of immunoblot analysis of NGF revealed that there was a positive correlation between uterine NGF expression and plasma concentrations of estradiol-17beta. To clarify the effects of estrogen and progesterone on NGF, NTRK1, and TNFRSF1B expression, adult female golden hamsters were ovariectomized and treated with estradiol-17beta and/or progesterone. Immunoblot analysis and immunohistochemistry indicated that estradiol-17beta stimulated expression of NGF and its two receptors in the uterus. Treatment with progesterone also increased NGF and NTRK1 expression in the uterus. However, no additive effect of these steroids on expression of NGF and its receptors was observed. Changes in uterine weights induced by estradiol-17beta and/or progesterone showed the same profile with that of NGF, suggesting that a proliferative act of NGF may be involved in uterine growth. These results suggest that NGF may play important roles in action of steroids on uterine function.     

Immunohistochemical detection of androgen receptors in the canine uterus throughout the estrus cycle

Serum androgen levels in the bitch increase during proestrus and remain elevated until metestrus. To find out whether androgens can have a direct impact on the canine uterus, androgen receptors (AR) were identified immunohistochemically in uterine tissue. Androgen receptor distribution in the uterine horns, body and cervix was described during different cycle stages, during pregnancy and in the postpartum period. Nuclear staining for AR was observed in cells of the surface epithelium, glandular ducts, basal glands and stroma of the endometrium, and in myometrial smooth muscle cells. In addition, cytoplasmic staining was observed in epithelial cells from proestrus to early metestrus, when the cells were secretory active, and in stroma cells during pregnancy, suggesting a role for androgens in decidualization. During pregnancy and in the postpartum period nuclear staining for AR was nearly absent. During the estrus cycle stroma cells stained with higher intensities for AR than epithelial cells, supporting the idea that stroma cells mediate some effects of steroid hormones on epithelial cells in the genital tract. In contrast with earlier findings on estrogen receptor-alpha and progesterone receptors, no significant changes in androgen receptor expression were observed during the estrus cycle. Few correlations were found between the staining for AR and serum levels of the sex steroids. The present findings suggest that there is a basal expression of AR in the canine uterus throughout the estrus cycle that may not be influenced by sex steroid hormones.     

Histochemical demonstration of estrogen and progesterone binding in endometriotic tissue and in uterine endometrium: A comparative study

Estrogen and progesterone binding to endometriotic and endometrial tissue was studied histochemically using estradiol and progesterone fluorochrome derivatives (E2-bovine serum albumin-fluorescein isothiocyanate and progesterone-bovine serum albumin-tetramethylrhodamine isothiocyanate). Thirty endometriotic samples from 21 women were studied, together with endometrial specimens obtained simultaneously from 14 of the women. In 77% of the endometriotic samples binding of the estrogen conjugate was indicated by specific fluorescence in more than half of the epithelial cell population, and in 20% in less than half. The corresponding figures for the progesterone conjugate binding were 75 and 18%, respectively. Blocking studies indicated a reasonable degree of ligand specificity. In endometrial tissue the corresponding figures were 64 and 29%, respectively, for binding of the estrogen conjugate and 54 and 38%, respectively, for binding of the progesterone conjugate. In 7 of 13 cases where evaluable samples of both tissues had been obtained, the relative proportion of fluorescent cells, with either reagent, was similar in the two tissue types. Our results suggest that the cytoplasm of epithelial cells in endometriotic tissue and in uterine endometrium contains specific binding sites for both estrogen and progesterone. The binding pattern of the two conjugates in endometriotic tissue was unrelated to the menstrual phase.     

Hormonal regulation of vitellogenin genes: an estrogen-responsive element in the Xenopus A2 gene and a multihormonal regulatory region in the chicken II gene

Expression of the vitellogenin genes in avian and amphibian liver is regulated by estrogens. The DNA elements mediating estrogen induction of the various vitellogenin genes of chicken and Xenopus encompass one or more copies of a 13-mer palindromic sequence called the estrogen-responsive element (ERE). Here we show that upon incubation with the purified estrogen receptor (ER) from calf uterus the Xenopus vitellogenin A2 gene yields a DNase-I footprint over the ERE between -331 and -319. This element does not mediate the response to glucocorticoids or progestins in T47D cells. The three guanine residues in each half of the palindrome are protected against methylation by dimethylsulfate after incubation with ER, but not with glucocorticoid (GR) or progesterone (PR) receptors. In contrast, the chicken vitellogenin II gene exhibits multihormonal regulation by estrogens, progestins, and glucocorticoids in T47D and MCF7 cells. Regulation is mediated by the DNA region between -721 and -591 that contains four binding sites for hormone receptors, as demonstrated by DNase-I footprints and methylation protection experiments. The two distal and most proximal binding sites are recognized by ER, GR, and PR, whereas the central binding site is only bound by ER and GR. At suboptimal concentrations, estrogens and progestins or glucocorticoids act synergistically. In experiments using a DNA fragment containing an ERE adjacent to a glucocorticoid-responsive element/progesterone-responsive element, ER and PR bind synergistically to their corresponding sites, perhaps explaining the functional synergism of both hormones. Thus, two very different regulatory elements are used to mediate estrogen induction of related genes in chickens and amphibians.     

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.     

Autocrine role of estrogens in the augmentation of luteinizing hormone receptor formation in cultured rat granulosa cells

The effects of estrogens on gonadotropin-stimulated luteinizing hormone (LH) receptor formation were examined in primary cultures of rat granulosa cells. Granulosa cells were cultured for 3 days with increasing concentrations of follicle-stimulating hormone (FSH) in the presence or absence of native and synthetic estrogens. Follicle-stimulating hormone stimulated LH receptor formation in a dose-dependent fashion, and estrogens enhanced the FSH-stimulated LH receptor content by decreasing the apparent ED50 of FSH. At 6.25 ng/ml FSH, the enhancement in LH receptor was estrogen dose dependent, with an ED50 value of about 3 X 10(-9) M for 17 beta-estradiol. The increased LH receptor content seen in cells treated with FSH and estrogen was correlated with increased cAMP production by these cells in response to LH stimulation. Time course studies revealed enhancement of FSH-stimulated LH receptor induction at 48 and 72 h of culture. Granulosa cells were also cultured with FSH for 2 days to induce functional LH receptors, then further cultured for 3 days with LH in the presence or absence of estrogens. At 30 ng/ml LH, increasing concentrations of estrogens maintained LH receptor content in a dose-dependent fashion, with their relative estrogenic potencies in keeping with reported binding affinities to estrogen receptors. An autocrine role of estrogens on LH receptor formation was further tested in granulosa cells treated with FSH and an aromatase substrate (androstenedione) to increase estrogen biosynthesis. Cotreatment with semipurified estrogen antibodies partially blocked the FSH stimulation of LH receptors, whereas nonimmune serum was ineffective. Also, inclusion of diethylstilbestrol prevented the inhibitory effect of the estrogen antibodies. Thus, local estrogens in ovarian follicles may play an autocrine role in granulosa cells to enhance LH receptor formation and to increase granulosa cell responsiveness to the LH surge, with subsequent ovulation and adequate corpus luteum formation.     

Estrogen receptors in the rat uterus. Retention of hormone-receptor complexes.

The retention pattern and biochemical characteristics of estrogen receptors in the nuclei of uterine cells were studied as a function of time after the in vivo injection of estradiol (E2) to immature female rats. One hour after the injection of 0.1 mug of tritiated E2, approximately 0.20 pmol per uterus of receptor bound hormone is retained in uterine nuclei. This dose of E2 produces a maximal uterotrophic response. Six hours after E2 administration, uterine nuclei retain 0.04-0.08 pmol of hormone per uterus. Hormone receptor complexes extracted from uterine nuclei 1, 3, and 6 h after in vivo injection of hormone have similar structural and binding characteristics. Receptors extracted at all three times sediment at 5S in high salt gradients and have a dissociation binding constant of approximately 3 nM for E2. The wash-out curves of receptors as a function of salt concentration are identical for uterine nuclei from animals treated for 1 or 6 h with estradiol, suggesting that the nature of the nuclear binding of receptors is not altered during this time interval. Experiments utilizing the injection of unlabeled estradiol, followed by an in vitro exchange procedure with tritiated estradiol, indicated that the total nuclear estrogen receptor sites, i.e., filled and vacant, decreased similarly.     

Quantification of cytosolic steroid receptors in secretory and non-secretory epithelial cells of the canine prostate

Radiolabelled methyltrienolone, dihydrotestosterone and estradiol were used as ligands to identify and quantify androgen and estrogen receptors in freshly dispersed cells from the canine prostate. Soluble extracts (cytosols) were obtained from secretory and non-secretory epithelial cells separated on the basis of their density in Percoll gradients. For both cell types, as well as for the whole prostate, Scatchard plot analyses were linear and showed a single class of high affinity binding sites: Kd values of 3.6 +/- 2.2 X 10(-9) M and 3.0 +/- 1.2 X 10(-10) M were measured for the androgen and estrogen receptors, respectively. The number of binding sites for the cytosolic androgen receptor, expressed per mg of protein or per mg of DNA, was 2.4- to 6.7-fold higher in the non-secretory cells compared to the secretory cells. However, these two cell types contained a similar number of specific sites for the estrogens. The specificities of the androgen and estrogen receptors were shown to be identical for the two cell types: the binding of [3H]R1881 was strongly inhibited by unlabelled R1881, 5 alpha-androstane-3 alpha, 17 beta-diol and dihydrotestosterone, while 5 alpha-androstane-3 beta, 17 beta-diol, estradiol and estrone did not displace bound R1881. The addition of triamcinolone acetonide did not alter the binding of R1881 in extracts of either cell type or in the whole prostate. The binding of [3H]estradiol to the estrogen receptor was highly specific since a strong displacement was only observed with estradiol (83%).     

The expression of vascular endothelial growth factor and its receptors (flt1/fms, flk1/KDR, flt4) and vascular endothelial growth inhibitor in the bovine uterus during the sexual cycle and their correlation with serum sex steroids

The present study was conducted to demonstrate of the immunohistochemical localization of vascular endothelial growth factor (VEGF) and its receptors (flt1/fms, flk1/KDR and flt4) as well as vascular endothelial growth inhibitor (VEGI) and to determine the correlation of VEGF and its receptors and VEGI with serum sex steroids (estrogen and progesterone) in the bovine uterus during the sexual cycle. The stage of the estrous cycle in 30 Holstein cattle was assessed based on the gross and histological appearance of the ovaries and uterus and on blood steroid hormone levels. Tissue samples obtained from the uterus were fixed in 10% formaldehyde for routine histological processing. During both follicular and luteal phases, positive cytoplasmic and membrane staining was achieved for VEGF and its receptors (flt1/fms, flk1/KDR and flt4) as well as VEGI in the luminal and glandular epithelial cells, the connective tissue and smooth muscle cells, and the vascular endothelial cells and smooth muscle cells in the uterus. The intensity, proportional and total scores determined for VEGF and its receptors (flt1/fms and flt4) as well as VEGI were greater in the luminal and glandular epithelial cells compared to the connective tissue and smooth muscle cells (P < 0.05). Furthermore, the number and intensity of the flk1/KDR positive cells were greater among the connective tissue cells compared to the luminal and glandular epithelial cells (P < 0.05). As a result, it was determined that the expression of VEGF and its receptors as well as VEGI in the bovine uterus during the follicular and luteal phases varied with different cell types. This suggests that depending on the stage of the sexual cycle, these factors may mediate the establishment of an appropriate environment for the nutritional supply and implantation of the embryo primarily due to the stimulation of angiogenesis but also through the increase in the secretory activity of the epithelial cells in the uterus. Furthermore, this indicates that ovarian steroid hormones play a significant role in regulating the expression of VEGF and its receptors as well as VEGI.     

Calmodulin enhances the stability of the estrogen receptor

The estrogen receptor mediates breast cell proliferation and is the principal target for chemotherapy of breast carcinoma. Previous studies have demonstrated that the estrogen receptor binds to calmodulin-Sepharose in vitro. However, the association of endogenous calmodulin with endogenous estrogen receptors in intact cells has not been reported, and the function of the interaction is obscure. Here we demonstrate by co-immunoprecipitation from MCF-7 human breast epithelial cells that endogenous estrogen receptors bind to endogenous calmodulin. Estradiol treatment of the cells had no significant effect on the interaction. However, incubation of the cells with tamoxifen enhanced by 5-10-fold the association of calmodulin with the estrogen receptor and increased the total cellular content of estrogen receptors by 1.5-2-fold. In contrast, the structurally distinct calmodulin antagonists trifluoperazine and CGS9343B attenuated the interaction between calmodulin and the estrogen receptor and dramatically reduced the number of estrogen receptors in the cell. Neither of these agents altered the amount of estrogen receptor mRNA, suggesting that calmodulin stabilizes the protein. This hypothesis is supported by the observation that, in the presence of Ca2+, calmodulin protected estrogen receptors from in vitro proteolysis by trypsin. Furthermore, overexpression of wild type calmodulin, but not a mutant calmodulin incapable of binding Ca2+, increased the concentration of estrogen receptors in MCF-7 cells, whereas transient expression of a calmodulin inhibitor peptide reduced the estrogen receptor concentration. These data demonstrate that calmodulin binds to the estrogen receptor in intact cells in a Ca2+-dependent, but estradiol-independent, manner, thereby modulating the stability and the steady state level of estrogen receptors.     

Comparative analysis of estrogen receptors covalently labeled with an estrogen and an antiestrogen in several estrogen target cells as studied by limited proteolysis

Estrogen receptors covalently labeled with the estrogen affinity label [3H]ketononestrol aziridine (KNA) or with the antiestrogen affinity label [3H]tamoxifen aziridine (TAZ) were subjected to limited proteolysis with trypsin, alpha-chymotrypsin, and Staphylococcus aureus V8 protease and then analyzed on 10-20% sodium dodecyl sulfate-polyacrylamide gradient gels followed by fluorography. The similar molecular weights of intact receptors (Mr 66,000 daltons) and the proteolytic digest patterns indicate extensive homology among estrogen receptors from MCF-7 human breast cancer cells, GH4 rat pituitary cells and rat uterus when liganded with estrogen or antiestrogen. Each protease generated a distinctive ladder of estrogen receptor fragments, and the fragmentation patterns were virtually identical for estrogen receptors labeled with estrogen (KNA) or antiestrogen (TAZ). Each protease yielded a relatively "resistant" receptor fragment of about 28,000-35,000 daltons. Trypsin and chymotrypsin at higher concentrations generated a much smaller 6,000-8,000 dalton digest product that still contained the [3H]KNA- or [3H]TAZ-labeled receptor binding site. Moreover, the receptor digest patterns were similar for estrogen receptors from the three different target cells. Our studies suggest considerable structural relatedness among these three estrogen receptors and also indicate that these two affinity labels bind to a similar, perhaps identical, region of the receptor molecule.