High Doses Of Estradiol Research Articles

Estrogen increases hepatic lipase levels in inbred strains of mice: a possible mechanism for estrogen-dependent lowering of high density lipoprotein.

We have shown mouse to be an useful animal model for studies on the estrogen-mediated synthesis and secretion of lipoproteins since, unlike in rats, low density lipoprotein receptors are not upregulated in mice. This results into the elevation of plasma levels of apolipoprotein (apo) B and apoE, and lowering of apoA-1-containing particles. The mechanisms of apoB and apoE elevation by estrogen have been elucidated, but the mechanism of lowering of plasma levels of HDL is still not known. Among other factors, apoA-I, cholesterol ester transfer protein (CETP), scavenger receptor B1 (SR-B1), and hepatic lipase are potential candidates that modulate plasma levels of HDL. Since estrogen treatment increased hepatic apoA-I mRNA and apoA-I synthesis, and mouse express undetectable levels of CETP, we tested the hypothesis that estradiol-mediated lowering of HDL in mice may occur through modulation of hepatic lipase (HL). Four mouse strains (C57L, C57BL, BALB, C3H) were administered supraphysiological doses of estradiol, and plasma levels of HDL as well as HL mRNA were quantitated. In all 4 strains estradiol decreased plasma levels of HDL by 30%, and increased HL mRNA 2-3 fold. In a separate experiment groups of male C57BL mouse were castrated or sham-operated, and low and high doses of estradiol administered. We found 1.4-2.5 fold elevation of HL mRNA with concomitant lowering of HDL levels. Ten other mouse strains examined also showed estradiol-induced elevation of HL mRNA, but the extent of elevation was found to be strain-specific. Based on these studies, we conclude that hepatic lipase is an important determinant of plasma levels of HDL and that HL mRNA is modulated by estrogen which in turn may participate in the lowering of plasma levels of HDL.

Megakaryocyte population in human bone marrow increases with estrogen treatment: a role in bone remodeling?

Skeletal effects of conventional hormone replacement therapy (HRT) are predominately antiresorptive, while high doses of estrogen have anabolic effects. The mechanisms mediating these effects are unclear but may involve cells in the bone marrow. We have investigated the in vivo effects of estrogen on the megakaryocyte (MK) population in bone marrow in 10 postmenopausal women before and after 2 years of conventional HRT, in 11 women after long-term, high-dose estradiol therapy, and in 2 premenopausal and 4 postmenopausal women who had received no previous estrogen treatment. Transiliac crest biopsies were halved and either decalcified and paraffin wax embedded for immunolocalization studies or dehydrated and embedded in LR White resin for histology. MKs were identified morphologically, and the bone marrow cell population and MK number quantified by cell counting in a defined area of view (1 mm 2) from 5 randomly selected fields of bone marrow. Compared with pretreatment values, significantly higher MK numbers were found after conventional HRT treatment (before treatment, mean ± SEM; 7.3 ± 1.1 vs. after treatment, 18.0 ± 1.6/5 mm 2; p < 0.0001), while the greatest MK number was associated with long-term, high-dose estradiol treatment (32.8 ± 2.1/5 mm 2; p < 0.0001). Total bone marrow cell number did not differ significantly between groups. Immunolocalization studies revealed more intense estrogen receptor (ER)β expression in MKs in the high-dose estradiol-treated group but similar levels of weak ERα staining in MKs in the control and high-dose estrogen-treated groups. Positive immunoreactivity for transforming growth factor (TGF)β1, 2, and 3 and TGFβ receptor I, II, and III was detected in MKs, with more intense staining being demonstrated in the high-dose estradiol-treated group, particularly for TGFβ2 and TGFβRI and II. Our results demonstrate an increase in the MK population in bone marrow from women treated with estrogen. The ability of MKs to express ERs and synthesise TGFβ, a potent mitogen in osteoblast differentiation, suggests that these cells may play a role in mediating estrogen-induced effects on bone.

Induction of glucose-regulated protein 78 in rat uterine glandular epithelium during uterine sensitization for the decidual cell reaction.

Endometrial receptivity for implantation and sensitization for decidualization in rodents is a transient state under the control of the ovarian steroids estrogen and progesterone. It is unclear, however, what molecular events mediate the onset of uterine receptivity. Messenger RNA differential display was performed on endometrial RNA from ovariectomized rats differentially sensitized for decidualization. Maximally sensitized uteri were at the equivalent of Day 5 of pseudopregnancy, and temporally nonsensitized uteri at Day 4 or 6; hormonally nonsensitized uteri were from animals on Day 5 treated with low or high doses of estradiol on Day 4. A cDNA with endometrial expression restricted to maximally sensitized uteri was isolated, cloned, and sequenced. The cDNA matched the sequence for glucose-regulated protein 78 (GRP78), a heat shock 70-related protein that resides in the lumen of the endoplasmic reticulum (ER) and has roles in several cellular processes including multimeric protein assembly, the degradation of proteins, and the storage and regulation of ER luminal calcium. Northern blot analysis indicated a dramatic increase in GRP78 mRNA levels restricted to the sensitized, Day 5 endometrium, suggesting a role in the onset of the sensitized phase. In situ hybridization and immunohistochemistry experiments localized the up-regulation of GRP78 within the receptive endometrium to the glandular epithelium.

Effects of oestrogen upon nitric oxide synthase NADPH-diaphorase activity in the hypothalamo–neurohypophysial system of the rat

An understanding of the interaction between oestrogen and the nitric oxide synthase/nitric oxide system is important for determining the roles of nitric oxide in central nervous control of osmotic homeostasis and certain aspects of reproduction. The effects of oestrogen on nitric oxide synthase and nitric oxide synthase activity were investigated in the magnocellular neurosecretory system. Ovariectomized female rats were injected subcutaneously with 17β-estradiol benzoate either 10 μg daily for 4 days (short-term low-dose) or 200 μg daily for 21 days (long-term high-dose). In the neurohypophysis the density of NADPH-diaphorase staining—a marker for nitric oxide synthase activity—was increased after both short-term low-dose and long-term high-dose estradiol treatment, but no difference in nitric oxide synthase immunoreactivity was observed after either treatment. In the magnocellular supraoptic and paraventricular nuclei, short-term low-dose oestrogen treatment did not induce any detectable changes in nitric oxide synthase gene expression, the proportion of nitric oxide synthase-immunoreactive neurons, or the proportion of NADPH-diaphorase-positive neurons. Long-term high-dose oestrogen treatment also had no effect on nitric oxide synthase gene expression or immunoreactivity, but caused a reduction of the proportion of NADPH-diaphorase-positive neurons in the supraoptic nucleus and a reduction in the intensity of this histochemical staining. Qualitatively similar changes were observed in the magnocellular part of the paraventricular nucleus. The results provide, for the first time, evidence of a complex interaction between oestrogen and nitric oxide synthase in the neuroendocrine system in which nitric oxide synthase activity is regulated differently in the magnocellular cell bodies and axonal terminals and in which the activity of the enzyme rather than its expression is controlled.

Secretion of plasminogen activator by cultured rat endometrial stromal cells from uteri differentially sensitized for the decidual cell reaction.

Endometrial stromal cells from rat uteri differentially sensitized for the decidual cell reaction in vivo and which undergo differing degrees of decidualization in vitro were cultured and plasminogen activator (PA) in the medium determined. The cells were obtained by enzymatic dispersion from the uteri of ovariectomized, steroid-treated rats at the equivalent of day 4, 5, or 6 of pseudopregnancy or on day 5 from rats treated on day 4 with 0, 0.3, or 1.0 microgram estradiol (low, intermediate, or high dose of estradiol, respectively) and cultured for 24, 48, or 72 hr. For cells from day 4, 5, and 6 uteri cultured under control conditions, PA activity in the medium was greatest for day 5 cells, which were from uteri maximally sensitized for decidualization both in vivo and in vitro. By contrast, for cells from low-, intermediate-, and high-estradiol uteri, PA activity in the medium was greatest for the high-estradiol cells; these cells do not undergo decidualization in vivo or in vitro to the same extent as intermediate-estradiol cells. Indomethacin, an inhibitor of prostaglandin (PG) synthesis, reduced PGE2 accumulation to nondetectable amounts and for most cultures decreased PA activity in the medium, suggesting that endogenous PG production regulated in part PA secretion under control conditions. The addition of PGE2 with indomethacin increased PA activities above those under control conditions, but activities were still lower for day 4 and 6 cells compared with day 5 cells, and for low- and intermediate-estradiol cells compared with high-estradiol cells. This indicates that the differences in PA secretion are not explainable by differences in PGE2 production. Northern blot analysis of RNA from cells cultured for 72 hr under control conditions did not reveal significant differences in steady-state concentrations of mRNA for urokinase-type PA or plasminogen activator inhibitor 1, but those for tissue-type PA were lower in day 6 cells compared with day 4 and 5 cells. It is concluded that PA activity secreted by the cultured endometrial stromal cells, although controlled in part by the endocrine milieu to which they were exposed prior to culture, does not simulate decidualization in vitro and, therefore, that PA activity is not a marker for decidualization in vitro.

Reproductive Behavioral Responsiveness to Noradrenergic Stimulation in Developing Guinea Pigs

The stimulatory effects of ovarian hormones on sexual receptivity in guinea pigs may be mediated by norepinephrine. Juvenile females rarely exhibit steroid-induced receptivity and also respond poorly to the lordosis-enhancing action of α-noradrenergic receptor stimulation. This experiment was designed to chart the development of behavioral responsiveness to the α-noradrenergic agonist, clonidine, and to test the hypothesis that higher doses of estradiol and/or clonidine are required to stimulate lordosis in juvenile compared to adult guinea pigs. Ovariectomized females received estradiol benzoate (10 μg or 50 μg SC) 40–48 h before administration of clonidine (1 mg/kg or 5 mg/kg IP) or saline at 14–17-day intervals. Regardless of treatment, few animals (0–36%) displayed lordosis at 20, 34, or 48 days of age. At 65 days of age, in both estradiol dose groups significantly more clonidine- (1 mg/kg) than saline-injected animals displayed lordosis (80–91 vs. 0–33%, respectively). Clonidine (5 mg/kg) was ineffective at all ages. These data do not support the hypothesis that behavioral responsiveness to α-noradrenergic receptor stimulation in immature females can be elicited by increasing the doses of estradiol and/or clonidine. These results suggest the occurrence of a maturational change in the neural systems governing noradrenergic involvement in steroid-induced sexual behavior in guinea pigs.

Effects of estradiol and an aromatase inhibitor on progesterone production in human cultured luteal cells

The human corpus luteum produces both estradiol and progesterone. It is well known that there are both autocrine and paracrine systems for the regulation of the corpus luteum and that estradiol regulates the progesterone production of the corpora lutea of some other species. To assess the direct effects of estrogen on human luteal function, we performed cell culture experiments.A low concentration of estradiol, almost equal to the amount of estradiol produced by human cultured luteal cells, directly stimulated progesterone production. 4-Cyclohexylaniline, an aromatase inhibitor, significantly reduced both progesterone production and estradiol production.Levels of estradiol higher than the levels that cultured human luteal cells themselves produced significantly reduced basal progesterone production and also significantly reduced human chorionic gonadotropin (hCG), forskolin and dibutyryl-cyclic AMP-stimulated progesterone production. According to these data, high doses of estradiol produced a luteolytic action which widely inhibited the steroidogenesis process.In conclusion, our results indicated that estradiol in part regulates progesterone production physiologically and blocks progesterone production in a pharmacological or pathological state in the human corpus luteum.

Effect of Estradiol on Serum Triglyceride Lipoprotein Levels and Fatty Acid Composition in Castrated Rats

Triglyceride content and fatty acid composition of rat serum lipoproteins showed specific variations after castration and estradiol treatment. Triglyceride levels decreased in VLDL after castration and in LDL and HDL after low doses of estradiol. High doses of estradiol enhanced triglyceride levels in VLDL and decreased them in LDL. Fatty acid composition showed a complex pattern: after castration, monoenoic acids decreased and essential fatty acids increased in all lipoprotein classes, enhancing the EFA/NEFA and EFA/ME ratios. Both doses of estrogen lowered these ratios in VLDL and LDL, but decreased them in HDL with high doses and enhanced them in HDL with low doses.

A surprisingly ameliorating effect of high doses of estradiol in primary osteomyelofibrosis: a case report

Objectives: To report a case of a postmenooausal woman with primary (idiopathic) osteomyelofibrosis who showed a considerable improvement under the effect of high doses of estradiol given for the control of severe intractable menopausal symptoms. Methods: This study was carried out by a case report. Results: Astonishingly, under the effect of high doses of estradiol, both clinical and laboratory indices of the disease were not only kept stable but also showed a noticeable improvement. Hemoglobin increased from a value of 8.4 gm/dl in 1989 to 10.7 gm/dl in 1996. Blood transfusion was no longer necessary, the patient had had no infections whatsoever and was only afraid of not being allowed to continue using these high estrogen doses, which seemed indispensable and central to her well-being. Conclusions: High doses of estrogens could possibly be given the credit for ameliorating the natural sinister course of primary osteomyelofibrosis.

Effects of Estradiol on the Serotonin Secretion and Turnover in the Hypothalamus of Male Tilapia,Oreochromis mossambicus, in Vitro

The effects of estradiol on the secretion and turnover of serotonin in the hypothalamic fragments of male tilapia,Oreochromis mossambicus,were studied using a static incubation system. The quantitative analysis of serotonin and its related metabolite, 5-hydroxyindoleacetic acid, were performed by high-performance liquid chromatography with electrochemical detection. The hypothalamic fragments were incubated with 17β-estradiol at a concentration of 2 × 10−8, 8 × 10−8, 2 × 10−7, 4 × 10−7, or 4 × 10−6g/ml. The low dose of estradiol, 2 × 10−8g/ml, had no effect on the concentration of serotonin and 5-hydroxyindoleacetic acid or serotonin turnover in the hypothalamic incubation media. The moderate doses of estradiol 8 × 10−8and 2 × 10−7g/ml, increased the concentrations of serotonin and 5-hydroxyindoleacetic acid in the hypothalamic incubation media, but had no effect on the serotonin turnover. The high doses of estradiol, 4 × 10−7and 4 × 10−6g/ml, did not alter the serotonin concentration in the hypothalamic incubation media, but increased the 5-hydroxyindoleacetic acid concentration and serotonin turnover. These results demonstrate that the moderate dose of estradiol increases the serotonin activity by increasing the serotonin concentration, whereas the high dose of estradiol increases the serotonin activity by increasing the ratio of 5-hydroxyindoleacetic acid and serotonin. However, the serotonin concentration is homeostatically maintained in the extracellular fluid of hypothalamus under the high dose of E2treatment.

Long-term estrogen therapy abolishes acute estrogen-induced coronary flow augmentation in postmenopausal women

Postmenopausal estrogen replacement therapy (ERT) may reduce the clinical manifestations of coronary heart disease by favorably modulating coronary vasoreactivity. Intravenous ethinyl estradiol acutely increases coronary flow in postmenopausal women not receiving ERT. Because several vasoactive agents induce vasomotor tolerance when administered on a long-term basis, we hypothesized that long-term ERT attenuates the acute coronary vasomotor effects of intravenous ethinyl estradiol. To test this hypothesis, coronary hemodynamics were determined before and 15 minutes after intravenous ethinyl estradiol (35 μg) in 10 postmenopausal women who were receiving long-term conjugated ERT (group 1) and 10 who had never received ERT (group 2). Estradiol administration in group 1 was not associated with significant changes in coronary flow or resistance. However, women in group 2 exhibited a 28.6% ± 6.5% ( p < 0.001) increase in coronary flow and a 19.9% ± 3.5% ( p = 0.008) decrease in resistance. These results demonstrate that long-term ERT significantly attenuates the response of coronary arteries to the acute vasomotor effects of a high dose of estradiol. This response may be caused by long-term estrogen-induced coronary flow augmentation or to the development of vasomotor tolerance to estrogen. (Am Heart J 1997;133:323-8.)

Temporal- and hormone-dependent changes in uterine sensitization for the decidual cell reaction and decidualization in vitro of rat endometrial stromal cells.

The ability of endometrial stromal cells from nonsensitized rat uteri to undergo decidualization in vitro was investigated. Cells were obtained by enzymatic dispersion from uteri of ovariectomized, steroid-treated rats at the equivalent of day 4, 5 or 6 of pseudopregnancy, or on day 5 from rats treated with 0, 0.3 or 1.0 microgram oestradiol (low, intermediate or high doses of oestradiol, respectively) on day 4, and cultured for 24, 48 or 72 h. Decidualization in vivo, as assessed by uterine mass 5 days after the unilateral intrauterine injection of 100 microliters sesame oil, was maximal for rats receiving the deciduogenic stimulus on day 5 and treated with the intermediate dose of oestradiol. Under control conditions in vitro, alkaline phosphatase (ALP) activity, the increase in ALP activity with time, and prostaglandin E2 (PGE2) accumulation in the medium were greatest for cells from maximally sensitized uteri. Indomethacin, an inhibitor of PG synthesis, reduced PGE2 accumulation to barely detectable amounts, and decreased ALP activity, especially in cells from maximally sensitized uteri, indicating that endogenous PG production contributed to the increase in ALP activity in these cells. The addition of PGE2 with indomethacin increased ALP activities. However, ALP activities were lower for cells derived from nonsensitized uteri when compared with cells from maximally sensitized uteri. These results suggest that endometrial stromal cells from nonsensitized uteri have a reduced capacity to undergo decidualization in vitro, and that this reduced capacity is not explained by differences in PGE2 production.

Continuous exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin inhibits the growth of surgically induced endometriosis in the ovariectomized mouse treated with high dose estradiol.

The objectives of this study were to evaluate the effects of repeated injection of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the growth of endometriosis and the formation of adhesions in the mouse. Sexually mature female B6C3F1 mice (n = 19) were ovariectomized and subcutaneously implanted with a silastic capsule containing estradiol (E2). Uterine tissues in full thickness (I x I mm2, 5 pieces) from the left uterine horn were surgically autotransplanted to 5 different locations in the peritoneal cavity. One week later the animals were randomly assigned to four groups and treated with: vehicle alone (corn oil). 10.50 or 100 ng/kg/day TCDD for 28 days. Two days after last dose a necropsy was performed to measure the diameter of endometrial implants and to score the adhesions. Endometrial implants were harvested for histology and blood was collected for assessment of E2 levels. The diameter of endometrial implants was significantly smaller in the TCDD treated groups compared to the controls (p < 0.05). Survival of endometrial implants, the formation and scores of adhesions, and serum E2 levels were not significantly different among the dosing groups. Histology of implants showed a significant regression of endometrial tissues in the animals receiving 50 and 100 ng/kg/day TCDD compared to the controls. We conclude that continuous exposure to TCDD resulted in regression of the endometriotic implants in the ovariectomized mice treated with a high dose exogenous F2.

Regulation of oxytocin, oestradiol and progesterone receptor concentrations in different uterine regions by oestradiol, progesterone and oxytocin in ovariectomized ewes.

The regulation of oxytocin, oestradiol and progesterone receptors in different uterine cell types was studied in ovariectomized ewes. Animals were pretreated with a progestogen sponge for 10 days followed by 2 days of high-dose oestradiol to simulate oestrus. They then received either low-dose oestradiol (Group E), low-dose oestradiol plus progesterone (Group P) or low-dose oestradiol, progesterone and oxytocin (via osmotic minipump; Group OT). Animals (three to six per time-point) were killed following ovariectomy (Group OVX), at oestrus (Group O) or following 8, 10, 12 or 14 days of E, P or OT treatment. In a final group, oxytocin was withdrawn on day 12 and ewes were killed on day 14 (Group OTW). Oxytocin receptor concentrations and localization in the endometrium and myometrium were measured by radioreceptor assay, in situ hybridization and autoradiography with the iodinated oxytocin receptor antagonist d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2(9)]-vasotocin. Oestradiol and progesterone receptors were localized by immunocytochemistry. Oxytocin receptors were present in the luminal epithelium and superficial glands of ovariectomized ewes. In Group O, endometrial oxytocin receptor concentrations were high (1346 +/- 379 fmol [3H]oxytocin bound mg protein-1) and receptors were also located in the deep glands and caruncular stroma in a pattern resembling that found at natural oestrus. Continuing low-dose oestradiol was unable to sustain high endometrial oxytocin receptor concentrations with values decreasing significantly to 140 +/- 20 fmol mg protein-1 (P < 0.01), localized to the luminal epithelium and caruncular stroma but not the glands. Progesterone treatment initially abolished all oxytocin receptors with none present on days 8 or 10. They reappeared in the luminal epithelium only between days 12 and 14 to give an overall concentration of 306 +/- 50 fmol mg protein-1. Oxytocin treatment caused a small increase in oxytocin receptor concentration in the luminal epithelium on days 8 and 10 (20 +/- 4 in Group P and 107 +/- 35 fmol mg protein-1 in Group OT, P < 0.01) but the rise on day 14 was not affected (267 +/- 82 in Group OT and 411 +/- 120 fmol mg protein-1 in Group OTW). In contrast, oestradiol treatment was able to sustain myometrial oxytocin receptors (635 +/- 277 fmol mg protein-1 in Group O and 255 +/- 36 in Group E) and there was no increase over time in Groups P, OT and OTW with values of 61 +/- 18, 88 +/- 53 and 114 +/- 76 fmol mg protein-1 respectively (combined values for days 8-14). Oestradiol receptor concentrations were high in all uterine regions in Group O. This pattern and concentration was maintained in Group E. In all progesterone-treated ewes, oestradiol receptor concentrations were lower in all regions at all time-points. The only time-related change occurred in the luminal epithelium in which oestradiol receptors were undetectable on day 8 but developed by day 10 of progesterone treatment. Progesterone receptors were present at moderate concentrations in the deep glands, caruncular stroma, deep stroma and myometrium in Group O. Oestradiol increased progesterone receptors in the luminal epithelium, superficial glands, deep stroma and myometrium. Progesterone caused the loss of its own receptor from the luminal epithelium and superficial glands and decreased its receptor concentration in the deep stroma and myometrium at all time-points. There was a time-related loss of progesterone receptors from the deep glands of progesterone-treated ewes between days 8 and 14. These results show differences in the regulation of receptors between uterine regions. In particular loss of the negative inhibition by progesterone on the oxytocin receptor by day 14 occurred only in the luminal epithelium, but is unlikely to be a direct effect of progesterone as no progesterone receptors were present on luminal epithelial cells between days 8 and 14.

Estradiol treatment transiently increases trabecular bone volume in ovariectomized rats

The effect of short-term estradiol treatment, administered from the time of ovariectomy, on increased bone turnover and subsequent bone loss was studied in the rat. Adult female Sprague-Dawley rats were ovariectomized and administered daily subcutaneous (s.c.) injections of 17β-estradiol at 8 μg/ kg per day (Low) and 20 μg/kg per day (High) or vehicle alone (Veh). Femoral trabecular bone volume (BV/TV) and trabecular number (Tb.N) in the distal femur were transiently increased at 6 days postoperation in a dose-dependent manner following estradiol administration [mean ± SEM: BV/TV (%), day 0, 6.6 ± 0.2; day 6, Veh 7.8 ± 0.4, Low 10.2 ± 2.2, High 12.8 ± 1.7 ( p < 0.05); Tb.N (/mm), day 0, 2.30 ± 0.24; day 6, Veh 2.89 ± 0.33, Low 3.4 ± 0.7, High 4.39 ± 0.34 ( p < 0.05)]. Estradiol prevented the ovariectomy-induced decrease in BV/TV and Tb.N between 9 and 15 days observed in Veh rats. Both serum alkaline phosphatase and urine hydroxyproline excretion were maintained at preoperative levels or lower from day 6 postoperation with high dose estradiol. Serum osteocalcin, however, rose above preoperative levels with estradiol at days 6 and 9, but returned to these values on days 15 and 21 postoperation. These results suggest that estradiol, administered from the time of ovariectomy, immediately suppressed markers associated with osteoblast proliferation/matrix synthesis and bone resorption. Mineralization does not appear to be so rapidly suppressed by estradiol with relatively high levels immediately following administration, resulting in a transient increase in trabecular bone volume and trabecular number.

Prevention of bone loss by percutaneous estradiol implants in ovariectomized rats

This study was conducted to investigate whether hydroxyapatite (HAP) is appropriate as a percutaneous drug carrier for estradiol (E2) for the suppression of bone loss. Ten-week-old female Sprague-Dawley rats were subjected either to bilateral ovariectomy (OVX) or to sham surgery (control). Ovariectomized rats were implanted percutaneously with E2-HAP disks containing low, medium or high doses of estradiol (50, 250, or 500 micrograms E2/rat, respectively). Ovariectomized rats without implant and OVX rats implanted only with HAP served as additional controls. All rats were sacrificed 90 days after surgery. At the end of the experiment, bone mineral density of the lumbar spine was measured by dual energy X-ray absorption, and serum E2 was assayed by radioimmunoassay. The bone mineral density of OVX and HAP-treated OVX rats decreased by 18% compared to sham surgery rats, but decreased by only 13, 7, and 3% in rats treated with 50, 250, and 500 micrograms E2/rat, respectively. The in vitro release of E2 from E2-HAP devices was determined by an HPLC method. Estradiol release from the HAP devices followed almost a zero-order kinetics. Estradiol remained intact in E2-HAP implants for up to six months when stored at 5, 25, and 40 degrees C. This study indicates that E2-HAP implants are effective in suppressing bone loss in the spine of OVX rats in a dose-dependent manner.

Progesterone enhances an estradiol-induced increase in Fos immunoreactivity in localized regions of female rat forebrain

In female rats, the onset of reproductive behavior depends on the sequential presence of estradiol followed by progesterone. Although treatment with high doses of estradiol has been shown to increase immunostaining for the Fos protein, an immediate early gene product that is expressed upon cellular activation, another report conflicts with this finding. However, the previous reports agree that subsequent treatment with progesterone has no apparent effect on Fos expression. In order to resolve this discrepancy and investigate possible effects of progesterone, we used Fos immunocytochemistry combined with computer-aided image analysis. In experiment one, we found that treatment with 5 micrograms of estradiol increased Fos immunoreactivity (Fos-IR) within a section of the medial preoptic area and the dorsal medial hypothalamus. Subsequent treatment with 500 micrograms of progesterone 1 hr before perfusion increased the intensity of the immunostaining within the medial preoptic area and the dorsal medial hypothalamus, although it had no significant effect on Fos-IR cell number. In experiment 2, a lower concentration of Fos antiserum was used in order to diminish the immunostaining sensitivity to a level in which no increase of Fos-IR cell number was observed after treatment with estradiol. Under these immunocytochemical conditions, subsequent treatment with progesterone increased the number of Fos-IR cells in the medial preoptic area, the dorsal medial hypothalamus and the steroid receptor-rich area lateral to the ventromedial hypothalamus. Thus, treatment with behaviorally effective doses of both estradiol and progesterone induces Fos expression in localized regions of female rat brain.

Breast cancer risk with oral contraceptives and oestrogen replacement therapy.

A detailed review of the international epidemiological literature is very reassuring in confirming that exogenous oestrogen in oral contraceptives and postmenopausal hormone replacement therapy does not induce breast cancer in women. However, there may sometimes be a promotional effect which accelerates the time of breast cancer diagnosis in certain subgroups of women. This promotional effect may occur in young oral contraceptive users, and in long term users of high dose oestradiol postmenopausal replacement formulations. Use of oral contraceptives while young may protect against the development of breast cancer after age 45. Physiological doses of conjugated equine oestrogens do not appear to increase breast cancer risk even with long-term postmenopausal use. Current data do not support an effect of progestogens in increasing or decreasing risk of breast cancer.

Cellular mechanisms of estrogen- and dopamine-induced control of glandular kallikrein in the anterior pituitary of the rat.

Glandular kallikrein (GK, a trypsin-like serine protease) exhibits estrogen induction and dopamine repression in rat pituitary lactotrophs. Steroid induction may reflect primary actions to increase selectively the synthesis of specific proteins, or may be part of broad cellular responses secondary to steroid-induced phenotype transitions. This study examined the cellular mechanisms underlying estrogen and dopaminergic control of lactotroph GK using a quantified immunocytochemical approach. Pituitaries from ovariectomized rats exhibited little GK staining. Estradiol treatment for 10 days produced dose-dependent increases in pituitary mass, the percentage of lactotrophs (indicating lactotroph proliferation) and the percentage of GK-positive cells. Also, GK staining intensity was dependent upon estradiol dose, increasing 4-fold between 5 micrograms and 50 micrograms/48 h. Dopamine receptor blockade with haloperidol (2.5 mg/kg/24 h) elicited weak GK immunostaining in 46% of the lactotrophs in the absence of estradiol, and markedly potentiated GK staining intensity elicited with low but not high doses of estradiol. The results suggest that GK induction is a primary estrogen effect, and is not secondary to a phenotype transition: the induction is enhanced by estrogen-induced lactotroph proliferation. Dopaminergic systems strongly inhibit GK induction by low estradiol levels. This dopaminergic modulation may shift the induction of lactotroph GK to physiological events associated with high estradiol levels or low dopaminergic tone.

Estradiol-Induced Knee Osteoarthrosis in Ovariectomized Rabbits

Experiments in animals have shown that estrogen is chondrodestructive. The existence of 17 beta-estradiol receptor in rabbit chondrocyte and canine cartilage suggests that estrogen is associated with the development of osteoarthrosis (OA). The increased frequency of knee OA in obese postmenopausal women, who are often associated with hyperestrogenism, suggests a link between estradiol and OA. The pathologic changes induced by nine and 12 weeks of intraarticular injection of high doses of estradiol (0.3 mg/kg body weight/day) and low doses of estradiol (0.06 mg/kg body weight/day) into knee joints of ovariectomized rabbits have been examined. In the high-dose group, loss of condyle surface congruity, thinning, fissuring, and fibrillation of the remaining cartilage surface were observed at Week 9. At Week 12, cartilage erosion extended to the calcified layer, exposing the subchondral bone. Scanning electron microscopy (SEM) further revealed numerous pits, which indicated formation of cysts on the cartilage surface. Injection of low dose estradiol, conversely, did not induce significant pathologic changes. The results demonstrated that the direct interaction of estradiol and rabbit cartilage was dose- and duration-dependent. Pathologic changes from the current animal model of knee OA closely resembled those of knee OA in humans. Thus, this model constitutes a potential tool for further studies of the early pathologic changes of OA and the possible prevention of cartilage degeneration by estrogen receptor inhibitors.