High-dose E2 Research Articles

The estrogen receptor α Σ3 mRNA splicing variant is differentially regulated by estrogen and progesterone in the rat uterus

The gene for estrogen receptor alpha (ER alpha) has been shown to be under complex hormonal control and its activity can be regulated by mRNA alternative splicing. Here we examined the regulation of ER alpha transcription and translation in the rat uterus by ovarian steroid hormones. We examined whether expression of ER alpha mRNA splice isoforms is hormonally regulated in ovariectomized (OVX) and cycling rats. Adult OVX female rats were treated daily with 17-beta estradiol (E2) (0.05 microg/rat or 5 microg/rat), progesterone (P4) (1 mg/rat) or a combination of both hormones for 4 days. Animals were killed 24 h after the last injection and uterine horns were removed. In order to determine whether ER alpha mRNA isoforms are differentially expressed under various physiological conditions, animals were evaluated at proestrus, estrus and diestrus. The ER alpha protein and mRNA were detected by immunohistochemistry and comparative RT-PCR analysis respectively. The presence of ER alpha mRNA isoforms was evaluated using a nested RT-PCR assay. In OVX control rats, ER alpha mRNA and protein levels were high, demonstrating a constitutive expression of the ER alpha gene in the uterus. When animals received P4 or the high dose of E2, a significant decrease in both ER alpha mRNA and protein was observed in the uterus. However, when rats were protein was treated with the low dose of E2, only the ER alpha down-regulated; no changes were observed in ER alpha mRNA expression. In addition to the full-length ER alpha mRNA, OVX control rat uteri expressed three shorter transcripts: sigma3, sigma4 and sigma3,4 (lacking exon 3, exon 4, or both 3 and 4 respectively). Surprisingly, when OVX animals were treated with P4, the low dose of E2 or a combination of both steroids, expression of the sigma3 isoform was completely abolished. During the estrous cycle, all ER alpha mRNA splicing variants were detected at proestrus and estrus. However, in diestrus, significant low levels of the sigma3 isoform were observed. In summary, our results suggest a dose-dependent relationship between E2 concentrations and the level of control in the ER alpha transcription-translation cascade. Moreover, the alternative splicing of the ER alpha primary transcript is influenced by the hormonal milieu, suggesting that these events could affect the estrogen responsiveness of the rat uterus during the estrous cycle.

ERβ in normal and malignant breast

Both ERα and ERβ are expressed in not only normal breast of the rodent, cow, monkey and human, but also in breast cancer. Cells that express ERα are found within the luminal epithelium, but not in the myoepithelium or stroma in the human breast. ERβ, on the other hand, is expressed not only in the luminal epithelial cells, but also in myoepithelial cells, stromal cells and in passenger lymphocytes. This widespread distribution of ERβ suggests multiple roles for ERβ in the mammary gland. We have shown that in the rodent mammary gland ERβ is the dominant ER, and that, in response to E2, ERα but not ERβ is downregulated in the early G1 phase of the cell cycle. Cells that contain ERα receive the signal to proliferate from E2, and within 4 hours of that signal ERα is lost from the nucleus. The cells then go through a complete cycle and ERα reappears in daughter cells. ERβ levels do not change in cell nuclei during the cell cycle. This pattern of ER regulation holds true in human breast cancer since ERα is never co-localized with proliferation markers in breast cancer samples. This means that under the conditions of a constant high level of E2, ERα does not reappear in the nucleus. A similar situation exists during pregnancy when there is a constant high level of E2 and there is no ERα in the mammary epithelium. This resistance to the proliferative response to E2 in the presence of a constant high dose of E2 probably explains the very successful use of high-dose E2 in the treatment of breast cancer. ERβ, on the other hand, appears to have a differentiative role not a proliferative role in the mammary gland, and the lactating rodent mammary gland of ERβ-/- mice does not express gap junction and adhesion proteins, typical indicators of fully differentiated cells. In recent years there have been several publications showing that ERβ is expressed in human breast cancer, and conclusions and speculations about a causative role for ERβ in breast cancer development and/or progression have been made. We have studied 500 frozen breast biopsies in collaboration with Prof. RC Coombes, London, in order to clarify the role of ERβ in normal and malignant breast. In this study we measured ERα and ERβ proteins by several techniques (immunohistochemistry, western blotting, ligand binding in sucrose gradients, and RT-PCR) in various human samples obtained from both benign breast and malignant breast. We found that ERβ is the predominant estrogen receptor in the normal mammary gland and in benign breast disease. There is very little ERα in the normal mammary gland. This low expression of ERα is one of the striking differences between rodents and humans. This is in stark contrast to ERβ, which is expressed in 80% of epithelial cells and is also present in the stroma. We found that ERα is abundantly expressed in invasive and in situ ductal carcinoma but not in medullary cancer. ERβ is also expressed in breast cancer, both ductal and medullary. In this study we also found that, in the human breast, the major ER in breast stroma is ERβ. This surprising finding has necessitated several new lines of investigation about the function of ERβ in the breast. It has long been thought that ERα in the stroma was responsible for secretion of growth factors in response to E2 and that these growth factors were responsible for epithelial cell proliferation. The discovery that it is ERβ that is present in the stroma might suggest a role of ERβ in growth factor secretion.

Optimization of estradiol supplementation during the luteal phase improves the pregnancy rate in women undergoing in vitro fertilization–embryo transfer cycles

To evaluate the influence of different E2 supplementation doses during the luteal phase on implantation and pregnancy rates in women undergoing intracytoplasmic sperm injection (ICSI) cycles. Prospective, randomized study. A private IVF unit. One hundred sixty-six women younger than 40 years who were undergoing IVF with long protocol controlled ovarian hyperstimulation (COH). A total of 231 cycles were investigated. Group 1 (P only) included 80 cycles, group 2 (P and 2 mg of E2) included 73 cycles, and group 3 (P and 6 mg of E2) included 78 cycles. Supplementation in the luteal phase with different doses of E2 (0, 2, or 6 mg/d). Serum E2 and P levels in the late luteal phase, and implantation rate and pregnancy rate (PR) were documented. The data were analyzed with regard to the entire study population and further stratified according to the E2 dose used. Significantly higher implantation rate and PR were recorded in those who received low dose E2 supplementation compared with no substitution (PR 23.1% vs. 32.8%). The best implantation and pregnancy results were found significantly in the group with high dose E2 supplementation (PR 51.3%). For women treated with a long GnRH analogue protocol for COH, addition of a high dose of E2 to daily P supplementation significantly improved the IVF-embryo transfer results.

The juvenile three-spined stickleback ( Gasterosteus aculeatus L.) as a model organism for endocrine disruption II—kidney hypertrophy, vitellogenin and spiggin induction

This study investigated the suitability of juvenile three-spined sticklebacks, Gasterosteus aculeatus L., for detecting both androgen- and oestrogen-induced endocrine disruption. The investigated endpoints were kidney hypertrophy and the induction of the protein markers spiggin and vitellogenin. Juveniles were exposed to steroid hormones 17β-oestradiol (E2: nominal 0.01, 1.0 and 10 μg/L), 17α-ethinylestradiol (EE2: nominal 0.05 μg/L) and 17α-methyltestosterone (MT: nominal 1.0 μg/L) from the day of hatching until the termination of the experiments between 39 and 58 days after hatching. E2 (10 μg/L) and MT were applied during different time windows: (a) 14 days after hatching only and (b) continuously with start 14 days after hatching. Kidney hypertrophy is an androgen-dependent secondary sexual character in adult male sticklebacks and corresponds to the production of the glue protein spiggin during the breeding season. The kidneys were hypertrophied and spiggin levels were elevated in juvenile sticklebacks after treatment with MT. Paradoxically, slightly elevated spiggin levels and kidney hypertrophy were observed also in fish treated with high dose E2. Levels of vitellogenin, the oestrogen-inducible yolk precursor protein, were elevated in juvenile sticklebacks after E2 medium and high dose and EE2 treatment. The tested endpoints are suitable for the study of endocrine disruption in juvenile sticklebacks, a fish species that is easy to handle in laboratory and relevant for temperate geographical regions.

Ovariectomy and 17β-estradiol modulate disease progression of a mouse model of ALS

The incidence of amyotrophic lateral sclerosis (ALS) is higher among men than women but rises in women after the menopause. Estrogens may play a protective role. Treatment with estrogens has been shown to be neuroprotective in models of several neurodegenerative diseases. We therefore determined the effect of ovariectomy on female G93A mSOD1 transgenic mice, and the effect of subsequent treatment with 17β-estradiol (E2). Ovariectomy led to a significant acceleration of disease progression of the mice, and high-dose E2 treatment significantly delayed disease progression of ovariectomized G93A mSOD1 transgenic mice. We conclude that treatment with E2 may also delay disease progression of post-menopausal women with ALS.

Differential effects of low and high-dose estradiol on airway reactivity in ovariectomized rats

The aim of this animal study was to test the hypothesis that low and high doses of 17beta-estradiol (E2) may differentially influence airway responsiveness. Ovariectomized female rats received either placebo or E2 (10 or 100 microg/kg per day) for 21 days. The concentration of inhaled acetylcholine (ACh) required to double pulmonary resistance (EC200 RL) was calculated as the in vivo index of airway responsiveness. Ex vivo airway responsiveness was evaluated by the cumulative concentration-response curve (CCRC) of isolated tracheal segments. Rats treated with low-dose E2 were less responsive to ACh than rats given either placebo or high-dose E2 (P=0.003). Ex vivo, low-dose E2 treatment decreased (P=0.01) and high-dose E2 increased the potency of ACh (P<0.001) compared to placebo. E2 treatment did not alter smooth muscle cross-sectional area or epithelium thickness. Accumulation of liquid within the tracheal mucosa was moderately enhanced by high-dose E2 treatment compared with animals given either placebo or low-dose E2 (P=0.03). We conclude that E2 treatment has differential, dose-dependent effects on airway responsiveness to acetylcholine.

Bypassing Estrogen

Ohtake et al. discovered an unexpected mechanism whereby dioxins can modulate estrogen signaling through the arylhydrocarbon receptor (AhR). Dioxins are widespread environmental contaminants that act as endocrine disrupters with both estrogenic and antiestrogenic activity. They have been implicated in the pathogenesis of endometriosis and possibly certain cancers. Both estrogens and dioxins bind to and activate nuclear receptors that function as transcription factors; estrogens act through the estrogen receptors ERα and ERβ, and dioxins act through the AhR-arylhydrocarbon nuclear translocator complex (AhR-Arnt, see Brosens and Parker). Ohtake et al. expressed reporter genes containing either ER or AhR-Arnt binding elements (ERE and XRE, respectively) in breast and uterine cancer cell lines and showed that AhR agonists stimulated transcription through the ERE in the absence of estrogens but inhibited 17β-estradiol (E2)-induced transcription. Transcriptional activation through the ERE by AhR agonists required ER, AhR, and Arnt. The authors used coimmunoprecipitation, glutathione- S -transferase pull-down assays, and chromatin immunoprecipitation analysis to demonstrate that ligand-activated AhR-Arnt associates with unliganded ER, recruits an ER coactivator, and promotes binding of the unliganded ER to the ERE. Dioxins stimulated estrogen-responsive genes and increased uterine weight and cell proliferation in the uterine glandular epithelium in ovariectomized mice, but had antiestrogenic effects in the presence of high doses of E2. These estrogenic effects were not observed in ovariectomized mutant mice lacking AhR or ERα. These data indicate that the estrogenic effects of dioxin may be mediated through ligand-independent activation of the ER through the dioxin-AhR-Arnt complex. F. Ohtake, K.-I. Takeyama, T. Matsumoto, H. Kitagawa, Y. Yamamoto, K. Nohara, C. Tohyama, A. Krust, J. Mimura, P. Chambon, J. Yanagisawa, Y. Fujii-Kuriyama, S. Kato, Modulation of oestrogen receptor signalling by association with the activated dioxin receptor. Nature 423 , 545-550 (2003). [Online Journal] J. J. Brosens, M. G. Parker, Oestrogen receptor hijacked. Nature 423 , 487-488 (2003). [Online Journal]

17β-estradiol and anti-estrogen ICI:Compound 182,780 regulate expression of lipoprotein lipase and hormone-sensitive lipase in isolated subcutaneous abdominal adipocytes

We sought to investigate the influence of 17β-estradiol (E2) on key enzymes of lipogenesis and lipolysis in subcutaneous (SC) abdominal adipocytes isolated from women. In addition, we wished to determine the influence of an anti-estrogen, ICI:compound 182,780 (anti-E), known to act via the estrogen receptor (ER), alone and in combination with E2. Adipose tissue was obtained from 17 women undergoing elective surgery, with a mean age of 47 years (range, 34 to 62), mean weight of 65.4 kg (range, 58.1 to 75.0), and mean body mass index (BMI) of 25 kg/m2 (range, 22 to 27). Isolated adipocytes were treated with varying doses of E2, anti-E, or E2 in combination with anti-E 10−8 mol/L for 48 hours. Following treatment, proteins were extracted and the effects on lipogenesis and lipolysis were assessed, using Western blotting to determine the relative expression of the key enzymes of these processes, lipoprotein lipase (LPL; 56 kd), and hormone-sensitive lipase (HSL; 84 kd), respectively. Glycerol release into the medium was also measured as an index of lipolytic activity. The protein expression studies demonstrated that E2 altered expression of LPL relative to control, with the highest dose significantly reducing LPL expression and the lower doses significantly increasing LPL expression (mean protein expression relative to control ± SE): E2 10−12 mol/L, 1.79 ± 0.16 (P <.001); E2 10−7 mol/L, 0.56 ± 0.08 (P <.05). In contrast, HSL expression was increased relative to control at the higher doses of E2 but was not significantly altered relative to control at the lower doses: E2 10−12 mol/L, 1.02 ± 0.14 (P >.05); E2 10−7 mol/L, 1.55 ± 0.17 (P <.01). Anti-E 10−8 mol/L alone reduced LPL protein expression relative to control (P <.05) and increased HSL protein expression relative to control (P >.05). In combination with E2 10−7 mol/L, anti-E 10−8 mol/L did not abrogate the inhibitory effect on LPL expression relative to control (P <.05). Furthermore, E2 10−7 mol/Lin combination with anti-E 10−8 mol/L, displayed a stimulatory effect on HSL expression relative to control (P <.01). Glycerol release studies following the higher doses of E2, and also following E2 10−7 mol/L in combination with anti-E 10−8 mol/L, provided support for the HSL protein expression studies. We conclude that the highest concentration of E2 (10−7 mol/L) significantly reduced LPL expression relative to control, while the lower concentrations significantly increased LPL expression relative to control. The highest concentration of E2 also significantly increased both HSL expression and glycerol release relative to control. The effects of anti-E suggest that the in vitro effects of E2 on lipogenesis and lipolysis occur, at least in part, through a receptor-mediated pathway. In addition, as recently observed in other tissues, ICI:compound 182,780 does not appear to behave as a pure anti-estrogen in isolated human adipocytes. Copyright 2003 Elsevier, Inc. All rights reserved.

Estradiol increases salt intake in female normotensive and hypertensive rats.

The objective of this study was to examine whether or not estradiol (E2) alters sodium intake in hypertensive and normotensive female rats. It was hypothesized that higher doses of E2 would increase sodium consumption and that this response would be greater in spontaneously hypertensive rats (SHR) compared with Wistar Kyoto (WKY) rats. The study involved female SHR and WKY (n = 12/group). All animals were ovariectomized. Six of twelve rats from each strain received three progressively larger doses of beta-estradiol propionate (each dose lasting 2 wk), whereas the other six rats from each strain received sham implants. Blood E2 levels were measured by radioimmunoassay after each 2-wk period, allowing a 10-day washout period before the next E2 dose. Rats had access to 0.0, 0.5, 1.0, and 1.5% NaCl solutions to drink throughout the experiment. There was a significant positive correlation between sodium intake and plasma E2 (r = 0.8, P < 0.001). Both strains avoided the 1.5% NaCl, and the increased sodium intake was achieved by an increase in consumption of the 0.5% NaCl. SHR females consumed more sodium than WKY females, which is similar to what has been observed in males of these strains. In conclusion, E2 was positively correlated with sodium intake in both strains of rat, with the hypertensive rats consuming more sodium than the normotensive rats.

The role of endothelial cells in the pathogenesis of ovarian hyperstimulation syndrome.

Ovarian hyperstimulation syndrome (OHSS) is an iatrogenic complication of treatment with fertility drugs. Using human lung microvascular endothelial cells (HUMEC-L) as an in-vitro model of OHSS, we have tested the hypothesis that the endothelium is a target of HCG in the pathogenesis of OHSS. Since OHSS is characterized by increased capillary permeability, we have investigated the production and action of vasoactive agents. When HUMEC-L were cultured with high doses of estradiol (E(2)), no significant changes were observed in the secretion of vascular endothelial growth factor (VEGF), interleukin (IL)-6 or IL-1 beta. However, the addition of HCG resulted in a significant increase in the secretion of VEGF and IL-6. Time-course experiments showed that VEGF was secreted within minutes of HCG addition, whereas IL-6 was significantly increased only after 48 h in culture. The secretion of IL-1 beta was unchanged by these hormonal conditions. The presence of HCG receptors was demonstrated in HUMEC-L in basal conditions as well as after the addition of E(2). The expression of VEGF receptors was also investigated. High doses of E(2) were unable to increase the expression of KDR, flt-1 and sfl-t, but the addition of HCG significantly upregulated the KDR concentration in endothelial cells, while no change was observed for flt. Permeability assays demonstrated that while E(2) alone did not change the arrangement of HUMEC-L in vitro, the presence of HCG caused changes in the actin fibres corresponding to increased capillary permeability. Anti-human VEGF antibodies were able to overcome these changes. In conclusion, these experiments show that the endothelium may be a primary target of HCG, causing an acute release of VEGF and a significant increase in IL-6 and resulting in an autocrine-paracrine action that may increase vascular permeability.

Effect of the ovarian hormones on GLUT4 expression and contraction-stimulated glucose uptake.

This study examined the roles of the female sex steroids, 17beta-estradiol (E(2)) and progesterone (Prog), on glucose uptake and GLUT4 protein expression. Female Sprague-Dawley rats were either sham operated (C) or ovariectomized and treated with placebo (O), E(2) (E), Prog (P), or both hormones at physiological doses (P + E) or the same dose of Prog with a high dose of E(2) (P + HiE) via timed-release pellets inserted at the time of surgery, 15 days before metabolic testing. On the morning of day 15, animals received a 300-microCi injection (ip) of 2-deoxy-[(14)C]glucose and then either exercised on a motorized treadmill for 30 min at 0.35 m/s or remained sedentary in their cages for the same period. Basal glucose uptake was not different between the treatment groups in either the red or white quadriceps. However, glucose uptake was decreased (P < 0.05) in O, P, and P + E rats during exercise in the red quadriceps compared with C rats, whereas E and P + HiE treatment restored glucose uptake. Glycogen content in skeletal muscle followed similar trends, with no differences seen in resting animals. Postexercise red quadriceps glycogen levels were higher (P < 0.05) in the E and P + HiE rats compared with O and P. Treatment of ovariectomized rats with progesterone (P rats) decreased (P < 0.05) GLUT4 content in the red quadriceps by 21% compared with C rats. These data demonstrate that estrogen-deficient animals have a decreased ability for contraction-stimulated glucose uptake and increased glycogen use during aerobic exercise. However, changes in contraction-stimulated glucose uptake could not be explained by altered transporter protein content, since the absence of E(2) had no effect on GLUT4 protein.

Vitellogenin mRNA regulation and plasma clearance in male sheepshead minnows, ( Cyprinodon variegatus) after cessation of exposure to 17β-estradiol and p-nonylphenol

Research was conducted to determine the kinetics of hepatic vitellogenin (VTG) mRNA regulation and plasma VTG accumulation and clearance in male sheepshead minnows ( Cyprinodon variegatus) during and after cessation of exposure to either 17β-estradiol (E2) or para-nonylphenol (NP). Adult fish were continuously exposed to aqueous measured concentrations of 0.089 and 0.71 μg E2 per l, and 5.6 and 59.6 μg NP per l for 16 days using an intermittent flow-through dosing apparatus. Fish were sampled on days 8 and 16 of exposure followed by sampling at discrete intervals for up to 96 days post-exposure. At each interval five fish were randomly sampled from each concentration and hepatic VTG mRNA and serum VTG levels for individual fish determined by slot blot and direct enzyme-linked immunosorbent assay (ELISA), respectively. Exposure to E2 and NP resulted in a dose dependent increase in hepatic VTG mRNA and plasma VTG over the course of the 16-day exposure period. Mean plasma VTG levels at day 16 were >100 mg/ml for both high doses of E2 and NP, and >20 mg/ml for the low exposure treatments. Within 8 days post-exposure, hepatic VTG mRNA levels returned to baseline in both high and low E2 treatments but remained elevated 2–4 fold in the NP treatments. Due to a shortened sampling period, a clearance rate for plasma VTG in the 5.6 μg NP per l treatment could not determined. In the 0.089, 0.71 μg E2 per l, and 59.6 μg NP per l treatments, VTG levels began decreasing within 4 days after exposure cessation and exhibited an exponential rate of elimination from plasma. Clearance rates for 0.71 μg E2 per l and 59.6 μg NP per l were not significantly different ( P=0.47), however, both demonstrated significantly higher rates of clearance ( P<0.02) than observed in the 0.089 μg E2 per l treatment. Our results indicate that hepatic VTG mRNA rapidly diminishes after cessation of estrogenic exposure in sheepshead minnows, but plasma VTG clearance is concentration and time dependent and may be detected at measurable levels for months after initial exposure to an estrogenic compound.

Sequential role of e2 and GnRH for the expression of estrous behavior in ewes.

Preovulatory GnRH secretion in ewes, measured in portal blood and cerebrospinal fluid, starts at the time of the LH surge, approximately 4 h after the onset of estrous behavior, and lasts as long as receptivity (36-48 h), which is much longer than the LH surge. This study tested the hypothesis that the extended GnRH secretion is involved in the maintenance of receptive behavior, prolonging the initial triggering effect of E2. Ovariectomized ewes were subjected to artificial estrous cycles and infused intracerebroventricularly either with a water soluble GnRH antagonist (Teverelix, Exp 1 and 2) or GnRH (Exp 3 and 4) after preovulatory E2 challenges of various intensity. The GnRH antagonist infused for 20 h (0.5 mg/ml, flow rate 3 microl/min) following a treatment with 2 x 30-mm E2 implants for 24 h (Exp 1) significantly reduced receptivity 36-48 h post E2 compared with vehicle infusion. By contrast, when the GnRH antagonist was infused with E2 implants still present (Exp 2: E2 for 48 h, GnRH antagonist given 24-44 h after E2 insertion, n = 14) receptivity was not affected. Administration of GnRH (0.5 mg/ml, flow rate 3 microl/min) when receptivity began to decline (Exp 3: 30-48 h after a 6-h 2 x 30-mm E2 implants n = 12) resulted in significantly higher receptivity scores at 48 and 52 h post E2 in GnRH treated animals compared with vehicle treated. GnRH infusion of ewes under subthreshold E2 treatment (Exp 4: GnRH 6-24 h after implantation of 1 x 30-mm E2 for 3 h, n = 12 in a cross-over design) significantly increased their receptivity compared with vehicle administration at 18 and 24 h post E2 insertion, but receptivity remained lower than when induced by high doses of E2. Our results demonstrate for the first time that GnRH is involved in the control of receptivity in a ruminant species and suggest that in the cycling ewe the sustained preovulatory GnRH secretion plays a physiological role in extending the duration of estrous behavior. They also indicate that it is possible to dissociate a direct effect of E2 on estrous behavior from its effect via stimulation of GnRH secretion.

Effect of ovarian hormones on mitochondrial enzyme activity in the fat oxidation pathway of skeletal muscle.

To examine the roles of 17beta-estradiol (E(2)) and progesterone (Prog) in lipid metabolism, skeletal muscle enzyme activities were studied in female Sprague-Dawley rats. Groups included sham-operated rats (C) and ovariectomized rats treated with placebo (O), E(2) (E), Prog (P), both hormones at physiological doses (P + E), or both hormones with a high dose of E(2) (P + HiE). Hormone (or vehicle only) delivery was via time-release pellets inserted at the time of surgery, 15 days before metabolic testing. Results demonstrated that carnitine palmitoyltransferase maximal activity was 19, 21, and 19% lower (P < 0.01) in O, P, and P + E rats, respectively, compared with C rats. Conversely, activity in E and P + HiE rats was 14 and 19% higher (P < 0.01) than in C. beta-Hydroxyacyl-CoA dehydrogenase (beta-HAD) maximal activity was 20% lower (P < 0.01) in O than in C rats; similarly, P and P + E rats were 18 and 19% lower, respectively (P < 0.01); however, treatment with E(2) returned beta-HAD activity to C levels. These results suggest that E(2) plays a role in lipid metabolism by increasing the maximal activity of key enzymes in the fat oxidative pathway of skeletal muscle.

Ovariectomy and 17beta-estradiol modulate the levels of Alzheimer's amyloid beta peptides in brain.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by accumulation of aggregated forms of the 40- and 42-amino acid Abeta peptides (Abeta40 and Abeta42). Estrogen replacement therapy (ERT) in postmenopausal women is associated with decreased risk for AD and/or delay in disease onset. The mechanism by which estrogen exerts this neuroprotective effect is elusive. 17beta-estradiol (E2) was shown to reduce the release of Abeta peptides by primary neuronal cultures of murine and human origin. To test whether estrogen can modulate the metabolism of Abeta peptides in vivo, four experimental sets of guinea pigs were used: intact animals, ovariectomized animals, and ovariectomized animals that received E2 at two different doses. Ovariectomy was associated with a 1.5-fold average increase in total brain Abeta levels as compared to intact controls. E2 treatment significantly reversed the ovariectomy-induced increase in brain Abeta levels. The high-dose E2 treatment did not lead to further decrease in brain Abeta beyond the one observed with the low-dose E2 treatment. Our results infer that cessation of ovarian estrogen production in postmenopausal women might facilitate Abeta deposition by increasing the local concentrations of Abeta40 and Abeta42 peptides in brain and suggest that modulation of Abeta metabolism may be one of the ways by which ERT prevents and/or delays the onset of AD in postmenopausal women.

Vitellogenin Association and Oocytic Accumulation of Thyroxine and 3,5,3′-Triiodothyronine in Gravid Fundulus heteroclitus

The association of the thyroid hormone (TH) thyroxine (T4) and its metabolically active metabolite 3,5,3′-triiodothyronine (T3) with serum vitellogenin (VTG) in gravid female and estrogenized male (E2+) Fundulus heteroclitus was investigated. In in vivo, time-course experiments, sera from gravid female fish exposed to [125I]T4 showed time- and dose-dependent increases in total [125I]TH content. The [125I]T4:[125I]T3 ratio was also affected by dose and time. In analysis of sera from female fish, >80% of detected radioactivity was associated with VTG (approximately 35%) and a second chromatographic peak (approximately 45%), a lipoprotein fraction possibly consisting of high-density lipoproteins. In experiments comparing estrogenized versus control male fish, the presence of VTG significantly increased the overall quantity and altered the profile of serum protein-associated [125I]TH. When serum VTG was present in the very large quantities typical of male fish treated with high doses of E2, the majority (59–70%) of detected radioactivity was associated with VTG. Both [125I]T4 and [125I]T3 were detected in extracts from oocytes collected during the in vivo female study. The total TH content and [125I]T4:[125I]T3 ratios in these extracts presented an accumulation profile that mirrored, in a delayed manner, the profiles observed in sera data. Furthermore, this accumulation was related to oocyte maturational state (i.e., size) and, correspondingly, VTG uptake. Together, these data suggest an important role for VTG as a vector of maternal transfer for both T4 and T3.

Biphasic effects of the natural estrogen 17beta-estradiol on hepatic cholesterol metabolism in intact female rats.

The protective influence of estrogens in cardiovascular disease is believed to be partly due to beneficial effects on cholesterol metabolism. Much of the experimental data are based on models in which synthetic estrogens have been used in pharmacological doses, and therefore, the physiological role of estrogens in cholesterol metabolism is uncertain. To evaluate this important issue, we performed experiments in intact female rats with use of the natural estrogen 17beta-estradiol (E2) administered either subcutaneously or orally. After physiological doses of E2 (< or =0.04 mg. kg(-1). d(-1)) were administered, plasma levels of high density lipoprotein (HDL) cholesterol and apolipoprotein (apo) A-I were increased. In the liver, 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol 7alpha-hydroxylase activities were increased, as well as cholesterol 7alpha-hydroxylase mRNA levels. These effects were abolished during treatment with higher doses of E2, whereas apo A-I mRNA increased in a dose-dependent way. After treatment with pharmacological doses of E2 (> or =0.2 mg. kg(-1). d(-1)), the number of hepatic low density lipoprotein receptors increased and plasma cholesterol was reduced. These effects were similar after both oral and subcutaneous administration of E2. Our results show that the responses to E2 are biphasic: plasma HDL, apo A-I, and hepatic enzyme activities governing bile acid and cholesterol synthesis increased only at physiological doses of E2. At pharmacological doses of E2, hepatic low density lipoprotein receptors are stimulated and plasma cholesterol is reduced. Therefore, under physiological conditions, E2 exerts its major effects on hepatic cholesterol metabolism through mechanisms other than stimulation of low density lipoprotein receptor expression.

Ovariectomy and 17beta-estradiol modulate the levels of Alzheimer's amyloid beta peptides in brain.

To test whether female gonadal hormone status and estrogen modulate the metabolism of Abeta peptides in vivo. AD is a neurodegenerative disorder characterized by accumulation of aggregated forms of the 40- and 42-amino acid Abeta peptides (Abeta40 and Abeta42). Estrogen replacement therapy in postmenopausal women is associated with decreased risk for AD or delay in disease onset or both. The mechanism by which estrogen exerts this neuroprotective effect is elusive. 17beta-estradiol (E2) was shown to reduce the release of Abeta peptides by primary neuronal cultures of murine and human origin. For this purpose, four experimental sets of guinea pigs were used: intact animals, ovariectomized animals (ovx), and ovariectomized animals that received E2 at two different doses (ovx+low-dose E2 and ovx+high-dose E2). Brain Abeta40 and Abeta42 levels were assessed using Abeta40 and Abeta42-specific ELISA assays. Prolonged ovariectomy resulted in uterine atrophy and decreased serum E2 levels and was associated with a pronounced increase in brain Abeta levels. Total brain Abeta in the ovx animals was increased by 1. 5-fold on average as compared to intact controls. E2 treatment of ovariectomized animals led to uterine hypertrophy and a dose-dependent increase in serum E2 levels. In addition, both doses of E2 significantly reversed the ovariectomy-induced increase in brain Abeta levels. The high-dose E2 treatment did not lead to a further decrease in brain Abeta beyond that observed with the low-dose E2 treatment. Our results infer that cessation of ovarian estrogen production in postmenopausal women might facilitate Abeta deposition by increasing the local concentrations of Abeta40 and Abeta42 peptides in brain. In addition, our finding that E2 treatment is associated with diminution of brain Abeta levels suggests that modulation of Abeta metabolism may be one of the ways by which estrogen replacement therapy prevents or delays the onset of AD or both in postmenopausal women.

Nitric-Oxide-Dependent Pial Arteriolar Dilation in the Female Rat: Effects of Chronic Estrogen Depletion and Repletion

In this study, we compared endothelial nitric oxide synthase (eNOS)-mediated cerebral vasodilating responses in intact female rats, chronically ovariectomized (OVX) rats, and OVX rats treated for 2 weeks with 17β-estradiol (E2). Under anesthesia, using intravital microscopy and a closed cranial window system, pial arteriolar diameter changes were monitored during sequential cortical suffusions of an eNOS-dependent dilator [acetylcholine (ACh)] and a direct NO donor [S-nitrosoacetylpenicillamine (SNAP)]. In separate rats from the same groups, we compared eNOS and caveolin-1 (CAV-1) protein abundance in pial arterioles (via immunofluorescence analyses). In untreated and low-dose E2-treated (1.0 μg · kg−1 · day−1) OVX rats, ACh-induced vasodilations were virtually absent. High-dose E2 treatment (100 μg · kg−1 · day−1) restored ACh-induced pial arteriolar dilations to levels seen in intact females. The vasodilations elicited by SNAP and ADO were unaffected by chronic estrogen changes, indicating no direct estrogen influence on vascular smooth muscle (VSM) reactivity. Pial arteriolar eNOS protein abundance was diminished by ovariectomy and restored by high-dose E2 treatment. Pial arteriolar CAV-1 expression was higher in OVX versus intact and E2-treated OVX females. These results suggest that long-term changes in estrogen directly influence brain eNOS functional activity. The estrogen-related changes in eNOS-dependent vasodilating function appear to be related, in part, to a capacity for E2 to increase eNOS protein expression and, in part, to an E2-associated diminution in endothelial CAV-1 expression.

Effects of High Doses of 17.BETA.-estradiol and Ovariectomy on 7,12-Dimethylbenz[a]anthracene-Induced Mammary Carcinomas and Dysplasias in Neonatally Androgenized Female Sprague-Dawley Rats.

Inbred, female Sprague-Dawley rats were neonatally androgenized at the age of 2 days by subcutaneous injections of 1.25 mg testosterone propionate and divided into 4 groups. At the age of 50 days, rats of all groups were given 20 mg 7, 12-dimethylbenz[a]anthracene (DMBA). Four weeks after the administration of DMBA, the rats in Group I remained intact, those in Group II were subjected to ovariectomy, while the animals in Groups III and IV were given 1 mg 17β-estradiol (E2) at 2-day intervals for 3 weeks. Additionally, the rats in Group IV were given 1 mg E2 at 2-day intervals for 3 weeks by intramuscular injection, starting from the 105th day after the administration of DMBA.The ovaries of the neonatally androgenized rats contained numerous large vesicular follicles and no corpora lutea, and the mammary glands of the animals in Groups I, III and IV consisted mainly of dilated acini containing milky secretions. In rats in Group I, mammary carcinomas and mammary dysplasias were induced. Rat mammary dysplasias were grossly characterized by gross cysts and solid masses. Solid masses were heteromorphic lesions and commonly consisted of several nodules with various microscopic features. These masses were subclassified into the 2 types of histological features that were predominant among the nodules: acinar adenosis and fibrotic adenosis. The induction of mammary carcinomas and mammary dysplasias was strongly suppressed by ovariectomy (Group II) as the development of mammary carcinomas and mammary dysplasias induced in neonatally androgenized rats are dependent on estrogens. The induction of mammary carcinomas was suppressed by injections of high doses of E2 for 21 days (Once in Group III and twice in Group IV). Conversely, the induction of mammary dysplasias was promoted by one time (Group III) but was suppressed by two times (Group IV) of E2. The results indicate that carcinoma cells and dysplasia cells induced by DMBA in neonatally androgenized rats differ in response to high doses of E2.