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Estrogen synthesis in vivo in the adult zebra finch: additional evidence that circulating estrogens can originate in brain.

Aromatase activity is abundant in limbic and nonlimbic neural structures in zebra finches, especially in the telencephalon near neural circuits that control singing behavior. Also, male songbirds can have high estrogen levels in blood. Because it is difficult to detect aromatase activity in other male tissues, we have postulated that the brain itself is the source of the estrogen present in the blood of males. Previously, we developed methods to measure estrogen synthesis in vivo by injecting [3H]androgen into the systemic circulation or directly into tissues and then determining the quantity of [3H]estrogen entering or leaving the brain. Our results support our hypothesis that the brain is the primary site of estrogen synthesis in males of this species. Here, we confirm that [3H]estrogen emanates from the brain after systemic [3H]androgen injection by showing that the presumptive estrogen in jugular plasma is significantly reduced by treatment with an aromatase inhibitor. In females, but not males, estrogens can be found in large amounts in carotid plasma after systemic androgen injection, presumably due to ovarian estrogen synthesis. However, carotid estrogen levels are variable in females, so it is difficult to determine if the female brain contributes estrogen to blood as is seen in males. The data presented here suggest that the adrenal of males is not an important site of aromatization, because [3H]estrogens are undetected in the adrenals of castrated or sham-castrated males after adrenal injections of [3H] androgen. Nevertheless, at least 2.8- to 17.3-fold more [3H]estrogen was present in the jugular than in the carotid of castrated males after systemic injection of [3H]androgen. Because androgen levels were in the physiological range, we conclude that the brain routinely enriches the estrogen content of blood in normal adult males of this species.

Adrenal Glands↗

Estrogen and skin: the effects of estrogen, menopause, and hormone replacement therapy on the skin.

UNLABELLED: Aging is associated with declining levels of several hormones, including estrogen. Although the effects of estrogen on the skin are still not fully understood, it is known that, in women, declining estrogen levels are associated with a variety of cutaneous changes, many of which can be reversed or improved by estrogen supplementation. Estrogens are C-18 steroids synthesized from cholesterol in the ovary premenopausally and in the peripheral tissue in postmenopausal women. Two estrogen receptors, alpha and beta, have been cloned and found in various tissue types. Studies of postmenopausal women indicate that estrogen deprivation is associated with dryness, atrophy, fine wrinkling, poor healing, and hot flashes. Epidermal thinning, declining dermal collagen content, diminished skin moisture, decreased laxity, and impaired wound healing have been reported in postmenopausal women. This article reviews the effects of declining estrogen levels on the skin and the effects of estrogen supplementation. LEARNING OBJECTIVE: At the conclusion of this learning activity, participants should be familiar with the pathways of estrogen synthesis, sites of estrogen receptors, age-dependent variations in serum estrogen concentration, the changes seen in postmenopausal skin, and the effects of estrogen supplementation.

Aging↗

Selective promoter usage of the human estrogen receptor-alpha gene and its regulation by estrogen.

Three promoters have been identified for the human estrogen receptor-alpha gene. The positions of promoters A and B are known whereas that of the recently identified promoter C is not. Cloning and hybridization experiments demonstrated that promoter C is located more than 21 kb upstream of promoter A. The use of the three promoters was examined in estrogen receptor-positive breast cancer cell lines, cell lines derived from other malignancies, and some normal tissues by RT-PCR and transient transfection. All estrogen-responsive breast cancer cell lines used all three promoters, apart from ZR-75 cells, which did not use promoter B in one of two sublines examined. Cell lines derived from other malignancies and other normal tissues that express lower levels of estrogen receptor-alpha showed more selective promoter usage. This suggests that the level of expression of estrogen receptor-alpha is determined by the number of promoters used, rather than the selective use of specific promoters. We also show that promoter C is used more widely than suggested by others. Analysis of a series of estrogen receptor-positive primary breast tumors showed that all three promoters were used in all the tumors. All three promoters were modulated by estrogen in estrogen-responsive breast cancer cell lines: all three promoters were down-regulated by estrogen in MCF-7 cells in which estrogens reduce receptor expression whereas all promoters used were upregulated in T47D, ZR-75, and EFM-19 cells in which estrogens increase receptor expression. This suggests that it is the repertoire of transcription factors present within a cell rather than the selective use of a specific promoter that determines whether estrogen receptor-alpha expression is increased or decreased by estrogen.

Breast Neoplasms↗

Effect of estrogen withdrawal on energy-rich phosphates and prediction of estrogen dependence monitored by in vivo 31P magnetic resonance spectroscopy of four human breast cancer xenografts.

The effect of estrogen withdrawal on energy metabolism was studied in four human breast cancer xenografts: the estrogen-dependent MCF-7 and ZR75-1 and the estrogen-independent ZR75/LCC-3 and MDA-MB-231. The tumors were grown in ovariectomized nude mice with a s.c. implanted estrogen pellet. After Gompertzian growth was verified, the estrogen pellet was removed from half of the animals. In vivo 31P magnetic resonance spectroscopy of the tumors was performed 1 day before and on days 2, 6, and 14 after estrogen removal. Estrogen withdrawal induced a significant increase in the nucleoside triphosphate:Pi ratio in the two estrogen-dependent xenografts, whereas this ratio remained unchanged in the estrogen-independent tumors. In ZR75/LCC-3 tumors a slight decrease in nucleoside triphosphate:Pi was observed following onset of estrogen stimulation after initial growth without estrogen. Extracts of freeze-clamped tumors prepared 14 days after estrogen removal were analyzed for ATP and phosphocreatine content. Our findings suggest a correlation between estrogen withdrawal and the steady-state concentrations of ATP, phosphocreatine, and Pi in human breast cancer xenografts. Discrimination analysis of the pretherapeutic spectra enabled us to identify the tumor line and the estrogen dependence of the tumors in 80-90% of all cases.

Animals↗

Plasma concentrations of unconjugated and conjugated estrogens and gonadotrophins following application of various estrogen preparations after oophorectomy and in the menopause.

In 55 patients after oophorectomy and 20 women after natural menopause an oral estrogen replacement therapy was performed with estrone-sulfate, estradiol 17-valerate, estriol-succinate, a combination of micronized estradiol and estriol (Estrifam, Trisequens), and natural conjugated estrogens. In 4 patients a 3 mg estradiol per 5 g ointment substance was applied on the abdominal skin. The interindividual variations of estrogen increments during therapy were considerably high. Oral intake of 2 mg estriol-succinate daily was followed by a 500% increase of total (conjugated + unconjugated) estriol. Concentrations of unconjugated estrogens were not altered by this dosage. Following oral application of the other above mentioned preparations, prominent rises--especially of unconjugated estrogens in plasma--were noted. The concentration peaks occurred within 3--6 h after application. Unconjugated estradiol-17 beta in plasma was comparable with values of the follicular phase of a normal menstrual cycle, unconjugated estrone, however, was nonphysiologically high. Consequently, the E1/E2 ratio was greater than one whereas it is normally below one. 12 h after oral estrogen application, plasma estrogens dropped to almost initial values, so that a second medication seems to be necessary in order to guarantee an adequate supplementation over the course of the day. The hormone values determined in this study did not show significant differences between patients after a natural menopause and after oophorectomy. There was a positive correlation between rising estrogen levels and suppressed gonadotrophins during replacement therapy. The occurrence of climacteric symptoms did not exclusively depend on low estrogen and high gonadotrophin levels. Good tolerance of estrogen therapy with significantly elevated estrogen concentrations in plasma can be obtained transcutaneously in the form of estrogen ointments. Such therapy might simulate the physiological estrogen pattern even better than oral application does because of delayed and diluted steroid flow to the liver.

Castration↗

High affinity binding of the estrogen receptor to a DNA response element does not require homodimer formation or estrogen.

We have developed an antibody-based DNA binding assay to study the effects of the absence or presence of an estrogen receptor agonist and two antagonists on the thermodynamic binding parameters for estrogen receptor binding to a consensus estrogen response element in vitro. We first demonstrate that antibodies raised against a region of the estrogen receptor directly adjacent to the DNA-binding domain do not interfere with the receptor's ability to preferentially bind to the vitellogenin estrogen response element in plasmid DNA. Exploiting this property to develop a quantitative DNA binding assay, we demonstrate that estrogen is not required for high affinity binding of the receptor for an oligonucleotide containing this element, nor do antiestrogens alter this interaction. In addition, we find that 1 mol of estrogen receptor is complexed with high affinity to 1 mol of vitellogenin estrogen response element, instead of two estrogen receptors/response element as would be predicted if estrogen receptor homodimer formation was required for high affinity DNA binding. Our data best fit a model in which the active estrogen receptor form is a monomer or heterodimer, but not a homodimer, and the regulatory role of estrogen on the estrogen receptor is the induction of protein-protein, but not protein-DNA, interactions.

Animals↗

Estrogen binding and estrogen receptor characterization (ERalpha and ERbeta) in the cholinergic neurons of the rat basal forebrain.

Estrogen is thought to enhance cognitive functions by modulating the production of acetylcholine in basal forebrain neurons; a system that projects to the cerebral cortex and hippocampus and plays a central role in learning and memory. To elucidate the mechanism of estrogen action in the cholinergic system, we utilized a combined in vivo autoradiography/immunocytochemistry technique to evaluate the distribution of estrogen binding sites in cholinergic neurons of the rat basal forebrain. The results of these studies revealed that a portion of the cholinergic neurons in the medial septum (41%), vertical (32%) and horizontal (29%) limbs of the diagonal band and in the substantia innominata/nucleus basalis (4%) contained estrogen receptors. Through the use of a double-label in situ hybridization/immunocytochemistry technique we have shown that estrogen receptor-alpha is the predominant estrogen receptor in the cholinergic neurons, with only a few cells containing estrogen receptor-beta. The results of these studies provide evidence that biologically active estrogen receptors are present in the basal forebrain cholinergic neurons of the adult rat brain, with estrogen receptor-alpha being the predominant receptor subtype. The demonstration that cholinergic neurons contain estrogen receptors is consistent with the possibility that estrogen directly modulates the activity of cholinergic neurons in rats and may provide insight as to how estrogen improves cognitive functions in women.

Animals↗

Critical in vivo roles for classical estrogen receptors in rapid estrogen actions on intracellular signaling in mouse brain.

Estrogen exerts classical genomic as well as rapid nongenomic actions on neurons. The mechanisms involved in rapid estrogen signaling are poorly defined, and the roles of the classical estrogen receptors (ERs alpha and beta) are unclear. We examined here the in vivo role of classical ERs in rapid estrogen actions by evaluating the estrogen-induced effects on two major signaling pathways within the brains of alphaER-, betaER-, and double alphabetaER-knockout (ERKO) ovariectomized female mice. Estrogen significantly (P < 0.05) increased the numbers of phospho-cAMP response element binding protein (phospho-CREB)-immunoreactive cells in specific brain regions of wild-type mice in a time-dependent manner beginning within 15 min. In brain areas that express predominantly ERbeta, this response was absent in betaERKO mice, whereas brain regions that express mostly ERalpha displayed no change in alphaERKO mice. In the medial preoptic nucleus (MPN), an area that expresses both ERs, the estrogen-induced phosphorylation of CREB was normal in both alphaERKO and betaERKO mice. However, estrogen had no effect on CREB phosphorylation in the MPN, or any other brain region, in double alphabetaERKO animals. Estrogen was also found to increase MAPK phosphorylation levels in a rapid (<15 min) manner within the MPN. In contrast to CREB signaling, this effect was lost in either alphaERKO or betaERKO mice. These data show that ERalpha and ERbeta play region- and pathway-specific roles in rapid estrogen actions throughout the brain. They further indicate an indispensable role for classical ERs in rapid estrogen actions in vivo and highlight the importance of ERs in coordinating both classical and rapid actions of estrogen.

Animals↗

Estrogen-mediated cytoplasmic and nuclear distribution of rat cardiovascular estrogen receptors.

We used either the synthetic estrogen R2858 (moxestrol) or estradiol-17 beta to characterize estrogen receptors in cytoplasmic (R2858) and nuclear (estradiol-17 beta) preparations from rat aorta and myocardium. Relative steroid specificity studies showed that only estrogens were effective inhibitors of R2858 or estradiol-17 beta binding to aortic and myocardial estrogen receptors, whereas androgens, progestins, and cortisol were ineffective inhibitors. Low ionic strength sucrose density gradient analyses showed that myocardial estrogen receptors that localized in the cytoplasmic fraction migrated as macromolecules with sedimentation coefficients of 8S to 9S. In contrast, two binding components of sedimentation coefficients 8S to 9S and 10S to 11S were characteristic of the estrogen receptors localized in aortic cytoplasmic preparations. High ionic strength sucrose density gradient analysis showed that aortic and myocardial estrogen receptors localized in the nuclear fraction migrated as macromolecules with sedimentation coefficients of 4S to 6S. Saturation analyses showed that aortic and myocardial cytoplasmic preparations from intact young mature male rats contained 50.6 +/- 12.9 (mean +/- SD) and 51.0 +/- 14.1 fmol receptor/mg DNA, respectively. The respective R2858 dissociation constants were 0.42 and 0.15 nM. Estrogen receptors could not be demonstrated in nuclear preparations from cardiovasculature of intact males. Estradiol-17 beta injection of intact young mature male rats caused "depletion" of aortic and myocardial cytoplasmic fraction estrogen receptors and resulted in the appearance of 51.9 +/- 21.0 and 36.9 +/- 9.5 fmol receptor/mg DNA in the corresponding nuclear fractions. The respective estradiol-17 beta dissociation constants were 1.56 and 0.71 nM. Increased estrogen receptor content of cardiovascular nuclear fractions of estradiol-17 beta injected male rats correlated well with the concomitant decreased cytoplasmic fraction receptor content. The ability of estradiol-17 beta to affect localization of cardiovascular estrogen receptors between cytoplasmic and nuclear fractions suggests these estrogen receptors are physiologically functional and indicates that estrogen may directly regulate cardiovascular cell function.

Animals↗

The E-SCREEN assay as a tool to identify estrogens: an update on estrogenic environmental pollutants.

Estrogens are defined by their ability to induce the proliferation of cells of the female genital tract. The wide chemical diversity of estrogenic compounds precludes an accurate prediction of estrogenic activity on the basis of chemical structure. Rodent bioassays are not suited for the large-scale screening of chemicals before their release into the environment because of their cost, complexity, and ethical concerns. The E-SCREEN assay was developed to assess the estrogenicity of environmental chemicals using the proliferative effect of estrogens on their target cells as an end point. This quantitative assay compares the cell number achieved by similar inocula of MCF-7 cells in the absence of estrogens (negative control) and in the presence of 17 beta-estradiol (positive control) and a range of concentrations of chemicals suspected to be estrogenic. Among the compounds tested, several "new" estrogens were found; alkylphenols, phthalates, some PCB congeners and hydroxylated PCBs, and the insecticides dieldrin, endosulfan, and toxaphene were estrogenic by the E-SCREEN assay. In addition, these compounds competed with estradiol for binding to the estrogen receptor and increased the levels of progesterone receptor and pS2 in MCF-7 cells, as expected from estrogen mimics. Recombinant human growth factors (bFGF, EGF, IGF-1) and insulin did not increase in cell yields. The aims of the work summarized in this paper were a) to validate the E-SCREEN assay; b) to screen a variety of chemicals present in the environment to identify those that may be causing reproductive effects in wildlife and humans; c) to assess whether environmental estrogens may act cumulatively; and finally d) to discuss the reliability of this and other assays to screen chemicals for their estrogenicity before they are released into the environment.

Animals↗

Estrogen conjugates and serum factors mediating the estrogenic trophic effect on MCF-7 cell growth.

Estrogens stimulate growth of MCF-7 breast cancer cells in monolayer culture. Possible interference of serum factors leading to an estrogen-insensitive cell growth was analyzed in various experiments carried out on serum batches producing no estradiol stimulation. Out of five estrogen conjugates, only 3-glucurono-estradiol partly suppressed the inhibition of hydroxytamoxifen; the conjugate also reduced the estrogen receptor content of the cells, probably by a down regulation process ("processing"). Moreover, prolonged subcultures in dextran-coated charcoal-treated serum attempting to remove possible intracellular estrogens produced no growth stimulation. Interference by hormone carriers of the serum was ruled out by the fact that two strong synthetic estrogens, moxestrol and diethylstilbestrol with weak binding affinity for these carriers, were unstimulatory. Reduction of the carrier concentration also failed to confer any estrogen sensitivity. This lack of effect of most estrogen conjugates and serum carriers seems to contradict the hypothesis of their interference leading to an estrogen-insensitive growth. Presence in the serum of potential inhibitors towards estrogen action was also examined. Dilution of sera inducing an estrogenic stimulated growth failed to show any growth increase, either in the absence or presence of estradiol, thus excluding the possibility of a major influence of an antagonism on growth control. Moreover, clonogenic assays in soft agar eliminated the hypothesis that a difference between "active" (stimulatory with estradiol) and "inactive" serum batches may result from distinct adherence properties rather than from real growth stimulation. All of these data are consistent with the concept that serum factors which are not of estrogenic nature mediate the trophic effect of estradiol; their absence in some serum batches may lead to an estrogen-insensitive cell growth.

Animals↗

Estrogens and anti-estrogens: key mediators of prostate carcinogenesis and new therapeutic candidates.

Despite the historical use of estrogens in the treatment of prostate cancer (PCa) little is known about their direct biological effects on the prostate, their role in carcinogenesis, and what mechanisms mediate their therapeutic effects on PCa. It is now known that estrogens alone, or in synergism with an androgen, are potent inducers of aberrant growth and neoplastic transformation in the prostate. The mechanisms of estrogen carcinogenicity could be mediated via induction of unscheduled cell proliferation or through metabolic activation of estrogens to genotoxic metabolites. Age-related changes and race-/ethnic-based differences in circulating or locally formed estrogens may explain differential PCa risk among different populations. Loss of expression of estrogen receptor (ER)-beta expression during prostate carcinogenesis and prevention of estrogen-mediated oxidative damage could be exploited in future PCa prevention strategies. Re-expression of ER-beta in metastatic PCa cells raises the possibility of using ER-beta-specific ligands in triggering cell death in these malignant cells. A variety of new estrogenic/anti-estrogenic/selective estrogen receptor modulator (SERM)-like compounds, including 2-methoxyestradiol, genistein, resveratrol, licochalcone, Raloxifene, ICI 182,780, and estramustine are being evaluated for their potential in the next generation of PCa therapies. Increasing numbers of patients self-medicate with herbal formulations such as PC-SPES. Some of these compounds are selective ER-beta ligands, while most of them have minimal interaction with ER-alpha. Although many may inhibit testosterone production by blockade of the hypothalamal-pituitary-testis axis, the most effective agents also exhibit direct cytostatic, cytotoxic, or apoptotic action on PCa cells. Some of them are potent in interfering with tubulin polymerization, blocking angiogenesis and cell motility, suppressing DNA synthesis, and inhibiting specific kinase activities. Further discovery of other compounds with potent apoptotic activities but minimal estrogen action should promote development of a new generation of effective PCa preventive or treatment regimens with few or no side-effects due to estrogenicity. Further advancement of our knowledge of the role of estrogens in prostate carcinogenesis through metabolic activation of estrogens and/or ER-mediated pathways will certainly result in better preventive or therapeutic modalities for PCa.

Estrogen Receptor Modulators↗

Neonatal exposure to estrogen differentially alters estrogen receptor alpha and beta mRNA expression in rat testis during postnatal development.

The biological actions of estrogens on target cells are mediated by two nuclear receptors: the estrogen receptor (ER) alpha and the recently characterized ER beta. In the male rat, the physiological role of estrogens involves multiple actions, from masculinization of brain areas related to reproductive function and sexual behavior to regulation of testicular development and function. Paradoxically, however, administration of high doses of estrogen during the critical period of neonatal differentiation results in an array of defects in the reproductive axis that permanently disrupt male fertility. The focus of this study was to characterize the effects and mechanism(s) of action of neonatal estrogenization on the pattern of testicular ER alpha and beta gene expression during postnatal development. To this end, groups of male rats were treated at day 1 of age with estradiol benzoate (500 microg/rat), and testicular ER alpha and ER beta mRNA levels were assayed by semi-quantitative RT-PCR from the neonatal period until puberty (days 1-45 of age). Furthermore, the expression of androgen receptor (AR) mRNA was evaluated, given the partially overlapping pattern of tissue distribution of ER alpha, ER beta and AR messages in the developing rat testis. In addition, potential mechanisms for neonatal estrogen action were explored. Thus, to discriminate between direct effects and indirect actions through estrogen-induced suppression of serum gonadotropins, the effects of neonatal estrogenization were compared with those induced by blockade of gonadotropin secretion with a potent LHRH antagonist in the neonatal period. Our results indicate that neonatal exposure to estrogen differentially alters testicular expression of alpha and beta ER messages: ER alpha mRNA levels, as well as those of AR, were significantly decreased, whereas relative and total expression levels of ER beta mRNA increased during postnatal/prepubertal development after neonatal estrogen exposure, a phenomenon that was not mimicked by LHRH antagonist treatment. It is concluded that the effect of estrogen on the expression levels of ER alpha and beta mRNAs probably involves a direct action on the developing testis, and cannot be attributed to estrogen-induced suppression of gonadotropin secretion during the neonatal period.

Animals↗

Estrogen receptor beta mediates rapid estrogen actions on gonadotropin-releasing hormone neurons in vivo.

The gonadal steroid estrogen exerts an important modulatory influence on the activity of multiple neuronal networks. In addition to classical genomic mechanisms of action, estrogen also exerts poorly understood rapid, nongenomic effects on neurons. To examine whether estrogen may exert rapid actions on intracellular signaling within gonadotropin-releasing hormone (GnRH) neurons in vivo,we examined the phosphorylation status of cAMP response element-binding protein (CREB) in these cells after the administration of 17-beta-estradiol to ovariectomized (OVX) mice. The percentage of GnRH neurons expressing phosphorylated CREB was increased more than sixfold (p < 0.05) in a time- and dose-dependent manner by estrogen, with the increase first observed 15 min after estrogen administration. A series of in vitro studies demonstrated that estrogen acted directly on native GnRH neurons to phosphorylate CREB, but that estrogen conjugated to bovine serum albumin was without effect. The role of classical estrogen receptors (ERs) was evaluated using ER knock-out mice in vivo. The effect of estrogen on CREB phosphorylation in GnRH neurons was normal in ERalpha knock-out mice but completely absent in ERbeta knock-out mice. Finally, studies in intact female mice revealed levels of CREB phosphorylation within GnRH neurons that were equivalent to those of estrogen-treated OVX mice. These observations demonstrate that ERbeta mediates the rapid, direct effects of estrogen on the GnRH neuronal phenotype, and that these actions persist under physiological conditions. They also provide the first evidence for a role of ERbeta in nongenomic estrogen signaling within the brain in vivo.

Animals↗

Proliferation, hormonal responsiveness, and estrogen receptor content of MCF-7 human breast cancer cells grown in the short-term and long-term absence of estrogens.

We have examined the effect of short-term and long-term growth in the absence of estrogens on the proliferation rate and estrogen and antiestrogen responsiveness of MCF-7 human breast cancer cells. The removal of phenol red, the pH indicator in tissue culture medium that is weakly estrogenic (Y. Berthois et al., Proc. Natl. Acad. Sci. USA, 83:2496-2500, 1986), immediately slows the cell proliferation rate, and MCF-7 cells grown in phenol red-free medium with charcoal dextran-treated serum for periods up to 1 mo maintain this reduced rate of cell proliferation. In these short-term phenol red-withdrawn cells, estradiol stimulates proliferation markedly and reproducibly, and antiestrogens inhibit estrogen-stimulated proliferation. Antiestrogens by themselves appear as partial agonists/antagonists; at low concentrations they stimulate proliferation weakly, but they show no stimulation at the high concentrations where they fully inhibit estrogen-stimulated proliferation. In contrast to the short-term phenol red-withdrawn cells, cells maintained for several months (5 to 6 mo) in the apparently complete absence of estrogens (no phenol red, with charcoal dextran-treated calf serum) show a markedly increased basal rate of cell proliferation; estradiol is unable to increase this rate of proliferation further, but antiestrogens are able to decrease proliferation. This change in growth pattern is associated with a 3-fold increase in cellular estrogen receptor levels. Despite their differing basal growth rates, cells grown in either the short-term (less than 1 mo) or long-term (greater than 6 mo) absence of estrogens both have progesterone receptor levels that are very low and, in both cases, estradiol increases progesterone receptor levels markedly. Thus, under long-term estrogen-free conditions, there is a dissociation between the stimulation of cell proliferation and of specific protein synthesis (progesterone receptor) by estrogen. The increase in the cell proliferation rate observed in cells grown in the long-term absence of estrogen may reflect altered regulation of growth factor production or altered sensitivity to growth factors in the medium or produced by the cells themselves. Hence, these breast cancer cells adapt significantly to long-term growth in estrogen-free conditions, an observation that may be relevant to understanding the growth of hormone-responsive human breast cancers in vivo.

Breast Neoplasms↗

Differential sensitivity of estrogen target tissues: implications for estrogen regulation of serum luteinizing hormone.

In the rat, it is generally accepted that the primary site of estrogen's stimulatory (positive) effects on serum LH is the preoptic area-anterior hypothalamus (POA/AH). In contrast, the primary site of estrogen's inhibitory (negative) effects on serum LH levels has not been conclusively identified. There is evidence to suggest both a medial basal hypothalamic (MBH) and an anterior pituitary site of action. The present studies utilized a unique characteristic of these estrogen effects to investigate their putative loci. Extensive dose-response curves of estrogen's induction of positive and negative feedback indicated that the negative feedback response was activated at a lower concentration of serum estradiol than the positive feedback response. The differential sensitivities of these two responses suggested that the tissues mediating them might also be differentially sensitive to estradiol. In a previous paper, we showed that receptor translocation is an index of estrogen sensitivity. We measured receptor translocation in response to a series of estradiol doses in the POA/AH, the MBH, and the pituitary. Dose-response curves for estrogen's effect on receptor translocation showed that the pituitary receptor translocation mechanism is activated at significantly lower levels of serum estradiol than that of either the POA/AH or the MBH. These results are consistent with the POA/AH as a site of estrogen's positive feedback effects. In addition, they suggest that negative feedback in the rat may be mediated via estrogen's action at the anterior pituitary. Estrogen's negative feedback effect on serum LH occurs at a serum level of estrogen at which no receptor is translocated in the MBH. Therefore, the pituitary, which does possess nuclear receptors at these estradiol dose levels, is more likely to be the primary mediator of estrogen's negative feedback effects. In another experiment, pituitary, but not hypothalamic, receptor was translocated to the nuclear fraction with an injection of 100 micrograms clomiphere (Clomid). Under these conditions serum LH is depressed, thus strengthening the hypothesis that, in the rat, estrogen action on the pituitary can cause suppression of serum LH independently of the hormone's action in the hypothalamus.

Animals↗

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.

9,10-Dimethyl-1,2-benzanthracene↗

Interaction of ring B unsaturated estrogens with estrogen receptors of human endometrium and rat uterus.

The present investigation was undertaken to compare the binding affinities (Ka) of the ring B unsaturated equine estrogens (equilin [Eq], equilenin [Eqn], 17 beta-dihydroequilin [17 beta-Eq], 17 beta-dihydroequilenin [17 beta-Eqn], 17 alpha-dihydroequilin [17 alpha-Eq], and17 alpha-dihydroequilenin [17 alpha-Eqn]) and the classic estrogens (estrone [E1], 17 beta-estradiol [17 beta-E2], and 17 alpha-estradiol [17 alpha-E2]) for estrogen receptors in human endometrium and rat uterus. In both species, the ring B unsaturated estrogens bind with cytosol and nuclear receptors with high affinity (Ka x 10(9) M-1). The relative binding affinities of these estrogens were measured by determining the amount of unlabeled estrogen required to reduce by 50% the specific binding of [3H]17 beta-Eq to endometrial cytosol receptors. The order of activity found was 17 beta-Eq greater than 17 beta-E2 greater than 17 beta-Eqn greater than E1 greater than Eq greater than 17 alpha-Eq greater than 17 alpha-E2 greater than 17 alpha-Eqn greater than Eqn. Essentially the same order of activity was observed when the apparent affinity constants of these estrogens for human and rat cytosol and nuclear receptors were determined by a competitive (inhibition) binding assay. Sucrose density gradient analysis indicated that these estrogens form protein complexes with cytosol and nuclear preparation that sediment at approximately 8S and 4S, respectively. The affinity constants for 17 beta-Eq were approximately two- to six-fold higher than E2 in both species. In a rat uterotropic assay, all nine estrogens were uterotropic. These data indicate that all ring B unsaturated estrogens present in conjugated equine estrogen preparations are biologically active and they express their biologic effects in the human endometrium by mechanisms similar to those described for the classic estrogens.

Animals↗