Estrogen and tamoxifen modulate cerebrovascular tone in ovariectomized female rats

Postmenopausal estrogen deficiency increases the incidence of cerebrovascular disease. However, hormone replacement therapy is associated with an increased cardiovascular risk. Tamoxifen is a selective estrogen receptor modulator with estrogenic effects on cardiovascular risk factors, but its long-term impacts on cerebral vasculature are unknown. We hypothesized that chronic 17beta-estradiol or tamoxifen treatment exerted similar effects in reducing cerebrovascular tension in ovariectomized rats. We therefore determine whether (1) chronic 17beta-estradiol treatment could influence vasomotor activities, (2) chronic tamoxifen therapy could exert an estrogen-like or estrogen-antagonistic effect, and (3) acute exposure to estrogen could mimic the effect of 17beta-estradiol. Isometric tension was measured in cerebral arteries from female rat groups: control, ovariectomy, ovariectomy plus 17beta-estradiol treatment, ovariectomy plus tamoxifen treatment, and ovariectomized rats treated with tamoxifen and 17beta-estradiol. Ovariectomy enhanced cerebrovascular contractions to endothelin-1 or CaCl2, but not to U46619 or phenylephrine. 17beta-Estradiol therapy reversed these effects. Chronic tamoxifen treatment exerted estrogen-like actions by reversing ovariectomy-induced enhancement of vessel tone without antagonizing the effect of chronic 17beta-estradiol treatment. Ovariectomy enhanced the relaxing potency of nicardipine, and 17beta-estradiol treatment prevented this effect. Acute exposure to 10(-9) mol/L 17beta-estradiol or 10(-8) mol/L tamoxifen did not modulate contractions in rings from nonoperated female rats. In conclusion, ovariectomy differentially enhances agonist-induced cerebrovascular tone, an effect that was reversed by estrogen therapy. Tamoxifen does not act as an estrogen antagonist; instead, it functions as an estrogen agonist during estrogen deficiency. Thus, tamoxifen may confer beneficial effects similar to estrogen in cerebrovascular vessels.     

Estrogen replacement raises rat CRP without evidence of complement activation

Given current controversies regarding anti- and pro-inflammatory effects of estrogen, there is a need to explore relationships between gonadal hormones and inflammation using appropriate animal models. It has been proposed that rats are not appropriate for such research since, contrary to the effect of estrogen in humans, earlier animal studies had reported that estrogen downregulates serum C-reactive protein (rCRP) levels in the rat. With these considerations in mind, we re-examined the effects of estrogen withdrawal and replacement on CRP expression and complement activation in the rat. F-344 rats underwent bilateral ovariectomy or sham surgery at 9-10 months of age. Four months later, ovariectomized rats were treated with traditional high-dose 17beta-estradiol (Hi-E2) capsules, lower-dose (Lo-E2) 17beta-estradiol capsules, or placebo capsules for 7 days prior to sacrifice. Levels of plasma rat C-reactive protein (rCRP) were significantly lower in ovariectomized vs. sham-operated animals (415.5 +/- 10.6 vs. 626.6 +/- 23.0 mg/L, p < 0.001). Estrogen replacement significantly raised rCRP levels in ovariectomized animals (690.0 +/- 28.0 mg/L in Lo-E2 and 735.5 +/- 35.8 mg/L in Hi-E2, respectively, p < 0.001). Plasma rCRP levels correlated significantly with both hepatic rCRP (r = 0.79, p < 0.001) and serum estradiol (r = 0.70, p < 0.001) levels. However, no significant differences were observed in indices of complement activation (C4b/c) or CRP-complement complex generation (rCRP-C3 complex). In the mature female rat, ovariectomy reduces and estrogen replacement raises rCRP. Effects of estrogen on plasma rCRP induction are mediated, at least in part, through hepatic mechanisms and do not appear to require or be associated with complement activation.     

Effects of the Japanese herbal medicine Keishi-bukuryo-gan and 17beta-estradiol on calcitonin gene-related peptide-induced elevation of skin temperature in ovariectomized rats

The effects of a Japanese herbal medicine, Keishi-bukuryo-gan, and 17beta-estradiol on calcitonin gene-related peptide (CGRP)-induced elevation of skin temperature were investigated in ovariectomized (OVX) rats. Ovariectomy not only potentiated CGRP-induced elevation of skin temperature and arterial vasorelaxation but also induced a lower concentration of endogenous CGRP in plasma and up-regulation of arterial CGRP receptors, suggesting that lowered CGRP in plasma due to ovarian hormone deficiency increases the number of CGRP receptors and consequently amplifies the stimulatory effects of CGRP to elevate skin temperature. Oral Keishi-bukuryo-gan (100-1000 mg/kg, once a day for 7 days) restored a series of CGRP-related responses observed in OVX rats by normalizing plasma CGRP levels in a dose-dependent manner as effectively as s.c. injection. 17Beta-estradiol (0.010 mg/kg, once a day for 7 days). However, Keishi-bukuryo-gan did not affect the lower concentration of plasma estradiol and the decreased uterine weight due to ovariectomy, although the hormone replacement of 17beta-estradiol restored them. These results suggest that Keishi-bukuryo-gan, which does not confer estrogen activity on plasma, may be useful for the treatment of hot flashes in patients for whom estrogen replacement therapy is contraindicated, as well as menopausal women.     

Ovariectomy selectively reduces the concentration of transforming growth factor beta in rat bone: implications for estrogen deficiency-associated bone loss.

Previous work showed that production of transforming growth factor beta (TGF-beta) by osteoblast-like rat UMR 106 cells was increased by 17 beta-estradiol at physiological concentrations. To determine whether ovariectomy alters the concentration of TGF-beta in rat long bones, female Sprague-Dawley rats were either sham-operated (n = 19) or ovariectomized (n = 19), pair-fed a semisynthetic diet for 6 weeks, and sacrificed. Tibial and femoral diaphyses were removed and extracted by demineralization. Ovariectomy lowered serum estrogen; did not alter body weight, serum magnesium, or serum 1,25-dihydroxyvitamin D; and produced only modest differences in serum calcium and phosphate concentrations. Hydroxyproline was higher and extractable protein was lower in bones from ovariectomized rats than in bones from sham-operated rats; calcium content did not differ between the two groups of animals. Ovariectomy lowered the concentration of TGF-beta in bone but did not change the concentration of insulin-like growth factors I or II compared with values in bone from control animals. The reduction of bone TGF-beta was evident 6 weeks after surgery but not at 3 weeks. Treatment of ovariectomized rats with estrogen eliminated the TGF-beta deficit. To determine whether 17 beta-estradiol increased TGF-beta production by normal bone cells, mouse osteoblasts were treated for 2 days with 17 beta-estradiol. The production of TGF-beta was increased almost 2-fold by 1 nM 17 beta-estradiol, and short-term treatment stimulated the intracellular accumulation of TGF-beta 1 mRNA. We conclude that ovariectomy reduces deposition of TGF-beta in rat bone and that diminished skeletal TGF-beta could play a role in the pathogenesis of bone loss, fractures, and microfractures that occur in estrogen-deficient states. Our results support the possibility that estrogen and bone TGF-beta may be necessary for normal maintenance of the skeleton in female rats.     

Estradiol replacement enhances working memory in middle-aged rats when initiated immediately after ovariectomy but not after a long-term period of ovarian hormone deprivation

The goal of the present study was to explore the effects of long-term hormone deprivation on the ability of subsequent estrogen replacement to affect cognition. Female rats, 12 months of age, underwent ovariectomies (n = 30) or sham surgeries (n = 10). Intact rats and 20 ovariectomized rats received cholesterol implants. Ten ovariectomized rats received implants containing 25% estradiol. Five months later, implants were replaced. Half of the ovariectomized rats with cholesterol implants received estradiol implants and half received new cholesterol implants. Rats with estradiol implants received new estradiol implants. Intact rats were ovariectomized and given estradiol implants. Beginning 1 wk later, working memory performance was assessed in an eight-arm radial maze across 24 d of acquisition and during eight additional trials in which a 2.5-h delay was imposed between the fourth and fifth arm choices. Estradiol replacement initiated immediately after ovariectomy at either 12 or 17 months of age significantly improved performance during acquisition and delay trials, compared with control treatment. When estradiol replacement was initiated at 17 months of age, 5 months after ovariectomy, no enhancements were evident. Uteri of rats that experienced delayed estradiol replacement weighed significantly more than uteri of ovariectomized controls but significantly less than uteri of rats that received immediate estradiol replacement. Uterine weight negatively correlated with mean errors during acquisition. These results indicate that whereas chronic estradiol replacement regimens positively affect working memory in middle-aged animals when initiated immediately after ovariectomy, estradiol replacement is not effective when initiated after long-term hormone deprivation.     

The effect of oral calcitonin on cartilage turnover and surface erosion in an ovariectomized rat model

OBJECTIVE: To investigate whether oral calcitonin treatment influences the increases in type II collagen (CII) degradation and related surface erosions of articular cartilage in ovariectomized rats. METHODS: Fifty rats were randomly allocated into 1 of the 5 following intervention groups: sham-operated, ovariectomy, ovariectomy plus subcutaneously implanted 17beta-estradiol pellet, ovariectomy plus 2 mg/kg salmon calcitonin plus 50 mg/kg 5CNAC (carrier), or ovariectomy plus 50 mg/kg 5CNAC. Each treatment was administered for 9 weeks after the ovariectomy. Blood samples for biochemical marker analysis were collected from fasting animals at baseline, on day 3, and after 1, 2, 4, 6, and 9 weeks. CII degradation was quantified by specific immunoassay, and the changes in severity scores of articular cartilage erosions were visualized and scored in histologic sections of the knees. RESULTS: Ovariectomy resulted in a marked increase in serum levels of C-telopeptide of type II collagen (CTX-II) (P < 0.001), which could be effectively reversed by 17beta-estradiol supplementation. Oral administration of calcitonin elicited similar decreases in serum levels of CTX-II (P < 0.001). Histologic scoring of cartilage erosion showed significantly less cartilage erosion in calcitonin-treated ovariectomized rats versus control ovariectomized rats that were untreated or treated with 5CNAC alone. (P < 0.01). CONCLUSION: The in vivo effects of calcitonin in rats suggest that calcitonin is able to counteract CII degradation and the accompanying structural disintegration of articular cartilage promoted by estrogen deficiency. Clinical assessment of the chondroprotective potential of calcitonin in postmenopausal women seems warranted.     

Age-related alterations in the number and function of pituitary lactotropic cells from intact and ovariectomized rats

We have sought to determine whether the reported age-related increase in in vivo and in vitro PRL secretion in intact and/or postpubertally ovariectomized female rats results from increased number and/or function of lactotropic cells Immunocytochemical staining with antirat-PRL serum, goat antirabbit serum, and peroxidase-antiperoxidase using H2O2 and 4-chloro-1-naphthol or 3'3'-diaminobenzidine hydrochloride as substrates was used to compare the percentages of lactotropes in primary suspensions of heterogeneous adenohypophyseal cells from groups of intact and 3-day ovariectomized mature (6 to 7 month) Wistar rats at estrus (E) or diestrus1-2 (D) with those from the corresponding old (22 to 24 month) rats in constant estrus (CE) or constant diestrus (CD). For both intact and ovariectomized old vs. mature rats, there were small (30-35%) but significant (P less than 0.005) increases in lactotrope number. Lactotrope number did not differ (P greater than 0.05) between E vs. D rats, or CE vs. CD rats, either before or after ovariectomy; moreover, ovariectomy per se did not alter (P greater than 0.05) lactotrope number in any of the groups. Electron microscopic examination of lactotropic cells derived from ovariectomized CE rats revealed ultrastructural changes compatible with estrogenic hyperstimulation; such alterations were not observed in cells from the corresponding E rats. Basal and 17 beta-estradiol (10(-10) M)-stimulated in vitro PRL secretion was measured for 4 days in primary cultures from each of the above groups of rats. After correction for lactotrope number, PRL secretion was greater (P less than 0.01) from cells of intact and ovariectomized CD vs. D rats, whereas 17 beta-estradiol-stimulated (P less than 0.001) but not basal (P greater than 0.05) PRL secretion was greater from the corresponding CE vs. E rats. There was no effect (P greater than 0.05) of reproductive status or of short term ovariectomy on in vitro PRL secretion. These data suggest that aging in the female rat is associated with alterations in both the number and function of pituitary lactotropic cells.     

Estrogen receptors in cultured rat uterine cells: induction of progesterone receptors in the absence of estrogen receptor processing

When cultured rat uterine cells were treated for up to 6 h with 5 nM 17 beta-estradiol, no decrease in the [3H] estradiol-binding capacity of the cells was observed (i.e. no processing). This was true whether the cells were treated directly with 5 nM [3H]estradiol or with 5 nM unlabeled 17 beta-estradiol followed by homogenization and exchange with [3H]estradiol in vitro. In additional experiments, intact cells were treated with medium containing 5 nM [3H]estradiol for 30 min, and then that medium was removed and replaced with medium containing 5 nM unlabeled 17 beta-estradiol. Receptor-bound estradiol in intact cells was totally exchangeable with estradiol in the culture medium (t1/2, approximately 90 min). Six-hour treatment of cells with 5 nM 17 beta-estradiol led to a 50% increase in the [3H]progesterone-binding capacity of the cells, while no loss of estrogen-binding capacity occurred. These results indicate that progesterone receptors can be induced by estrogen in the rat uterus in the absence of estrogen receptor processing.     

Aging reduces the efficacy of estrogen substitution to attenuate cardiac hypertrophy in female spontaneously hypertensive rats

Clinical trials failed to show a beneficial effect of postmenopausal hormone replacement therapy, whereas experimental studies in young animals reported a protective function of estrogen replacement in cardiovascular disease. Because these diverging results could in part be explained by aging effects, we compared the efficacy of estrogen substitution to modulate cardiac hypertrophy and cardiac gene expression among young (age 3 months) and senescent (age 24 months) spontaneously hypertensive rats (SHRs), which were sham operated or ovariectomized and injected with placebo or identical doses of 17beta-estradiol (E2; 2 microg/kg body weight per day) for 6 weeks (n=10/group). Blood pressure was comparable among sham-operated senescent and young SHRs and not altered by ovariectomy or E2 treatment among young or among senescent rats. Estrogen substitution inhibited uterus atrophy and gain of body weight in young and senescent ovariectomized SHRs, but cardiac hypertrophy was attenuated only in young rats. Cardiac estrogen receptor-alpha expression was lower in intact and in ovariectomized senescent compared with young SHRs and increased with estradiol substitution in aged rats. Plasma estradiol and estrone levels were lower not only in sham-operated but surprisingly also in E2-substituted senescent SHRs and associated with a reduction of hepatic 17beta-hydroxysteroid dehydrogenase type 1 enzyme activity, which converts weak (ie, estrone) into potent estrogens, such as E2. Aging attenuates the antihypertrophic effect of estradiol in female SHRs and is associated with profound alterations in cardiac estrogen receptor-alpha expression and estradiol metabolism. These observations contribute to explain the lower efficiency of estrogen substitution in senescent SHRs.     

Estrogen restores brain insulin sensitivity in ovariectomized non-obese rats,but not in ovariectomized obese rats

Objective

We previously demonstrated that obesity caused the reduction of peripheral and brain insulin sensitivity and that estrogen therapy improved these defects. However, the beneficial effect of estrogen on brain insulin sensitivity and oxidative stress in either ovariectomy alone or ovariectomy with obesity models has not been determined. We hypothesized that ovariectomy alone or ovariectomy with obesity reduces brain insulin sensitivity and increases brain oxidative stress, which are reversed by estrogen treatment.

Materials/Methods

Thirty female rats were assigned as either sham-operated or ovariectomized. After the surgery, each group was fed either a normal diet or high-fat diet for 12 weeks. At week 13, rats in each group received either the vehicle or estradiol for 30 days. At week 16, blood and brain were collected for determining the peripheral and brain insulin sensitivity as well as brain oxidative stress.

Results

We found that ovariectomized rats and high-fat diet fed rats incurred obesity, reduced peripheral and brain insulin sensitivity, and increased brain oxidative stress. Estrogen ameliorated peripheral insulin sensitivity in these rats. However, the beneficial effect of estrogen on brain insulin sensitivity and brain oxidative stress was observed only in ovariectomized normal diet-fed rats, but not in ovariectomized high fat diet-fed rats.

Conclusions

Our results suggested that reduced brain insulin sensitivity and increased brain oxidative stress occurred after either ovariectomy or obesity. However, the reduced brain insulin sensitivity and the increased brain oxidative stress in ovariectomy with obesity could not be ameliorated by estrogen treatment.     

Estrogens act in rat hippocampus and frontal cortex to produce rapid, receptor-mediated decreases in serotonin 5-HT(1A) receptor function

Previously our laboratory has shown that 17beta-estradiol in vivo rapidly decreases R(+)-8-OH-DPAT-stimulated [(35)S]GTPgammaS binding (a measure of the initial biochemical event in the intracellular signaling pathway associated with 5-HT(1A) receptors) in the hippocampus, frontal cortex and amygdala. Studies were designed to determine if 17beta-estradiol also acts in vitro on estrogen receptors in the hippocampus and frontal cortex to decrease 5-HT(1A) receptor function. Hippocampus and frontal cortex were dissected from ovariectomized rats and incubated for up to 3 h with various estrogens and antiestrogens; membrane homogenates were prepared for R(+)-8-OH-DPAT-stimulated [(35)S]GTPgammaS binding assays. 17beta-Estradiol (10(-6) M) decreased the maximal response in the R(+)-8-OH-DPAT-stimulated [(35)S]GTPgammaS binding assay in a time-dependent manner (observed at 30, 60 and 120 min) in both hippocampus and frontal cortex. The hormone, however, did not alter the EC(50) of R(+)-8-OH-DPAT. When hippocampus and frontal cortex were incubated in graded concentrations of 17beta-estradiol for 1 h, the calculated EC(50) was approximately 2.5 x 10(-8) M in both brain regions. The nonestradiol estrogen diethylstilbestrol also decreased 5-HT(1A) receptor function while the less potent estrogens 17alpha-estradiol and estriol were inactive at 5 x 10(-8) M. The estrogen receptor antagonist ICI 182,780 potently and completely blocked the effects of 17beta-estradiol on 5-HT(1A) receptor function with an apparent K(B) of approximately 10(-9) M. These data demonstrate clearly that estrogens can act on estrogen receptors located in hippocampus and frontal cortex of ovariectomized rats to produce rapid heterologous decreases in 5-HT(1A) receptor function.     

Ovariectomy leads to a rapid increase in rat placental lactogen secretion

Serum placental lactogen (rPL) levels were measured by RIA in late pregnant rats subjected to a number of endocrine ablations. Adrenalectomy or unilateral ovariectomy had no significant effect on serum rPL levels. Hypophysectomy of the dam at midpregnancy resulted in a significant increase in serum rPL at late pregnancy. Bilateral ovariectomy of day-14 or -16 pregnant rats led to a rapid increase in serum rPL levels for 2 days followed by a gradual decrease as fetuses were reabsorbed or aborted. Adrenalectomy combined with ovariectomy led to sustained, elevated levels of serum rPL which were greater than those seen with bilateral ovariectomy alone. When progesterone (4 mg/rat X day) and estrone (500 ng/rat X day) or 17 beta-estradiol (100 or 200 ng/rat X day) were administered to ovariectomized pregnant rats, serum rPL remained elevated and the conceptuses were retained. In conclusion our studies have shown that ovarian and adrenal factors influence rPL secretion.     

Ovariectomy-induced increases in hypothalamic serotonin-1A receptor function in rats are prevented by estradiol

The present study investigated the effects of long-term estradiol withdrawal (ovariectomy) on hypothalamic serotonin-1A (5-HT(1A)) receptor signaling. Changes in neuroendocrine responses to the 5-HT(1A) agonist 8-OH-DPAT and levels of G(z) protein in the hypothalamus were used to examine 5-HT(1A) receptor signaling. Five days following ovariectomy, rats received daily injections of either 2 microg of beta-estradiol 3-benzoate or vehicle (subcutaneously) for 2, 4 or 14 days. Twenty-four hours after the last injection, and 15 min prior to sacrifice, rats were injected with (+/-)8-OH-DPAT (50 micro;g/kg, s.c.) or saline. Estradiol treatment did not alter basal corticotropin (ACTH) or oxytocin levels. Injection of (+/-)8-OH-DPAT produced significant increases in plasma ACTH and oxytocin levels. In the vehicle-treated rats, hormone responses to 8-OH-DPAT were enhanced in rats that received injections for 14 days compared with rats that received injections for either 2 or 4 days. Estradiol treatment for 4 or 14 days blunted this enhanced ACTH response to 8-OH-DPAT, whereas the oxytocin response to 8-OH-DPAT was only blunted after 14 daily injections of beta-estradiol 3-benzoate. The treatment with beta-estradiol 3-benzoate (2 microg/rat) did not reduce membrane-associated G(z) protein levels in the paraventricular nucleus of the hypothalamus. Hence, the inhibitory influence of a low dose of beta-estradiol 3-benzoate on 5-HT(1A) receptor signaling in the hypothalamus is not accompanied by a change in the levels of G(z) protein in the paraventricular hypothalamic nucleus. Results from the present study indicate a supersensitivity of 5-HT(1A) receptors after withdrawal of estradiol and suggest that estradiol suppresses 5-HT(1A) receptor signaling.     

Positive effects of 17beta-estradiol on insulin sensitivity in aged ovariectomized female rats

Aging is associated with insulin resistance, which represents a common factor in age-related diseases. We aimed to determine the role of 17beta-estradiol on insulin sensitivity and memory during aging using ovariectomized rats (2-26 months of age) treated with physiological doses of 17beta-estradiol. Our results indicate a lack of effect of 17beta-estradiol replacement on spatial memory assessed in a water maze. Conversely, estradiol treatment improved insulin sensitivity in aging rats. These data imply that relatively low doses of 17beta-estradiol may have beneficial effects on glucose homeostasis due to the protective effects of estrogen. However, estradiol treatment used in the present study did not prevent memory impairment associated with aging.     

Supplementation with vitamins E plus C or soy isoflavones in ovariectomized rats: effect on the activities of Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase and cholinesterases

Since a previous study demonstrated that ovariectomized rats present an activation of Na⁺, K⁺-ATPase and acetylcholinesterase (AChE) activities, in the present study we investigated the influence of vitamins E plus C or soy isoflavones on the effects elicited by ovariectomy on the activities of these enzyme in hippocampus of ovariectomized rats. We also determined the effect of the same compounds on the reduction of serum butyrylcholinesterase (BuChE) activity caused by ovariectomy. Female adult Wistar rats were assigned to one of the following groups: sham (submitted to surgery without removal of the ovaries) and ovariectomized. Seven days after surgery, animals were treated for 30 days with a single daily intraperitoneous injection of vitamins E (40 mg/kg) plus C (100 mg/kg) or saline (control). In another set of experiments, the rats were fed for 30 days on a special diet with soy protein or a standard diet with casein (control). Rats were sacrificed after treatments and the hippocampus was dissected and serum was separated. Data demonstrate that vitamins E plus C reversed the activation of Na⁺, K⁺-ATPase and AChE in hippocampus of ovariectomized rats. Conversely, soy protein supplementation reversed the increase of AChE activity, but not of Na⁺, K⁺-ATPase activity, caused by ovariectomized group. Neither treatment was able to reverse the reduction of serum BuChE activity. Furthermore, treatments with vitamins E plus C or soy were unable to reverse the decrease in estradiol levels caused by ovariectomy. Our findings show that the treatment with vitamins E plus C significantly reversed the effect of ovariectomy on hippocampal Na⁺, K⁺-ATPase and AChE activities. However, a soy diet that was rich in isoflavones was able to reverse just the increase of AChE. Neither treatment altered the reduction in serum BuChE activity. Taken together, these vitamins and soy may have a protective role against the possible brain dysfunction observed in some menopause women. Vitamins E plus C and soy isoflavones may be a good alternative as a novel therapeutic strategy.     

Estrogen treatment prevents osteopenia and depresses bone turnover in ovariectomized rats

Female Sprague-Dawley rats were subjected to bilateral ovariectomy (OVX) or sham surgery (control). Groups of ovariectomized (OVX) and control rats were injected daily with low, medium, or high doses of 17 beta-estradiol (10, 25, or 50 micrograms/kg BW, respectively). An additional group of OVX and control rats was injected daily with vehicle alone. All rats were killed 35 days after OVX, and their proximal tibiae were processed undecalcified for quantitative bone histomorphometry. Trabecular bone volume was markedly reduced in vehicle-treated OVX rats relative to that in control rats (12.1% vs. 26.7%). This bone loss was associated with a 2-fold increase in osteoclast surface and a 4-fold increase in osteoblast surface. The bone formation rate, studied with fluorochrome labeling, was also significantly elevated in vehicle-treated OVX rats (0.111 vs. 0.026 micron3/micron2.day). In contrast, treatment of OVX rats with the three doses of estradiol resulted in normalization of tibial trabecular bone volume and a decline in histomorphometric indices of bone resorption and formation. Our results indicate that estrogen treatment provides complete protection against osteopenia in OVX rats. The protective mechanism involves estrogenic suppression of bone turnover. These findings are consistent with the skeletal effects of estrogen therapy in postmenopausal women.     

Identification of 17 beta-estradiol as the estrogenic substance in Saccharomyces cerevisiae.

Saccharomyces cerevisiae possesses a high-affinity estrogen binding protein and an endogenous ligand that displaces [3H]estradiol from both the yeast binding protein and mammalian estrogen receptors. Semipurified preparations of this ligand have been shown to exhibit estrogenic activity in mammalian systems. We now describe the purification procedure and ultimate identification of the estrogenic substance in extracts of S. cerevisiae as 17 beta-estradiol. Organic solvent extracts of commercially obtained dried yeast were sequentially chromatographed on silica gel columns and then subjected to a series of reversed phase HPLC fractionations. Active ligand was monitored by [3H]estradiol displacement in a rat uterine cytosol assay. After seven chromatography steps, the purified and highly active ligand exhibited a single peak with retention times identical to those of 17 beta-estradiol on both HPLC and GC. The yeast material was identified as 17 beta-estradiol by its UV absorbance and mass spectrometric fragmentation pattern. In addition, radioimmunoassay confirmed the presence of approximately the same mass of 17 beta-estradiol (approximately equal to 800 ng/1.5 kg of yeast) as estimated both by a competitive binding assay with estrogen receptor and by mass spectrometry. Extraneous contamination by estradiol was excluded by repeat experiments with different batches of starting material and demonstration of estradiol by RIA in conditioned medium and cell pellets of laboratory-grown S. cerevisiae whereas non-conditioned medium did not possess the steroid. We conclude that 17 beta-estradiol is a yeast product.