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PubMed2005

Sex differences in hippocampal estradiol-induced N-methyl-D-aspartic acid binding and ultrastructural localization of estrogen receptor-alpha.

Estradiol increases dendritic spine density and synaptogenesis in the CA1 region of the female hippocampus. This effect is specific to females, as estradiol-treated males fail to show increases in hippocampal spine density. Estradiol-induced spinogenesis in the female is dependent upon upregulation of the N-methyl-D-aspartic acid (NMDA) receptor as well as on non-nuclear estrogen receptors (ER), including those found in dendrites. Thus, in the male, the inability of estradiol to induce spinogenesis may be related to a failure of estradiol to increase hippocampal NMDA receptors as well as a paucity of dendritic ER. In the first experiment, we sought to investigate this possibility by assessing NMDA receptor binding, using [(3)H]-glutamate autoradiography, in estradiol-treated males and females. We found that while estradiol increases NMDA binding in gonadectomized females, estradiol fails to modulate NMDA binding in gonadectomized males. To further investigate sex differences in the hippocampus, we conducted a second separate, but related, ultrastructural study in which we quantified ERalpha-immunoreactivity (ERalpha-ir) in neuronal profiles in the CA1 region of the hippocampus in intact males and females in diestrus and proestrus. Consistent with previous reports in the female, we found ERalpha-ir in several extranuclear sites including dendrites, spines, terminals and axons. Statistical analyses revealed that females in proestrus had a 114.3% increase in ERalpha-labeled dendritic spines compared to females in diestrus and intact males. Taken together, these studies suggest that both the ability of estrogen to increase NMDA binding in the hippocampus and the presence of ERalpha in dendritic spines may contribute to the observed sex difference in estradiol-induced hippocampal spinogenesis.

Animals↗
PubMed1991

Estradiol rapidly stimulates dopamine release from the posterior pituitary in vitro.

Dopamine (DA) from both the posterior pituitary (PP) and stalk-median eminence (SME) inhibits prolactin (PRL) secretion from the anterior pituitary. Estradiol participates in the regulation of PRL release, in part by modulating DA release from the SME. However, little is known concerning the effects of estradiol on the release of DA from the PP. The objective of this study was to examine whether estradiol rapidly affects the potassium-evoked release of endogenous DA from the PP and SME in vitro. Tissues were dissected from ovariectomized rats and allowed to equilibrate in media for 30 min. Two pulses of 28 mM K+, 3 min each, were then given 30 min apart. Test substances were added 20 min before the second stimulus. DA in the media was determined by HPLC. Estradiol, at a concentration of 1 and 10 nM, significantly stimulated the potassium-evoked DA release from the PP by 34 and 47%, respectively. This stimulation was specific since 17 alpha-estradiol, a biologically inactive isomer, and testosterone, were without effects. Estradiol did not alter DA release from either the SME or isolated neural lobes of the PP. Naloxone, an opioid receptor antagonist, abolished the estradiol-induced stimulation of DA release from the PP. In contrast, amphetamine, a DA-releasing agent, significantly increased DA release in the presence of naloxone. In conclusion, (1) estradiol stimulates DA release from the PP but not the SME or neural lobe; this effect is rapid and stereospecific, and (2) the effects of estradiol appear to be mediated via an opioid(s) peptide(s) from the intermediate lobe.

Amphetamine↗
PubMed1997

Short-term anti-ischemic effect of 17beta-estradiol in postmenopausal women with coronary artery disease.

BACKGROUND: Short-term administration of 17beta-estradiol improves effort-induced myocardial ischemia in female patients with coronary artery disease. 17Beta-estradiol also has direct and indirect coronary vascular smooth muscle relaxing properties. The aim of the present study was to evaluate the effect of short-term administration of 17beta-estradiol on pacing-induced myocardial ischemia by means of continuous monitoring of coronary sinus pH in 16 postmenopausal female patients with coronary artery disease. METHODS AND RESULTS: Patients underwent incremental atrial pacing starting at a rate of 100 bpm and increments of 20 bpm every 2 minutes up to 160 bpm before and 20 minutes after either 17beta-estradiol (1 mg sublingual, 9 patients) or placebo (sublingual, 7 patients). The time to the onset of myocardial ischemia during pacing was significantly increased by 17beta-estradiol (mean+/-SD, 254+/-36 versus 298+/-23 seconds; P<.02) but not by placebo (262+/-45 versus 256+/-34 seconds; P=NS) The pH shift was significantly reduced by 17beta-estradiol but not by placebo at every step of the pacing protocol. The maximum pH shift at peak pacing was significantly reduced by the administration of 17beta-estradiol by 0.022 pH units (95% CI, 0.001, 0.043; P<.04) but not by sublingual placebo (-0.002 pH units; 95% CI, -0.0073, 0.0021; P=NS). The maximum pH shift at maximum comparable pacing was also reduced by 17beta-estradiol by 0.015 pH units (95% CI, 0.012, 0.017; P<.001) but not by placebo (-0.0022 pH units; 95% CI, -0.006, 0.0015; P=NS). CONCLUSIONS: 17Beta-estradiol reduces the degree of pacing-induced myocardial ischemia in postmenopausal patients with coronary artery disease. The reduction of pacing-induced coronary sinus pH shift is consistent with an anti-ischemic effect of the hormone and is not due to preconditioning, as evidenced by the absence of improvement after placebo.

Cardiac Pacing, Artificial↗
PubMed1998

Estradiol therapy combined with progesterone and endothelium-dependent vasodilation in postmenopausal women.

BACKGROUND: Epidemiological studies indicate that estrogen replacement therapy decreases the risk of cardiovascular events in postmenopausal women. Estrogen may confer cardiovascular protection by improving endothelial function because it increases endothelium-dependent vasodilation. It is not known whether progesterone attenuates the beneficial effects of estrogen on endothelial function. METHODS AND RESULTS: Seventeen postmenopausal women with mild hypercholesterolemia were enrolled in a placebo-controlled, crossover trial to evaluate the effect of transdermal estradiol, with and without vaginal micronized progesterone, on endothelium-dependent vasodilation in a peripheral conduit artery. Brachial artery diameter was measured with high-resolution B-mode ultrasonography. To assess endothelium-dependent vasodilation, brachial artery diameter was determined at baseline and after a flow stimulus induced by reactive hyperemia. To assess endothelium-independent vasodilation, brachial artery diameter was measured after administration of sublingual nitroglycerin. During estradiol therapy, reactive hyperemia caused an 11.1+/-1.0% change in brachial artery diameter compared with 4. 7+/-0.6% during placebo therapy (P<0.001). Progesterone did not significantly attenuate this improvement. During combined estrogen and progesterone therapy, flow-mediated vasodilation of the brachial artery was 9.6+/-0.8% (P=NS versus estradiol alone). Endothelium-independent vasodilation was not altered by estradiol therapy, either with or without progesterone, compared with placebo. There was a modest decrease in total and LDL cholesterol during treatment both with estradiol alone and when estradiol was combined with progesterone (all P<0.001 versus placebo). In a multivariate analysis that included serum estradiol, progesterone, total and LDL cholesterol concentrations, blood pressure, and heart rate, only the estradiol level was a significant predictor of endothelium-dependent vasodilation. CONCLUSIONS: The addition of micronized progesterone does not attenuate the favorable effect of estradiol on endothelium-dependent vasodilation. The vasoprotective effect of hormone replacement therapy may extend beyond its beneficial actions on lipids.

Aged↗
PubMed2001

Effects of estradiol and its metabolites on glomerular endothelial nitric oxide synthesis and mesangial cell growth.

Reduced nitric oxide synthesis by glomerular endothelial cells and increased proliferation of glomerular mesangial cells is associated with glomerular remodeling that leads to accelerated glomerulosclerosis. Estradiol induces nitric oxide synthesis and slows the progression of renal disease. Because the estradiol metabolites 2-hydroxyestradiol and 2-methoxyestradiol are more potent than estradiol in inhibiting growth of vascular smooth muscle cells, which are phenotypically similar to mesangial cells, we compared the effects of estradiol, 2-hydroxyestradiol, and 2-methoxyestradiol on growth of glomerular mesangial cells and on basal nitric oxide synthesis by glomerular endothelial cells. In human glomerular mesangial cells, estradiol and its metabolites concentration-dependently (1 nmol/L to 10 micromol/L) inhibited serum (2.5%)-induced DNA synthesis, cell proliferation, and collagen synthesis with the order of potency being 2-methoxyestradiol > 2-hydroxyestradiol > estradiol. ICI182780 (100 micromol/L, an estrogen receptor antagonist) blocked the growth inhibitory effects of estradiol but not 2-hydroxyestradiol or 2-methoxyestradiol. Treatment with estradiol, but not 2-hydroxyestradiol and 2-methoxyestradiol, induced nitric oxide synthesis (P<0.05, assayed by the formation of (3)H-L-citrulline from (3)H-L-arginine) in human glomerular endothelial cells, and these effects were blocked by ICI182780 and L-NMA (a nitric oxide synthesis inhibitor). In conclusion, estradiol may attenuate glomerulosclerosis by inducing nitric oxide synthesis via an estrogen receptor-dependent mechanism and by conversion to 2-hydroxyestradiol and 2-methoxyestradiol, which inhibit glomerular mesangial cell proliferation independent of estrogen receptors.

2-Methoxyestradiol↗
PubMed2005

Neonatal androgenization of hypogonadal (hpg) male mice does not abolish estradiol-induced FSH production and spermatogenesis.

BACKGROUND: Testicular development is arrested in the hypogonadal (hpg) mouse due to a congenital deficiency in hypothalamic gonadotropin-releasing hormone (GnRH) synthesis. Chronic treatment of male hpg mice with estradiol induces FSH synthesis and secretion, and causes testicular maturation and qualitatively normal spermatogenesis. As estradiol negative feedback normally inhibits FSH production in the male, this study tested whether this paradoxical response to estradiol in the male hpg mouse might be due to inadequate masculinisation or incomplete defeminization in the neonatal period. Previous studies have demonstrated that treatment of hpg mice with testosterone propionate in the immediate neonatal period is necessary to allow full reproductive behaviors to be expressed following suitable endocrine stimulation at adult ages. METHODS: Hpg mice were treated with 100 mug testosterone propionate or vehicle on postnatal day 2. At 35 days of age, subgroups of these mice were treated with silastic implants containing estradiol or cholesterol. Reproductive behavior was scored in tests with steroid-primed female mice, then testicular development was assessed histologically, and measures of pituitary FSH content made at 85 days of age. RESULTS: The neonatal testosterone propionate treatment successfully defeminized female litter mates, as revealed by impaired vaginal opening and deficiencies in lordosis behavior, and it allowed appropriate male reproductive behavior to be expressed in a proportion of the hpg males when tested at an adult age. However, neonatal androgen supplementation did not block or even reduce the subsequent actions of estradiol in increasing pituitary FSH content, nor did it affect the ability of estradiol to induce qualitatively normal spermatogenesis. CONCLUSION: The ability of the hpg male to show a "female" neuroendocrine response to estradiol is not a result of inadequate androgenization during neonatal development, and thus the actions of estradiol revealed in this rodent model are not an artefact of incomplete sexual differentiation, but reflect a physiological role of estradiol occurring during a specific early temporal window of male reproductive development.

Androgens↗
PubMed2006

Relaxin and beta-estradiol modulate targeted matrix degradation in specific synovial joint fibrocartilages: progesterone prevents matrix loss.

Relaxin, a 6-kDa polypeptide hormone, is a potent mediator of matrix turnover and contributes to the loss of collagen and glycosaminoglycans (GAGs) from reproductive tissues, including the fibrocartilaginous pubic symphysis of several species. This effect is often potentiated by beta-estradiol. We postulated that relaxin and beta-estradiol might similarly contribute to the enhanced degradation of matrices in fibrocartilaginous tissues from synovial joints, which may help explain the preponderance of diseases of specific fibrocartilaginous joints in women of reproductive age. The objective of this study was to compare the in vivo effects of relaxin, beta-estradiol, and progesterone alone or in various combinations on GAG and collagen content of the rabbit temporomandibular joint (TMJ) disc fibrocartilage, knee meniscus fibrocartilage, knee articular cartilage, and the pubic symphysis. Sham-operated or ovariectomized female rabbits were administered beta-estradiol (20 ng/kg body weight), progesterone (5 mg/kg), or saline intramuscularly. This was repeated 2 days later and followed by subcutaneous implantation of osmotic pumps containing relaxin (23.3 microg/kg) or saline. Tissues were retrieved 4 days later and analyzed for GAG and collagen. Serum relaxin levels were assayed using enzyme-linked immunosorbent assay. Relaxin administration resulted in a 30-fold significant (p < 0.0001) increase in median levels (range, approximately 38 to 58 pg/ml) of systemic relaxin. Beta-estradiol, relaxin, or beta-estradiol + relaxin caused a significant loss of GAGs and collagen from the pubic symphysis and TMJ disc and of collagen from articular cartilage but not from the knee meniscus. Progesterone prevented relaxin- or beta-estradiol-mediated loss of these molecules. The loss of GAGs and collagen caused by beta-estradiol, relaxin, or beta-estradiol + relaxin varied between tissues and was most prominent in pubic symphysis and TMJ disc fibrocartilages. The findings suggest that this targeted modulation of matrix loss by hormones may contribute selectively to degeneration of specific synovial joints.

Animals↗
PubMed2005

Estrogen and the brain: beyond ER-alpha, ER-beta, and 17beta-estradiol.

The brain of both sexes is a major target of estradiol and a site of estrogen synthesis during development and in the adult. In addition to the classical intranuclear estrogen receptors (ERs) ER-alpha and ER-beta, we have recently identified a novel, plasma membrane-associated ER that is neither ER-alpha nor ER-beta in the brain and uterus, which we have designated "ER-X". ER-X is a developmentally regulated estrogen-binding protein that is present in wild-type, ER-alpha gene-disrupted (alphaERKO) and ER-alpha-null mice. ER-X is re-expressed after ischemic brain injury and in adult transgenic mice with Alzheimer's disease. Although ER-X shares some homology with the C-terminal region of ER-alpha, it is not an alternative splicing variant of ER-alpha and may be a new gene. ER-X mediates 17alpha-estradiol and 17beta-estradiol activation of MAPK/ERK. In contrast, ER-alpha does not elicit ERK activation but, surprisingly, is inhibitory. The potential importance of 17alpha-estradiol, the preferred ligand of ER-X, for the brain is underscored by our findings by liquid chromatography/tandem mass spectrometry that the endogenous levels of 17alpha-estradiol are significantly elevated in the postnatal day-7 and adult mouse neocortex and hippocampus, as compared with 17beta-estradiol. That there is so much more 17alpha-estradiol than 17beta-estradiol in the brain suggests that this enantiomer would be readily available to the brain. In considering estrogens for postmenopausal treatment, one should consider all the ERs present in the brain, not just ER-alpha and ER-beta, but ER-X as well, and focus on ligands such as 17alpha-estradiol that may be more selective for this ER.

Animals↗
PubMed2003

Estradiol exacerbates hippocampal damage in a model of preterm infant brain injury.

We have developed a model for prenatal hypoxia-ischemia in which muscimol, a selective gamma-aminobutyric acid A (GABA(A)) receptor agonist, administered to newborn rats, induces hippocampal damage. In the neonatal rat brain, activation of GABA(A) receptors leads to membrane depolarization and neuronal excitation. Because of our previous detection of sex differences in this model and the considerable interest in the neuroprotective effects of estradiol in the adult brain, we now investigate the effect of pretreatment with high physiological levels of estradiol in our model of prenatal hypoxia-ischemia. We used unbiased stereology to assess neuron number in the hippocampal formation of control, muscimol-treated, and estradiol- plus muscimol-treated animals. Muscimol decreased neuron number in the hippocampus, with damage exacerbated by pretreatment with estradiol. A hippocampal culture paradigm was developed to mirror the in vivo investigation. We observed elevated cytotoxicity (using the lactate dehydrogenase assay) by 48 h after treatment with estradiol plus muscimol, but decreased cytotoxicity between 2 and 24 h after treatment. To determine whether the actions of estradiol on muscimol-induced damage were via the estrogen receptor, hippocampal cultures were pretreated with ICI 182,780, a selective estrogen receptor antagonist. Treatment with ICI 182,780 blocked the potentiating effect of estradiol on the late period of cytotoxicity, but had no effect on the protective actions of estradiol during the early period of cytotoxicity. There appears to be a biphasic action of estradiol in our model of neonatal brain injury that involves early nongenomic, nonreceptor-mediated protection, followed by late deleterious receptor-mediated effects.

Animals↗
PubMed2003

Evidence for a negative intrafollicular role for inhibin in regulation of estradiol production by granulosa cells.

Intrafollicular concentrations of inhibin A and estradiol vary inversely during development of dominant follicles in cattle. Thus, we hypothesized that inhibin has a negative autocrine or paracrine effect on estradiol production by granulosa cells. To examine this hypothesis, a homologous model system was used to test the effects of bovine antibovine inhibin antibodies, bovine inhibin, and a peptide fragment of bovine inhibin (bINH) on capacity of granulosa cells isolated from individual estrogen-active or -inactive dominant or subordinate follicles to produce estradiol during short-term (18 h) serum-free culture. Immunoblot analysis of media demonstrated that granulosa cells basally produce different molecular weight forms of inhibin, similar to those in bovine follicular fluid. Immunoneutralization of endogenous inhibin in culture with different doses (12.5-1000 microg) of highly purified bovine antibovine inhibin antibodies increased estradiol production 2- to 15-fold, compared with controls. Preadsorption of the anti-inhibin antibodies with bINH precursors or bovine pro-alpha(C) suppressed the capacity of anti-inhibin antibodies to enhance estradiol production by granulosa cells, compared with controls. Treatment of granulosa cells with an immunoaffinity-purified preparation of bINH suppressed basal estradiol production 60%, compared with controls. In contrast, treatment of granulosa cells with the bINH peptide increased estradiol production 14-fold, compared with controls. Based on these results, we concluded that both anti-inhibin antibodies and bINH blocked the suppressive local effects of basally produced inhibin on estradiol production during culture of granulosa cells and that inhibin has a negative autocrine or paracrine effect on the in vitro capacity of granulosa cells isolated from dominant or subordinate follicles to produce estradiol.

Animals↗
PubMed2006

Differential modulation of estrogen receptors (ERs) in ischemic brain injury: a role for ERalpha in estradiol-mediated protection against delayed cell death.

Estradiol enhances plasticity and survival of the injured brain. Our previous work demonstrates that physiological levels of estradiol protect against cerebral ischemia in the young and aging brain through actions involving estrogen receptors (ERs) and alterations in gene expression. The major goal of this study was to establish mechanisms of neuroprotective actions induced by low levels of estradiol. We first examined effects of estradiol on the time-dependent evolution of ischemic brain injury. Because estradiol is known to influence apoptosis, we hypothesized that it acts to decrease the delayed phase of cell death observed after middle cerebral artery occlusion (MCAO). Furthermore, because ERs are pivotal to neuroprotection, we examined the temporal expression profiles of both ER subtypes, ERalpha and ERbeta, after MCAO and delineated potential roles for each receptor in estradiol-mediated neuroprotection. We quantified cell death in brains at various times after MCAO and analyzed ER expression by RT-PCR, in situ hybridization, and immunohistochemistry. We found that during the first 24 h, the mechanisms of estradiol-induced neuroprotection after MCAO are limited to attenuation of delayed cell death and do not influence immediate cell death. Furthermore, we discovered that ERs exhibit distinctly divergent profiles of expression over the evolution of injury, with ERalpha induction occurring early and ERbeta modulation occurring later. Finally, we provide evidence for a new and functional role for ERalpha in estradiol-mediated protection of the injured brain. These findings indicate that physiological levels of estradiol protect against delayed cell death after stroke-like injury through mechanisms requiring ERalpha.

Animals↗
PubMed1984

Regulation of luteal cell lipoprotein receptors, sterol contents, and steroidogenesis by estradiol in the pregnant rat.

Although estradiol has been found to possess receptors in the luteal cell and to stimulate progesterone synthesis, its mechanism of action in the corpus luteum remains completely unknown. To determine whether estradiol modulates cellular uptake of lipoprotein substrate and intracellular cholesterol utilization, pregnant rats were hypophysectomized and hysterectomized on day 12 to reduce the luteal content of estradiol. They were treated with either 100 micrograms estradiol daily or with a 1-cm capsule filled with testosterone, which maintained luteal estradiol at levels found in intact pregnant rats. Blood was obtained 24, 48, and 72 h later for progesterone and cholesterol measurement. At 72 h, rats were killed, and corpora lutea (CL) were isolated for measurement of cholesteryl ester, free cholesterol, and [125I]iodo high density lipoprotein [( 125I]iodo-HDL)- and [125I]iodo-hCG-binding activities. In vivo treatment with estradiol or testosterone increased serum progesterone concentrations from 35 +/- 7 ng/ml in vehicle-treated rats to 128 +/- 21 and 118 +/- 16, respectively, and luteal weight from 2.1 +/- 0.2 mg/CL to 3.9 +/- 0.3 and 4.0 +/- 0.3 within 72 h. However, steroid treatment did not induce a change in luteal cell number, since the content of DNA per CL remained similar in all groups. It also did not modify levels of serum cholesterol. [125I]Iodo-HDL binding in luteal cells increased from 3.2 +/- 0.3 pg/cell in vehicle-treated rats to 9.9 +/- 0.8 and 7.9 +/- 0.6 after estradiol or testosterone treatment, while the luteal cell content of cholesteryl ester declined from 12.5 +/- 2.0 to 7.7 +/- 0.5 and 8.4 +/- 0.9 micrograms/CL, respectively. Thus, estradiol or testosterone increases luteal cell size but not cell number, depletes cholesteryl ester, and enhances HDL receptor content and progesterone synthesis. These results suggest that one possible mechanism by which estradiol and testosterone stimulate luteal cell steroidogenesis is by increasing the delivery of cholesterol substrate through a receptor-mediated process and by enhancing cholesterol utilization.

Animals↗
PubMed1985

Differential action of decidual luteotropin on luteal and follicular production of testosterone and estradiol.

Decidual tissue of the rat produces a hormone with physiological and biochemical characteristics similar to those of PRL. Because PRL affects both follicular and luteal production of testosterone and estradiol, it was of interest to determine whether decidual luteotropin affects basal and/or LH-stimulated ovarian secretion of steroids and whether it differentially affects follicular and luteal synthesis of testosterone and estradiol. The uteri of pseudopregnant adult rats were scratched on day 5 to induce decidual tissue formation. Pseudopregnant animals without decidua were used as controls. Rats were either hypophysectomized on day 8 or left intact. They were treated with 1.5 IU hCG/day or with vehicle between days 8-9. On day 9, blood was obtained from the ovarian vein, and both corpora lutea and large antral follicles were isolated and incubated in vitro. The presence of the decidua significantly suppressed both basal and hCG-stimulated ovarian secretion of estradiol, yet enhanced progesterone production. A similar inhibitory effect of decidual tissue on hCG stimulation of testosterone and estradiol was observed in the hypophysectomized rats. When the effect of decidua on follicles and corpora lutea was studied separately, it was found that follicles of rats with decidua produced significantly less testosterone and estradiol than follicles of rats without decidua. hCG administration to either intact or hypophysectomized rats markedly enhanced the follicular capacity to produce these two steroids. However, the degree of hCG stimulation of follicular steroidogenesis was significantly reduced by the presence of decidual tissue. In contrast, the decidua did not inhibit the in vitro steroidogenic capacity of corpora lutea. Luteal tissue of intact rats with or without decidua produced similar basal amounts of testosterone and estradiol and responded to a hCG challenge with comparable increases in the production of both steroids. After hypophysectomy, however, the responsiveness of corpora lutea to hCG stimulation differed in rats with or without decidual tissue. Whereas luteal cells of rats without decidual tissue gradually lost their responsiveness to hCG stimulation, luteal cells of rats with decidua remained highly responsive to hCG and produced high levels of testosterone and estradiol. In summary, the present investigation demonstrates that decidual luteotropin impairs ovarian secretion of estradiol and significantly inhibits the stimulatory effect of hCG on ovarian secretion of testosterone and estradiol.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Evidence that the [3H]estradiol-binding protein in pancreas is localized in exocrine cells.

Extracts of rat pancreas contain significant amounts of an [3H]estradiol-binding protein. The amount of steroid-binding activity that could be measured varied considerably depending on the tonicity of the homogenizing medium. High speed supernatants of homogenates initially prepared in isotonic buffer contained about 10% of the binding activity as homogenates prepared in hypotonic buffer. Extraction with hypotonic buffer of pellets obtained by the isotonic procedure yielded most of the remaining [3H]estradiol-binding activity. In an attempt to avoid errors resulting from incomplete homogenization and to detect possible changes in intracellular distribution of [3H]estradiol-binding activity, pancreata were initially homogenized in isotonic buffer and centrifuged at high speed (100,000 X g; 1 hr). The pellet was then extracted with hypotonic buffer and centrifuged again at high speed, and both supernatants were analyzed for [3H]estradiol-binding and amylase activities. Two or 14 days after treatment of male rats with streptozotocin, no apparent decline or redistribution of [3H]estradiol-binding activity to the cytosol was noted despite extensive alteration of beta-islet cells, as determined by electron microscopic examination of sections of these pancreata and significant loss of insulin, as measured by RIA. Amylase activity was unaffected 2 days after streptozotocin treatment, but was depressed to about 1% of control levels at 14 days. Administration of insulin to the latter group of animals resulted in return of amylase to normal levels and a modest increase (approximately 50%) in [3H]estradiol-binding activity. Since amylase levels remained unchanged 2 days after streptozotocin treatment, during which time beta-islet cells were irreversibly altered, and amylase activity was restored to normal levels by insulin treatment after its depletion in chronically treated animals, it follows that neither amylase nor the [3H]estradiol-binding protein could have been associated with beta-islet cells. This was consistent with the observation that M cells (a tumor line of beta-cells only) and 14B cells (a cloned variant of this insulinoma) had neither detectable amounts of amylase nor [3H]estradiol-binding activity. To determine whether estrogen-binding activity was associated with any other type of islet cell, islets of Langerhans were isolated by the sedimentation procedure of Lacy and Kostianovsky. In this procedure, several washing steps are employed to separate the suspended acinar cells from the denser islets that sediment rapidly. During this isolation procedure, the cells from each wash were analyzed for protein, [3H]estradiol-binding protein, and amylase a

Amylases↗
PubMed1985

Bipotential actions of estrogen on progesterone biosynthesis by ovarian cells. II. Relation of estradiol's stimulatory actions to cholesterol and progestin metabolism in cultured swine granulosa cells.

Although both inhibitory and stimulatory actions of estradiol on swine granulosa cells have been described, the bases for these inconsistent effects are not clear. We have tested properties of ovarian follicles and in vitro culture conditions that result in consistently stimulatory effects of estradiol on progesterone biosynthesis. Stimulatory actions of estradiol (in contrast to inhibitory effects) were critically dependent upon the density of granulosa cells in culture and the size and maturational status of the parent Graafian follicles. Granulosa cells isolated from small, rather than medium or large sized, swine follicles exhibited the greatest peak response to estradiol, although half-maximally stimulatory concentrations (ED50) of estradiol were similar (mean, 81 ng/ml). Granulosa cells from atretic follicles also secreted increased quantities of progesterone in response to estradiol, but the ED50 for estrogen stimulation was significantly higher (ED50 = 322 ng/ml estradiol) than that of comparable healthy follicles (ED50 = 109 ng/ml). This estrogen-responsive system was used to test the mechanisms subserving estrogen's trophic actions on granulosa cells. Estradiol significantly enhanced the activity of 3 beta-hydroxysteroid dehydrogenase with consequently increased production of progesterone and 20 alpha-hydroxypregn-4-en-3-one. Estrogen also augmented functional cholesterol side-chain cleavage activity in a dose- and time-dependent fashion with a resultant increase in pregnenolone biosynthesis. Moreover, parallel observations documented concordant dose responses for the synthesis of all three major progestins by pig granulosa cells. The trophic actions of estrogen on the steroidogenic pathway were associated with enhanced hydrolysis of endogenous cholesteryl ester stores but were not significantly antagonized by inhibition of de novo cholesterol biosynthesis. We conclude that suitable follicle selection and appropriate in vitro culture conditions provide a consistently estrogen-responsive granulosa-cell system, in which estradiol modulates certain key aspects of progestin and cholesterol metabolism. These trophic actions of estrogen are likely to prepare granulosa cells for the increased rates of progesterone biosynthesis ultimately required by fully differentiated luteal cells.

20-alpha-Dihydroprogesterone↗
PubMed1989

Estradiol regulation of sterol carrier protein-2 independent of cytochrome P450 side-chain cleavage expression in the rat corpus luteum.

A major action of estradiol in the corpus luteum of the pregnant rat is to increase the supply of cholesterol substrate for progesterone production by stimulating both cholesterol synthesis and uptake. To determine whether this steroid also affects cholesterol metabolism and transport, estradiol's action on the expression of cytochrome P450 side-chain cleavage enzyme (P450scc) and the cholesterol transport protein, sterol carrier protein-2 (SCP2), was examined. Mitochondria isolated from corpora lutea of estradiol-treated rats secreted significantly more progestagen than mitochondria of control corpora lutea. Several findings indicate that estradiol enhances cholesterol transport and availability to the P450scc rather than affects the expression of this enzyme: 1) the difference in mitochondrial progestagen synthesis induced by estradiol was obliterated by the presence of 25-hydroxycholesterol; 2) immunoblotting of P450scc indicated no stimulatory effect of estradiol on the amount of enzyme; and 3) levels of P450scc mRNA were not increased by estradiol. Whereas estradiol had no stimulatory effect on P450scc it caused a mark (3-fold) increase in the mitochondrial content of SCP2. Thus, the increase in luteal progestagen synthesis stimulated by estradiol appears to be associated with an increase in mitochondrial SCP2 and is independent of luteal P450 content or message.

Animals↗
PubMed1991

Marked seasonal changes in response to the negative feedback action of estradiol on luteinizing hormone secretion in the female Japanese monkey.

The seasonal changes in response to the negative feedback action of estradiol on LH secretion were studied in two groups of ovariectomized Japanese monkeys (Macaca fuscata fuscata; n = 6). Blood samples were collected two to three times a week, and changes in serum concentrations of LH, estradiol, and progesterone were examined. Before ovariectomy (OVX), serum LH was maintained at about 320 pg/ml regardless of the season (breeding season: autumn and winter). After OVX and simultaneous treatment sc with a Silastic implant of estradiol during the first breeding season, serum LH increased rapidly and remained high. As spring arrived, serum LH decreased abruptly and remained low. The removal of implants during the subsequent nonbreeding season resulted in an increase in serum LH similar to that during the previous breeding season after OVX and estradiol treatment. After the reinsertion of implants during the same nonbreeding season, serum LH reverted to previous low levels. As autumn arrived (second breeding season), serum LH rose again and remained high under the presence of implants. In the first subset of animals (n = 3), the implants were left in place until the subsequent mid-nonbreeding season when they were removed, and reinserted during the same non-breeding season. The findings closely corresponded to the previous results. In these animals, the implants were removed during the third breeding season, which resulted in a significant increase in serum LH. The second subset of animals (n = 3) underwent removal of implants during the second breeding season, which resulted in a further increase in serum LH, but the difference was not significant. In both subsets of animals, the timing of the increase and decrease in serum LH under the presence of implants virtually coincided with the onset and end of the breeding season in ovary-intact animals. Serum estradiol after OVX was maintained at levels similar to those in intact animals (80-100 pg/ml) under the presence of implants, but removal of implants resulted in decrease in serum estradiol to around 30 pg/ml within 1 week. Serum progesterone after OVX was maintained at low levels (less than 0.2 ng/ml) regardless of the presence of estradiol implants. These results clearly indicate a marked seasonal difference in serum LH concentrations in ovariectomized monkeys in response to constant serum estradiol. It is suggested that seasonal breeding of the Japanese monkey is governed by biannual changes in the response of the hypothalamo-hypophysial axis to the negative feedback action of estradiol.

Animals↗
PubMed1975

Specific, high-affinity binding of 17beta-estradiol in cytosols from several brain regions and pituitary of intact and castrated adult male rats.

Specific 17beta-estradiol binding capacities of cytosols from several brain regions and pituitary were determined in intact and castrated adult male rats. The binding capacity of the pituitary was approximately 10 times higher than that of any of the 5 brain region studied. Of these brain regions, the highest 17beta-estradiol binding capacities were present in the anterior hypothalamus followed by progressively lower capacities in the posterior hypothalamus, amygdala, midbrain, and cerebral cortex. The specific 17beta-estradiol binding capacity of cytosol from the anterior hypothalamus was significantly higher in castrated males than in intact rats. No such difference was found in any of the other tissues studied. Using sucrose gradient ultracentrifugation, an 8S sedimentation coefficient was found for the specific estradiol binding macromolecules present in cytosols from pituitary as well as anterior and posterior hypothalamus of castrated rats. The affinity for estradiol of cytosols from anterior and posterior hypothalamus was very high, with the mean association constants being 2.9 and 2.4 X 10(10) M-1, respectively. In competition experiments the 17beta-estradiol binding molecules present in cytosols from pituitary and anterior hypothalamus showed a higher affinity for 17beta-estradiol than for either estrone or estriol. In both tissues these 17beta-estradiol binding molecules showed a moderate affinity for the anti-estrogens MER-25 and cis-clomiphene citrate as well as for the androgen 3beta-androstranediol, but almost no affinity for 3alpha-androstanediol, 5alpha-dihydrotestosterone, testosterone, or corticosterone. These findings suggest that a true cytoplasmic receptor for estradiol exists in the male rat brain and pituitary which may play an important role in regulating reproductive function.

Amygdala↗