Vasorelaxant action of 17 -estradiol in rat uterine arteries: role of nitric oxide synthases and estrogen receptors

The uterine vasculature plays an important role during pregnancy by providing adequate perfusion of the maternal-fetal interface. To this end, substantial remodeling of the uterine vasculature occurs with consequent changes in responsiveness to contractile agents. The purpose of our study was to characterize the vasorelaxant effects of estrogens on vascular smooth muscles of the rat uterine artery during pregnancy and to evaluate the involvement of estrogen receptors (ESR) and nitric oxide synthases (NOS). To do so, we measured NOS expression in the whole uterine and mesenteric circulatory bed by Western blotting. Vasorelaxant effects of 17beta-estradiol (17beta-E(2)) were assessed on endothelium-denuded uterine arteries with wire myographs in the absence and presence of pharmacological modulators [nitro-L-arginine methyl ester (L-NAME), ICI-182780, tamoxifen]. All experiments were performed on arteries from nonpregnant (NP) and late pregnant (P) rats. In the uterine vasculature of the latter group, NOS3 (endothelial NOS) expression was increased, while NOS1 (neuronal NOS) was reduced compared with NP rats. Expression of the NOS2 (inducible NOS) isoform was undetectable in the two groups. Both 17beta-E(2) and 17alpha-E(2) induced uterine artery relaxation, but the latter evoked lower responses. Endothelium-denuded arteries from NP rats showed larger relaxation with 17beta-E(2) than P rats. This larger relaxation disappeared in the presence of L-NAME. The ESR antagonist ICI-182780 did not affect acute relaxation with 17beta-E(2) and 17alpha-E(2). Moreover, membrane-nonpermeant 17beta-E(2):BSA (estradiol conjugated to bovine serum albumin) did not induce any vasorelaxation. Our results indicate that estrogens exert direct acute vasorelaxant effects in smooth muscles of the rat uterine artery that are mediated by mechanisms independent of ESR activation, but with some stereospecificity. Part of this effect, in NP rats only, is due to nitric oxide produced from muscle NOS1.     

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.     

Interaction of pepsin with aromatic amino acids and their derivatives immobilized to Sepharose

A new LC-MS/MS method for the separation, identification and quantification of residues of 17alpha-estradiol (17alpha-E2) and 17beta-estradiol (17beta-E2) in bovine serum is reported. Deuterium-labelled 17beta-estradiol was used as internal standard. The method was in-house validated in accordance with European Union criteria and adopted in a proficiency study organised by the Community Reference Laboratory (CRL-RIVM, Bilthoven, The Netherlands). The analytes were extracted from serum using acetate buffer, purified by C18 solid-phase extraction (SPE) and chromatographed on a C18 LC column. They were then ionized in a heated nebulizer (HN) interface operating in negative ion mode, where only intact deprotonated molecules, [M-H](-), were generated at m/z 271 and 274 for 17alpha/17beta-E2 and 17beta-E2-d(3), respectively. The decision limits obtained (CCalpha, i.e., critical concentration alpha) were 0.06 ng/mL and 0.03 ng/mL, respectively for 17alpha-E2 and 17beta-E2. Detection capability (CCbeta, i.e., critical concentration beta) values were 0.08 ng/mL and 0.04 ng/mL, respectively, for 17alpha-E2 and 17beta-E2. Precision, accuracy and specificity were satisfactory, recovery ranged from 86.3% to 93.2% and the method resulted sensitive for the required purposes. This method is currently in use for Official Control purposes.     

Activation of 'immediate-early' genes by estrogen is not sufficient to achieve stimulation of DNA synthesis in rat uterus

17 beta-estradiol, a long acting estrogen that is mitogenic for rat uterus in vivo, or the short acting estrogens estriol and 16 alpha-estradiol, not mitogenic on their own, were injected into adult, castrated rats and their effect on uterine gene expression and rate of DNA synthesis were compared. All three compounds increased steady-state mRNA concentration of c-fos, c-jun and c-myc proto-oncogenes to comparable levels (2 hrs after treatment), whereas only 17 beta-estradiol was found to stimulate significantly DNA synthesis (20-22 hrs later). Based on the different retention time of the tested estrogens in rat tissues, it is concluded that a short exposure to the hormone is sufficient to render uterine cells competent to progress through the cell cycle, via activation of 'immediate-early' genes expression, but that stimulation of DNA synthesis requires further changes, achieved via a prolonged exposure of the cells to the estrogenic stimulus.     

17alpha-estradiol-induced VEGF-A expression in rat pituitary tumor cells is mediated through ER independent but PI3K-Akt dependent signaling pathway

17alpha-E(2), a weak estrogen exhibited both agonistic and antagonistic effects, and caused a time- and dose-dependent induction of VEGF-A mRNA expression in GH3 rat pituitary tumor cells. This effect was unaffected by the presence of the pure estrogen receptor antagonist ICI 182,780 but was specifically blocked by a protein synthesis inhibitor puromycin. Inhibition of phosphatidylinositol-3 kinase (PI3K) activity by wortmannin decreased the effect of 17alpha-E(2) on VEGF-A mRNA expression. This inhibitor also blocked the increase in phosphorylation of Akt induced by exposure to 17alpha-E(2). In contrast, exposure to the MAP kinase inhibitor, U0126, had no impact on 17alpha-E(2)-induced VEGF-A mRNA expression. Taken together, these studies indicate that like potent estrogens 17alpha-E(2) up-regulates VEGF-A mRNA expression in estrogen responsive GH3 rat pituitary tumor cells, but this induction is not mediated through a classical estrogen receptor pathway. PI3K-Akt signaling pathway is required for the induction of VEGF-A mRNA in GH3 cells by 17alpha-E(2).     

Molecular characterization of a B-ring unsaturated estrogen: implications for conjugated equine estrogen components of premarin

Conjugated equine estrogens (CEEs) are routinely used for hormone replacement therapy (HRT), making it important to understand the activities of individual estrogenic components. Although 17beta-estradiol (17beta-E2), the most potent estrogen in CEE, has been extensively characterized, the actions of nine additional less potent estrogens are not well understood. Structural differences between CEEs and 17beta-E2 result in altered interactions with the two estrogen receptors (ERalpha and ERbeta) and different biological activities. To better understand these interactions, we have determined the crystal structure of the CEE analog, 17beta-methyl-17alpha-dihydroequilenin (NCI 122), in complex with the ERalpha ligand-binding domain and a peptide from the glucocorticoid receptor-interacting protein 1 (GRIP1) coactivator. NCI 122 has chemical properties, including an unsaturated B-ring and 17alpha-hydroxyl group, which are shared with some of the estrogens found in CEEs. Structural analysis of the NCI 122-ERalpha LBD-GRIP1 complex, combined with biochemical and cell-based comparisons of CEE components, suggests that factors such as decreased ligand flexibility, decreased ligand hydrophobicity and loss of a hydrogen bond between the 17-hydroxyl group and His524, contribute significantly to the reduced potency of CEEs on ERalpha.     

Evaluation of neuroactive steroid levels by liquid chromatography-tandem mass spectrometry in central and peripheral nervous system: effect of diabetes

The nervous system is a target for physiological and protective effects of neuroactive steroids. Consequently, the assessment of their levels in nervous structures under physiological and pathological conditions is a top priority. To this aim, identification and quantification of pregnenolone (PREG), progesterone (PROG), dihydroprogesterone (DHP), tetrahydroprogesterone (THP), testosterone (T), dihydrotestosterone (DHT), 5alpha-androstan-3alpha, 17beta-diol (3alpha-diol), 17alpha- and 17beta-estradiol (17alpha-E and 17beta-E) by liquid chromatography and tandem mass spectrometry (LC-MS/MS) has been set up. After validation, this method was applied to determine the levels of neuroactive steroids in central (i.e., cerebral cortex, cerebellum and spinal cord) and peripheral (i.e., brachial nerve) nervous system of control and diabetic rats. In controls only the brachial nerve had detectable levels of all these neuroactive steroids. In contrast, 17alpha-E in cerebellum, 17alpha-E, 17beta-E, DHP and THP in cerebral cortex, and 17alpha-E, 17beta-E and DHP in spinal cord were under the detection limit. Diabetes, induced by injection with streptozotocin, strongly affected the levels of some neuroactive steroids. In particular, the levels of PREG, PROG and T in cerebellum, of PROG, T and 3alpha-diol in cerebral cortex, of PROG, DHT and 3alpha-diol in spinal cord and of PREG, DHP, THP, T, DHT and 3alpha-diol in brachial nerve were significantly decreased. In conclusion, the data here reported demonstrate that the LC-MS/MS method allows the assessment of neuroactive steroids in the nervous system with high sensitivity and specificity and that diabetes strongly affects their levels, providing a further basis for new therapeutic tools based on neuroactive steroids aimed at counteracting diabetic neuropathy.     

Vitellogenin-inducing activities of natural, synthetic, and environmental estrogens in primary cultured Xenopus laevis hepatocytes

Vitellogenin (VTG)-inducing activities of natural estrogens (E1: estrone, E2:17beta-estradiol, E3: estriol, alpha-E2: 17alpha-estradiol), synthetic estrogens (EE2: 17alpha-ethynyl estradiol, DES: diethylstilbestrol,), phytoestrogen (GEN: genistein), and xeno-estrogens (BPA: bisphenol A, NP: nonylphenol, OP: octylphenol) were investigated by an assay system using primary-cultured hepatocytes of Xenopus laevis. An enzyme-linked immunoabsorbent assay (ELISA) was able to detect VTG at a minimum detection limit of 0.06 ng/mL. Relative estrogenic activities of the compounds were determined from their dose-response curves. The activities relative to E2 activity were 138% for DES, 121% for EE2, 6.1% for E3, 0.33% for E1, 0.29% for alpha-E2, 0.037% for GEN, 0.008% for BPA, 0.005% for NP, and 0.002% for OP. Comparison with data reported for other bioassay systems revealed that there were significant interspecies-and cell-type-differences in the activities of DES, E3, E1 and alpha-E2. BPA was found to have a substantial antagonistic activity (approximately 0.8% of tamoxifen activity) under the influence of physiological concentrations of E2. Complex-effects of endocrine disrupters on aquatic animals will be discussed.     

Estrogens and menopause: pharmacology of conjugated equine estrogens and their potential role in the prevention of neurodegenerative diseases such as Alzheimer's

Menopause marks the start of a new phase in a woman's life that is associated with a decrease in circulating estrogen levels. Although the average age of women has increased from 50 to nearly 85 years, the average age at menopause has remained essentially constant at 50 years. Thus, women now spend nearly a third of their lives in an estrogen deficient state. This normal aging process in women is associated with increasing health problems such as osteoporosis, cardiovascular disease, neurodegenerative diseases, and cancer. Estrogen replacement therapy (ERT) has been shown to play an important beneficial role in the health and well being of postmenopausal women. Several estrogen preparations are available and among these conjugated equine estrogens (CEE) are most frequently used. The drug CEE, is a complex natural urinary extract of pregnant mare's urine and contains at least 10 estrogens in their sulfate ester form and these are the ring B saturated estrogens: estrone (E(1)), 17beta-estradiol (17beta-E(2)), 17alpha-estradiol (17alpha-E(2)), and the ring B unsaturated estrogens equilin (Eq), 17beta-dihydroequilin (17beta-Eq), 17alpha-dihydroequilin (17alpha-Eq), equilenin (Eqn), 17beta-dihydroequilenin (17beta-Eqn), 17alpha-dihydroequilenin (17alpha-Eqn), and Delta(8)-estrone (Delta(8)-E(1)). All of these estrogens in their unconjugated form are biologically active and can interact with recombinant human estrogen receptor alpha (ERalpha) and beta (ERbeta) with 17beta-estradiol and 17beta-dihydroequilin having the highest affinity for both receptors. A number of the ring B unsaturated estrogens had nearly twofold higher affinity for the ERbeta. The pharmacokinetics of these estrogens in postmenopausal women indicate that the unconjugated estrogens compared to their sulfated forms are cleared more rapidly. The 17-keto estrogens are metabolized to the more potent 17beta-reduced products which are cleared at a slower rate. In postmenopausal women, the extent of 17beta-activation is much higher with the ring B unsaturated estrogens than with ring B saturated estrogens. Oxidized LDL and oxidative stress are thought to contribute to both atherosclerosis and neurodegenerative disorders. Neurons in particular are at a high risk from damage resulting from oxidative stress. In vivo and in vitro studies indicate that the oxidation of LDL isolated from postmenopausal women was inhibited differently by various estrogens and other antioxidants. The unique ring B unsaturated estrogens were the most potent while the red wine component t-resveratrol was the least potent.Studies were designed to explore the cellular and molecular mechanisms that may be involved in the neuroprotective effects of CEE components. The data indicate that the neurotoxic effects of oxidized LDL and glutamate can be inhibited by various estrogens, with the ring B unsaturated estrogens being the most active. These effects are involved in the inhibition of DNA fragmentation and up-regulation of anti-apoptotic protein Bcl-2 and down-regulation of pro-apoptotic protein Bax. These combined data suggest that some of the neuroprotective benefits associated with long-term estrogen therapy may occur by the above mechanism(s). Because estrogens such as the Delta(8)-estrogens are relatively less feminizing than the classical estrogen 17beta-estradiol, they may be important in the development of more neuro-specific estrogens that will be useful in the prevention of neurodegenerative diseases, such as Alzheimer's and Parkinson disease, in both men and women.     

Differential interactions of estrogens and antiestrogens at the 17 beta-hydroxy or counterpart function with the estrogen receptor

The action of diethylpyrocarbonate on lamb uterine estrogen receptor produced an homogeneous population of the receptor (approximately 55%) which still bound triarylethylene antiestrogens such as 4-hydroxytamoxifen with a high affinity but bound classical potent estrogens such as estradiol or diethylstilbestrol with a very low affinity. To specify the structural features of the ligands involved in the decrease of ligand affinity upon modification of the estrogen receptor, we determined the relative affinity constants of 17 steroidal estrogens or antiestrogens (deriving from estradiol by a 7 alpha- or 11 beta-substitution) and 14 nonsteroidal estrogens or antiestrogens (all including the 1,2-trans-diphenylethylene structure of diethylstilbestrol) for native and diethylpyrocarbonate-modified estrogen receptors. Then the ratio of the relative affinity constant for the native receptor to that for the modified receptor (rho) was calculated for each ligand, to compare the variation in the affinity of the ligand upon modification of the receptor to that of 4-hydroxytamoxifen (rho = 1). The results showed that the strong decrease of ligand affinity upon modification of the receptor displayed by classical estrogens (rho greater than or equal to 200) is strictly dependent on the presence of the 17 beta-hydroxyl group in steroidal compounds or its alpha-4- and beta-4-counterparts in diethylstilbestrol-related compounds. However, for the 7 alpha- or 11 beta-derivatives of estradiol displaying potent antiestrogenic properties, the relative decrease in affinity was much more limited (rho less than or equal to 19). For 11 beta-derivatives displaying a relative estrogenic activity weaker than that of estradiol itself, an average decrease in affinity was observed (23 less than or equal to rho less than or equal to 62). With the diethylstilbestrol-related compounds, bearing or not the alpha-4-hydroxyl and/or the beta-4-hydroxy functions and showing either weak relative estrogenic or antiestrogenic properties, the relative variation in affinity was weak (0.6 less than or equal to rho less than or equal to 24). These results indicate that the interaction of 7 alpha- or 11 beta-substituted steroidal antiestrogens and of 1,2-trans-diphenylethylene or triphenylethylene derivatives, displaying either weak relative estrogenic or antiestrogenic properties with the receptor, differs at the 17 beta-hydroxy or at the alpha-4-/beta-4-hydroxy functions from that of potent estrogens. They suggest that the strong decrease in the relative affinity of ligands upon receptor modification may reflect the high efficiency of the ligands to activate the receptor properly.(ABSTRACT TRUNCATED AT 400 WORDS)     

Positive and negative effects of estradiol-17 beta in the rat uterus

Estrogens could act as effectors or inhibitors of protein synthesis in the rat uterus, depending on the doses given to animals. A single injection of estradiol-17 beta to immature female rats led to the increase in protein synthesis and in enzyme activities involved in DNA synthesis. Four injections, given once daily, resulted in the inhibition of enzyme activity and synthesis of all proteins but one. The 105 kD protein which showed a gradual increase with the duration of estrogen treatment could be responsible for the negative action of estrogens on uterine growth.     

Transplacental estrogen responses in the fetal rat: increased uterine weight and ornithine decarboxylase activity

The synthetic estrogens, diethylstilbestrol (DES) and ethynylestradiol (EE2), are more potent than 17 beta-estradiol (E2) in inducing uterine weight gain in the neonatal rat, due to the binding of E2 to serum alpha-fetoprotein (AFP). However, all three hormones are equipotent in inducing neonatal uterine ornithine decarboxylase (ODC) activity. The present study assessed estrogen potency in fetal rats. Pregnant CD rats were injected sc daily on gestation days (GD) 16-20 with DES, EE2, or E2 in sesame oil. Both DES and EE2, but not E2, significantly increased uterine weight at birth, to more than twice that of controls. In addition, implants which continuously release E2 only slightly increased uterine weight at birth. Alternatively, dams were given a single estrogen injection on GD 20 and were sacrificed at various times after injection. Peak fetal uterine ODC activity occurred at 6-8 hours after maternal injection for all three estrogens. E2 had a relative potency about tenfold less than either DES or EE2 in stimulating fetal ODC activity, in contrast to equal potencies of the three estrogens in the postnatal rat uterus. Similar patterns were found following direct fetal injection with E2 or DES. In summary, these data demonstrate a transplacental induction of fetal uterine ODC activity and uterine weight gain by both DES and EE2. In addition, the lack of correlation between these endpoints in response to E2 suggests that they may be useful as selective indicators of potential toxicity of both natural and synthetic estrogens.     

Rat luminal cell nuclear area changes correlated with uterine growth responses induced by a low dose infusion or injection of estradiol-17 beta

Rat uterine luminal epithelial cells (LEC) responded differently when exposed to an injection of 1.0 microgram estradiol-17 beta (E2) compared to a continuous infusion of E2 at the rate of 1.0 microgram/24 hours. After injection or beginning infusion, LEC mean nuclear area significantly decreased by 4 h, then increased thereafter. After injection, nuclear area distributions were determined at each time point. The percentage of large nuclei (greater than 40 mu 2) decreased by 4h postinjection and remained a relatively small proportion of the population, while the percentage of nuclei of 20-30 mu 2 areas increased throughout the experiment. During infusion, the percentage of large nuclei decreased by 4h after pump implantation, then increased. Only infusion induced sustained, increased uterine protein content, DNA synthesis and ornithine decarboxylase activity. This study suggests that E2 treatment modality induces differences in nuclear size in target cells as well as in biochemical parameters.     

Estrogen induces the Akt-dependent activation of endothelial nitric-oxide synthase in vascular endothelial cells

Although estrogen is known to activate endothelial nitric oxide synthase (eNOS) in the vascular endothelium, the molecular mechanism responsible for this effect remains to be elucidated. In studies of both human umbilical vein endothelial cells (HUVECs) and simian virus 40-transformed rat lung vascular endothelial cells (TRLECs), 17beta-estradiol (E2), but not 17alpha-E2, caused acute activation of eNOS that was unaffected by actinomycin D and was specifically blocked by the pure estrogen receptor antagonist ICI-182,780. Treatment of both TRLECs and HUVECs with 17beta-E2 stimulated the activation of Akt, and the PI3K inhibitor wortmannin blocked the 17beta-E2-induced activation of Akt. 17beta-E2-induced Akt activation was also inhibited by ICI-182,780, but not by actinomycin D. Either treatment with wortmannin or exogenous expression of a dominant negative Akt in TRLECs decreased the 17beta-E2-induced eNOS activation. Moreover, 17beta-E2-induced Akt activation actually enhances the phosphorylation of eNOS. 17beta-E2-induced Akt activation was dependent on both extracellular and intracellular Ca(2+). We further examined the 17beta-E2-induced Akt activity in Chinese hamster ovary (CHO) cells transiently transfected with cDNAs for estrogen receptor alpha (ERalpha) or estrogen receptor beta (ERbeta). 17beta-E2 stimulated the activation of Akt in CHO cells expressing ERalpha but not in CHO cells expressing ERbeta. Our findings suggest that 17beta-E2 induced eNOS activation through an Akt-dependent mechanism, which is mediated by ERalpha via a nongenomic mechanism.     

The analysis of estrone and 17beta-estradiol by stir bar sorptive extraction-thermal desorption-gas chromatography/mass spectrometry: application to urine samples after oral administration of conjugated equine estrogens

The development of a sensitive and solvent-free method for the measurement of estrone (E(1)) and 17beta-estradiol (17beta-E(2)) in human urine samples is described. The deconjugated estrogens were derivatized in situ with acetic acid anhydride and the derivatives were extracted directly from the aqueous samples using stir bar sorptive extraction (SBSE). The compounds containing a secondary alcohol function are further derivatized by headspace acylation prior to thermal desorption and gas chromatography/mass spectrometry (GC/MS). A number of experimental parameters, including salt addition, temperature and time, were optimized to increase the recovery of E(1) and 17beta-E(2) by SBSE. The derivatization reactions were also optimized to obtain the highest yields of the acylated estrogens. Detection limits of 0.02 and 0.03 ng mL(-1) were obtained for E(1) and 17beta-E(2), respectively. The method was applied to determine the effect of conjugated equine estrogen intake on the excretion of E(1) and 17beta-E(2) in human urine samples. Increased levels of the endogenous estrogens were detected after administering a standard dose of Premarin to a female volunteer. Routine monitoring of estrogen levels is recommended to avoid a high urinary excretion of E(1) and 17beta-E(2), nowadays enlisted as endocrine disrupting chemicals (EDCs), during hormone replacement therapy.     

Differential effects of ovarian steroids and triphenylethylene compounds on macromolecular uptake and thymidine incorporation in the mouse uterus

In the rodent uterus, estrogen elicits a biphasic response i.e. an early phase (Phase I) and a late phase (Phase II). Estradiol-17 beta (E2) and estriol (E3), as well as triphenylethylene (TPE) compounds, CI-628 and clomiphene citrate (CC), were used to characterize Phase I and Phase II responses in uterine preparation for implantation in the mouse. While uterine macromolecular uptake (vascular permeability), a Phase I response, was studied in progesterone (P4)-primed animals, uterine [3H]thymidine incorporation (DNA synthesis), a Phase II response, was investigated with and without P4-priming. In the P4-primed uterus, all compounds, except CC, significantly increased uterine macromolecular uptake as determined by interstitial tissue accumulation of [125I]bovine serum albumin [( 125I]BSA). DNA synthesis as determined by cellular incorporation of [3H]thymidine was modulated by P4, estrogens and TPE compounds in a cell-type specific and temporal manner. As a single injection and in the absence of P4, E2 induced [3H]thymidine incorporation in the luminal and glandular epithelium at 18 and 24 h. E3 was inferior to E2 in this response. On the other hand, treatment with P4 for 1 day or 4 days induced [3H]thymidine incorporation primarily in stromal cells. However, stromal cell incorporation was potentiated when P4 treatment was combined with estrogens or TPE compounds. These results reveal the relative importance of Phase I and cell-type specific Phase II responses in uterine preparation for implantation.     

Replenishment and nuclear retention of oestradiol-17 beta receptors in rat uteri during postnatal development.

1. Uteri of 6--10-day-old rats do not show a late growth response to oestrogen (increase in rate of DNA synthesis and cell division) exhibited by fully competent (20 days or older) uteri. We posed the question whether the lack of the late growth response is due to an inability to replenish the cytoplasmic pool of oestrogen receptors or to curtailed retention of oestrogen binding in the nucleus. Uterine nuclear and cytoplasmic receptors were measured by a [3H]oestradiol-17 beta exchange assay, at 1, 3, 6, 14 and 24 h after oestrogen injection. 2. The replenishment of cytoplasmic oestrogen receptors showed a similar pattern in the uteri of 6 and 10-day-old (partially responsive) and in 20-day-old (fully responsive) rats. 3. Oestrogen was retained longer in uterine nuclei obtained from 5 and 10-day-old rats than in uterine nuclei of 20 and 25-day-old rats. 4. Oestrogen receptors resistant to 0.4 M KCl extraction (residual receptors) were found in uterine nuclei of 6 and 25-day-old rats after oestrogen injection at all the times tested. The concentration of these residual receptors during the late period (6--24 h after injection) was not significantly different in uterine nuclei of 6-day-old and 25-day-old rats. 5. We conclude that neither lack of oestrogen receptor replenishment nor curtailed retention of oestrogen binding in the nucleus is the factor which limits the complete responsiveness to oestrogen in uteri of rats during postnatal development.     

Comparative effects of estradiol-17beta and estriol on uterine RNA polymerases I, II, and III in vivo.

The early and later effects of estradiol-17β and estriol on the RNA polymerase activities of uterine nuclei obtained from ovariectomized rats were compared. At 4 hr of hormone action both estradiol-17β and estriol stimulated the activity of polymerase I, but not the activities of polymerases II and III. At 24 hr, however, the effect of estriol had disappeared, whereas estradiol-17β stimulated all three polymerase activities. These results indicate that estrogen-induced growth of the uterus occurs in two phases, initiation and maintenance. Estriol initiates uterine growth, but does not maintain the process. Estradiol-17β, in contrast, does both. The differences in the effects of the two estrogens may reside in their different binding affinities.     

Specific inhibition of estrogen receptor alpha function by antisense oligodeoxyribonucleotides

We have tested the effect of a range of antisense oligodeoxyribonucleotides (ODN) directed against the human estrogen receptor alpha (ERalpha) on ERalpha protein expression and function. Antisense ERalpha ODN transfected into the ERalpha-positive human breast carcinoma cell line MCF7-K2 showed variable responses dependent on the oligo used. The most active antisense ODN (oligo 7) decreased the levels of ERa protein by 61% as measured by Western blot analysis. Exogenous 17beta-estradiol (17beta-E2), but not 17alpha-E2, augmented this effect, with a threshold effect at 10(-8) M 17beta-E2. The inhibitory effect of antisense ERa oligo 7 was confirmed by measurement of functional ERalpha protein. 3H-17beta-E2 binding to MCF7 cell extracts was inhibited to approximately 40% of control values in the presence of oligo 7. Antisense-transfected MCF7-K2 cell cultures produced a further 30% binding reduction in the presence of exogenous 17beta-E2. An inhibitory effect on 17beta-E2-dependent cell function was confirmed by the demonstration that ERalpha oligo 7-transfected MCF7-K2 cells failed to exhibit 17beta-E2-stimulated cell proliferation. Exogenous 17beta-E2 enhanced the inhibitory effect of the antisense ODN by increasing ODN transfection efficiency but without ERalpha catabolism via the proteosomal pathway, suggesting an effect of 17beta-E2 on the plasma membrane and the existence of different ERalpha degradation pathways in the MCF7-K2 cell subclone. As 17beta-E2 had no effect on ERalpha protein degradation, we conclude that the observed reduction of ERalpha protein levels is due solely to the presence of the antisense ERalpha ODN. Antisense ERalpha ODN molecules, therefore, may form the basis of effective therapies against ERalpha-dependent malignancies.     

Nuclear translocation of estrogen-receptor complex and stimulation of RNA synthesis by estrogens of different biological potencies in the female rat pituitary

Stimulation of RNA synthesis and of nuclear translocation of estrogen-receptor complexes was investigated in isolated nuclei of anterior pituitaries of castrated female rats after injection with estrogens of different biological potencies. The assay system for the estimation of total RNA synthesis was validated and data suggest that incorporation of [3H]UMP into acid-precipitable material is consistent with RNA synthesis. An increase in RNA synthesis was seen 30 min after application of either 17 beta-estradiol, estriol or 1,3-diacetyl-17 alpha-ethinyl-7 alpha-methyl-1,3,5,(10)estratriene-17,3-ol (DMEE). RNA synthesis was maximal 90 min after estrogen application. Thereafter, RNA synthesis decreased slowly and reached pretreatment levels 3, 8 and 30 h after application of estriol, 17 beta-estradiol and the diacetyl derivative of ethinyl-estradiol, respectively. All estrogens were found to stimulate rapidly nuclear translocation of estrogen-receptor complexes. Peak levels of nuclear receptor contents were reached 30 min after administration of estrogens. A concomitant depletion of cytosol receptor levels was noted. Nuclear retention of estrogen-receptor complexes paralelled duration of enhanced RNA synthesis and correlated with biological potencies of the steroids. Data of present experiments combine to suggest that long-term nuclear retention is a requisite for expression of biological activity of estrogens at the anterior pituitary. Furthermore, the degree of biological activity seems to be associated with duration of stimulation of RNA synthesis, amount of estrogen-receptor complexes translocated to the nucleus, and duration of nuclear retention.