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PubMed1999

Estradiol suppresses mesangial cell type I collagen synthesis via activation of the MAP kinase cascade.

We have previously shown that estradiol suppresses the synthesis of type I collagen by murine mesangial cells grown in the presence of serum via activation of the transcription factor activator protein-1 (AP-1). We hypothesized that estradiol upregulates AP-1 via activation of the mitogen-activated protein (MAP) kinase cascade, a signal transduction pathway that regulates AP-1 activity. Estradiol (10(-10) to 10(-7) M) upregulated the MAP kinase pathway in murine mesangial cells grown in the presence of serum in a dose-dependent manner. Activation was evident by 1 min, peaked at 10 min, and was completely dissipated by 2 h. In contrast, estradiol had no significant effect on total (phosphorylated + unphosphorylated) p44 extracellular signal-related protein kinase (ERK) or p42 ERK. Nuclear extracts isolated from mesangial cells treated with estradiol showed increased binding to a consensus sequence AP-1 binding oligonucleotide in gel shift assays. In contrast, nuclear extracts from cells exposed to PD-98059, a highly selective inhibitor of MAP kinase-ERK kinase 1 (MEK1) and MEK2, showed reduced binding. In addition, PD-98059 antagonizes the enhanced binding induced by estradiol. Estradiol (10(-9) M) suppressed mesangial cell type I collagen synthesis (37.8 +/- 2.4%, expressed as a percentage of control values, P < 0.001 vs. control). In contrast, PD-98059 increased type I collagen synthesis (344.6 +/- 98.8, P < 0.01) and reversed the suppression of type I collagen synthesis induced by estradiol. The effects of estradiol, PD-98059, and PD-98059 plus estradiol on type I collagen protein synthesis were closely paralleled by their effects on steady-state levels of mRNA for the alpha(1) chain of type I collagen. These data suggest that estradiol suppresses type I collagen synthesis via upregulation of the MAP kinase cascade, leading to stimulation of AP-1 activity.

Angiotensin II↗
PubMed2001

Cardiovascular protective effects of 17beta-estradiol metabolites.

17beta-estradiol (estradiol), the most abundant endogenous estrogen, affords cardiovascular protection. However, in a given cohort of postmenopausal women, estradiol replacement therapy provides cardiovascular protection in only a subset. The reasons for this variable action can only be understood once the mechanisms by which estradiol induces its cardiovascular protective effects are known. Because most biological effects of estradiol are mediated via estrogen receptors (ERs) and the heart and blood vessels contain both ER-alpha and ER-beta, the prevailing view is that ERs mediate estradiol-induced cardiovascular protection. However, recent findings that estradiol protects against vascular injury in arteries of mice lacking either ER-alpha or ER-beta seriously challenges this concept. Thus other non-ER mechanisms may be operative. Endogenous estradiol is enzymatically converted to several nonestrogenic metabolites, and some of these metabolites induce potent biological effects via ER-independent mechanisms. Therefore, it is conceivable that the cardiovascular protective effects of estradiol are mediated via its endogenous metabolites. On the basis of the evidence cited in this review, the cardiovascular protective effects of estradiol are both ER dependent and independent. The purpose of this article is to review the evidence regarding the cardiovascular protective effects of estradiol metabolites and to discuss the cellular, biochemical, and molecular mechanisms involved.

Animals↗
PubMed1987

Morphological evidence that luteinizing hormone-releasing hormone neurons participate in the suppression by estradiol of pituitary luteinizing hormone secretion in ovariectomized rats.

Morphological characteristics of LHRH neurons identified by immunocytochemistry were studied using light and electron microscopy in female rats in which estradiol was replaced at the time of ovariectomy ('pseudo-intact' rats) or 3 weeks after ovariectomy (long-term ovariectomized, estradiol-treated). While estradiol levels were equivalent in these two groups, the rise in LH after ovariectomy was prevented by the immediate administration in the pseudo-intact rats, while the augmented plasma LH levels present three weeks following ovariectomy were only reduced by 50% as a result of delayed estradiol treatment. The LHRH content of the medial basal hypothalamus (MBH) including the median eminence (ME) was greater in pseudo-intact females than in untreated long-term ovariectomized control females or long-term ovariectomized, estradiol-treated females, both 1 and 14 days after estradiol exposure. Immunocytochemistry revealed fewer LHRH-immunopositive neuronal processes coursing throughout the MBH and terminating in the ME of long-term ovariectomized, estradiol-treated rats compared to those in pseudo-intact rats. However, within individual neurovascular terminals in the ME, image analysis revealed that the area of reaction product was greater in long-term ovariectomized, estradiol-treated animals. Equivalent amounts of LHRH were assayed in the MBH within each group of animals by several LHRH antisera regardless of their different binding requirements (R42, IJ29 and A-R743), suggesting that the predominant moiety present in neuronal terminals is the fully mature decapeptide. In contrast, in the preoptic area-anterior hypothalamus (POA-AH) these antisera assayed amounts of LHRH that varied as a function of binding characteristics, although the quantities did not vary with the estradiol treatment schedule. Immunocytochemical results paralleled these assay data; antisera requiring an interior sequence of amino acids (A-R743 and A-R419) detected approximately 3 times as many immunoreactive perikarya in the POA-AH as did an antiserum requiring the free amidated C terminal (IJ29). The estradiol treatment schedules had no effect on the total number of LHRH-immunopositive neurons detected by each antiserum or the distribution of LHRH-immunopositive neuronal perikarya. These data support the hypothesis that the predominant moieties present in neuronal cell bodies are precursor forms. The fine-structural characteristics of LHRH-immunopositive neuronal cell bodies are consistent with greater secretory and biosynthetic activity in LHRH neurons of long-term ovariectomized, estradiol-treated rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Regulation of melatonin's activity in the female rat brain by estradiol: effects on neurotransmitter release and on iodomelatonin binding sites.

The effects of ovariectomy and of 17 beta-estradiol (estradiol) treatment in vivo and in vitro on the ability of melatonin to inhibit dopamine release from the female rat hypothalamus and on [125I]-iodomelatonin binding sites in the brains and hypothalami of female rats were investigated. In long-term (2-4 weeks) ovariectomized (OVX) female rats, the inhibitory effect of melatonin in vitro on dopamine release from the hypothalami was abolished. After implantation of estradiol capsules, the ability of melatonin to inhibit dopamine release from the hypothalamus was reinstated and resembled that observed in intact female rats at estrus and in females exhibiting spontaneous persisting estrus. Similar reinstatement of the responsiveness to melatonin was observed in hypothalami of OVX rats shortly (2 h) after a single subcutaneous injection of estradiol (200 micrograms/animal). Incubation of hypothalami of OVX rats in vitro for 50-90 min with estradiol (0.1-1 nM) resulted in a partial (up to 50%), time and steroid concentration-dependent reinstatement of the ability of melatonin to inhibit the induced dopamine release. Conversely, incubations with estradiol in vitro reduced the ability of melatonin to inhibit dopamine release from the hypothalami of intact rats at proestrus. Such incubations had no effect on the release of dopamine from hypothalami of rats at estrus or of short-term OVX (3 days) rats. The changes in the responsiveness of the hypothalamus to melatonin were accompanied by profound changes in the binding of [125I]-iodomelatonin, to synaptosomes isolated from whole brains and from hypothalami of the OVX female rats. In OVX rats, the densities of the binding sites in the brains and particularly in the hypothalami decreased to 18 and 24% respectively, of the values observed in control females at estrus. The apparent dissociation constant of the remaining sites was significantly lower (ca. 90 nM) than that observed in the intact controls (ca. 300 nM). An almost complete reinstatement of the [125I]-iodomelatonin binding sites was observed in synaptosomes prepared from the hypothalami of OVX rats shortly (2 h) after a single subcutaneous injection of estradiol, or after incubation with estradiol in vitro. The estradiol-mediated reinstatement of [125I]-iodomelatonin binding sites was less pronounced in synaptosomes prepared from whole brains. The results clearly show that estradiol directly modulates the responses of the dopaminergic neurosecretory system in the hypothalamus to melatonin. This phenomenon may be primarily associated with the estradiol-induced changes in the density and function of melatonin receptors in the hypothalamus.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Regional specificity of gamma-aminobutyric acid receptor regulation by estradiol.

In vitro quantitative autoradiography and the microdissection technique of Palkovitz were used to examine the effects of estradiol-17 beta on GABAA receptors and on glutamic acid decarboxylase in discrete areas of rat brain. Under the conditions examined, estradiol did not affect glutamic acid decarboxylase activity. However, treatment with estradiol decreased GABAA receptor binding in a majority of areas that contain high levels of intracellular estradiol receptors and in a number of areas that contain few or no estradiol receptors. Within one brain area, the ventromedial nucleus of the hypothalamus, the estradiol effect was mapped and found to occur within the estradiol-sensitive ventrolateral portion and the surrounding dendritic plexus. Time- and dose-response relationships were region specific suggesting that estradiol might influence GABAA-receptor binding through multiple mechanisms. Estradiol does not appear to interact directly with GABAA receptors since addition of estradiol to the assay system did not affect binding. Our findings suggest that one way estradiol might affect neuroendocrine and other centrally mediated processes is through effects on GABAA-receptor binding.

Animals↗
PubMed2000

Chronic 17beta-estradiol replacement increases nitric oxide-mediated vasodilation of guinea pig coronary microcirculation.

BACKGROUND: Estrogen is cardioprotective of the coronary circulation by mechanisms incompletely understood. This study determined the effect of chronic 17beta-estradiol replacement on dilator responses to acetylcholine and sodium nitroprusside of the isolated coronary microcirculation. METHODS AND RESULTS: Adult female guinea pigs were ovariectomized, and a 21-day-release pellet containing 0.0, 0.1, 0.25, 0.5, or 1.0 mg 17beta-estradiol was implanted subcutaneously. Serum estradiol concentrations ranged from 3.9 to 74.9 pg/mL, increasing with the dose of estradiol. After 19 to 20 days, the animals were euthanized, and their hearts were removed and perfused with buffer at constant flow on an isolated heart apparatus. Both perfusion pressure and contractile force were measured in prostaglandin F(2alpha)-constricted hearts. Vasodilation to the cumulative addition of the endothelium-dependent agonist acetylcholine (10(-9) to 10(-5) mol/L) and the nitric oxide (NO) donor sodium nitroprusside (10(-9) to 10(-5) mol/L) was measured before and after NO synthesis inhibition by nitro-L-arginine (LNA, 10(-4) mol/L). Baseline coronary resistance was unaltered by estradiol, although LNA increased resistance in estradiol-treated hearts more than in ovariectomized controls. Chronic 17beta-estradiol increased sensitivity (measured by -log EC(50) values) but not maximal response to acetylcholine compared with ovariectomized controls. Differences were abolished by LNA at all doses of estradiol. Sodium nitroprusside-induced dilation was unaffected by estradiol replacement. CONCLUSIONS: Chronic 17beta-estradiol replacement, at doses producing hormone levels within the physiological range, enhances dilator sensitivity of the coronary microcirculation through enhanced NO production by the endothelium, independent of changes in NO sensitivity of the vascular smooth muscle. Thus, estradiol enhances NO production as a protective mechanism of the coronary microcirculation.

Animals↗
PubMed1987

The long-term effect of oral and percutaneous estradiol on plasma renin substrate and blood pressure.

The long-term effect of percutaneous and oral estrogen replacement therapy on blood pressure, plasma renin substrate, and serum estrogens was examined in a 2 year placebo-controlled study with 110 early postmenopausal women. The women were allocated to four treatment groups: (1) oral cyclical combination of 2 mg estradiol valerate and cyproterone acetate, (2) oral placebo, (3) percutaneous 17 beta-estradiol, supplemented by 200 mg oral progesterone during the second year, or (4) percutaneous placebo cream. Systolic and diastolic blood pressure remained unchanged in both hormone treatment groups, whereas the diastolic blood pressure tended to increase in both placebo groups. Plasma renin substrate increased during oral treatment with estradiol, but remained unchanged with percutaneous estradiol. No correlation was found between blood pressure and plasma renin substrate. During percutaneous administration of estradiol, the serum concentrations of estrone and estradiol continued to rise after 3 months and reached a plateau at 6 months of therapy. Serum estrone but not estradiol showed the same pattern during oral estradiol therapy. No further changes in any of the measured variables were observed in the women treated with percutaneous estradiol after addition of cyclical oral progesterone. We conclude that both oral and percutaneous treatment with estradiol may provide protection against the age-related increase in diastolic blood pressure observed in early postmenopausal women, and that the metabolic steady state is not attained until after 3 months of estradiol therapy.

Administration, Cutaneous↗
PubMed1995

17 beta-Estradiol inhibits Ca2+ influx and Ca2+ release induced by thromboxane A2 in porcine coronary artery.

BACKGROUND: We wished to investigate the possible mechanism of the protective effect of estrogen replacement on coronary atherosclerosis observed in postmenopausal women. METHODS AND RESULTS: Cytosolic Ca2+ concentration ([Ca2+]i) and contraction were measured simultaneously in fura 2-loaded porcine coronary arterial strips stimulated by the thromboxane A2 analogue U46619 and high-K+ depolarization in the presence and absence of 17 beta-estradiol. Pretreatment with 17 beta-estradiol (30 nmol/L to 30 mumol/L) inhibited the sustained elevation of [Ca2+]i and the sustained contraction induced by 300 nmol/L U46619. Higher concentrations of 17 beta-estradiol (1 to 100 mumol/L) also inhibited the U46619-induced transient increase in [Ca2+]i and contraction in the absence of extracellular Ca2+. In the strips precontracted by 90 mmol/L K+, 17 beta-estradiol (30 mumol/L) inhibited the increases in [Ca2+]i and contraction to resting levels. In contrast, 30 mumol/L 17 beta-estradiol only partially inhibited the U46619-induced sustained contraction, despite complete inhibition of the sustained increase in [Ca2+]i. Verapamil (10 mumol/L) also strongly inhibited the sustained increase in [Ca2+]i induced by 300 nmol/L U46619, with a partial inhibition of the U46619-induced sustained contraction. A subsequent addition of 30 mumol/L 17 beta-estradiol did not show an additional inhibitory effect on either the [Ca2+]i or the tension after the addition of verapamil. 17 beta-Estradiol (10 mumol/L) also inhibited the increase in [Ca2+]i and the contraction induced by cumulative addition of Ca2+ in the strips pretreated with 90 mmol/L K+. However, 17 beta-estradiol did not change the slope of the [Ca2+]i-tension curves. 17 beta-Estradiol (10 mumol/L) had no effect on the levels of cAMP and cGMP in the coronary strips. CONCLUSIONS: 17 beta-Estradiol inhibits the contraction of coronary vascular smooth muscle mainly inhibiting Ca2+ influx without changing Ca2+ sensitivity of contractile elements. The Ca2+ channel blocker-like action of 17 beta-estradiol may explain at least a part of the antiatherosclerotic effect of estrogen.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed2003

Catecholamines block the antimitogenic effect of estradiol on human glomerular mesangial cells.

Local sequential conversion of estradiol to hydroxyestradiols and methoxyestradiols by CYP450 and catechol-O-methyltransferase, respectively, contributes to the antimitogenic effects of estradiol on glomerular mesangial cell growth via estrogen receptor-independent mechanisms. Catecholamines are also substrates for catechol-O-methyltransferase and therefore, might abrogate the renoprotective effects of estradiol by inhibiting formation of methoxyestradiols. To test this hypothesis, we investigated the antimitogenic effects of estradiol on human glomerular mesangial cell proliferation and collagen synthesis in the presence and absence of catecholamines. Norepinephrine, epinephrine, and isoproterenol abrogated the inhibitory effects of estradiol on cell number, DNA synthesis, and collagen synthesis. For example, serum-induced DNA synthesis was inhibited from 100% to 62+/-1.9% by 0.1 micromol/L estradiol, and these inhibitory effects were reversed to 91+/-1.9% by 1 micromol/L epinephrine, 90.7+/-3.3% by 1 micromol/L isoproterenol, 87.5+/-2.8% by 10 micromol/L norepinephrine, and 92+/-1% by 10 micromol/L OR486 (catechol-O-methyltransferase inhibitor). The interaction of catecholamines with estradiol was not affected by phentolamine or propanolol, alpha- and beta-adrenoceptor antagonists, respectively. Similar to estradiol, the antimitogenic effects of 2-hydroxyestradiol were abrogated by epinephrine, isoproterenol, and OR486. In contrast to estradiol and 2-hydroxyestradiol, the antimitogenic effects of 2-methoxyestradiol were not attenuated by epinephrine, isoproterenol, or OR486. Norepinephrine, epinephrine, and isoproterenol inhibited the conversion of both estradiol and 2-hydroxyestradiol to 2-methoxyestradiol. Our findings suggest that catecholamines within the glomeruli might abrogate the antimitogenic effects of estradiol by blocking the conversion of 2-hydroxyestradiol to 2-methoxyestradiol.

2-Methoxyestradiol↗
PubMed2002

Methoxyestradiols mediate the antimitogenic effects of locally applied estradiol on cardiac fibroblast growth.

Estradiol inhibits cardiac fibroblast growth and may protect against cardiac remodeling associated with heart disease. However, the mechanisms by which estradiol attenuates cardiac fibroblast growth remain unclear. Because cardiac fibroblasts express cytochrome P450s (CYP450s) and catechol-O-methyltransferase (COMT) capable of converting estradiol to hydroxyestradiols and methoxyestradiols, respectively, and because hydroxyestradiols and methoxyestradiols (estradiol metabolites with little affinity for estrogen receptors) are potent inhibitors of cardiac fibroblast growth, we hypothesized that the antimitogenic effects of estradiol are mediated via hydroxyestradiols and/or methoxyestradiols. The inhibitory effects of estradiol (1 to 100 nmol/L) on serum-stimulated (3)H-thymidine incorporation (DNA synthesis), (3)H-proline incorporation (collagen synthesis), and cell number (proliferation) were enhanced (P<0.005) by CYP450 inducers 3-methylcholanthrene (10 micromol/L) and phenobarbital (10 micromol/L). Moreover, the inhibitory effects of estradiol were blocked by the CYP450 inhibitor 1-aminobenzotriazole (10 micromol/L) and the COMT inhibitors quercetin (10 micromol/L) and OR486 (10 micromol/L). In contrast to estradiol, the modulators of CYP450 and COMT were poor ligands for estrogen receptors (binding affinity less-than-or-equal 0.0001% versus estradiol). In cardiac fibroblasts, both quercetin and OR486 inhibited the metabolism of hydroxyestradiol to methoxyestradiol and blocked the inhibitory effects of hydroxyestradiol on cardiac fibroblast proliferation and DNA and collagen synthesis. The abrogating effects of quercetin and OR486 on the metabolism and antimitogenic effects of 2-hydroxyestradiol were mimicked by 20 micromol/L norepinephrine and isoproterenol, substrates for COMT. Our findings provide evidence that estradiol can inhibit cardiac fibroblast growth via an estrogen receptor--independent pathway that involves the local metabolism of estradiol to methoxyestradiols.

Animals↗
PubMed2005

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.

Aging↗
PubMed1990

The estradiol-induced surge of gonadotropin-releasing hormone in the ewe.

Previous studies suggest two roles for estradiol in inducing the LH surge in ewes: a neural action to evoke a sudden release of GnRH and a pituitary action to maximize response to GnRH. We tested two hypotheses: a follicular phase estradiol rise induces a GnRH surge; and the surge-inducing action of estradiol does not vary with season. In the breeding season, ewes in the midluteal phase of the estrous cycle were ovariectomized and treated with implants producing luteal phase levels of estradiol and progesterone, and an apparatus was surgically installed for later sampling of pituitary portal blood. At the normal time of luteolysis (1 week later), progesterone implants were removed, simulating luteal regression. Ewes were divided into two groups: estradiol implants also removed (n = 6) and estradiol implants added 16 h after progesterone removal to produce a rise in estradiol to levels that mimic those that circulate in the late follicular phase (n = 6). In anestrus, the estradiol rise treatment was replicated in ewes (n = 5) after an artificial luteal phase produced by sequential insertion and subsequent removal of progesterone implants. Regardless of season, the LH surge induced by estradiol was invariably accompanied by a massive GnRH surge, ranging from 73- to 394-fold over presurge values. The GnRH and LH surges began together, but the GnRH surge continued well beyond the surge of LH. There was no seasonal difference in time course or amplitude of the GnRH surge. Control ewes not treated with estradiol exhibited regular pulses of LH and GnRH every 1-2 h, but no surge of either hormone. We conclude that, regardless of season, a rise in estradiol to late follicular phase levels initiates a large and abrupt GnRH surge coincident with the onset of the LH surge. The LH surge ends despite continued elevation of GnRH.

Anestrus↗
PubMed1975

Antiestrogen action in the uterus: biological ineffectiveness of nuclear bound estradiol after antiestrogen.

These studies attempt to analyze the basis of the estrogenic and antiestrogenic action of three nonsteroidal clomophene-type compounds as monitored by their ability to bind to immature rat uterine cytoplasmic estrogen receptor, transfer receptor sites to the nucleus, and elicit estrogenic responses (increased uterine weight and induction of the synthesis of a specific uterine protein, called induced protein, or "IP"), and by their ability to antagonize the effects of estradiol on these receptor interactions and uterine responses. Both CI-628 (CI) and U-11, 100A (UA) [50 mug] elicit slight IP induction at 1-2 hand give pronounced uterine weight increases at 24 h but feeble increases at 72 h (3 single daily injections). Both bind to cytosol, and effect the transfer of receptor sites to the nucleus, which may account for the estrogenicity of these compounds. Both CI and UA give rapid (by 2-4 h), prolonged (for over 24 h), and complete blockage of estradiol-stimulated treatment abolishes short-term estradiol-stimulated uterine weight increase and antagonizes the 72 h estradiol-stimulated uterine weight response to the level attributable to the antiestrogen alone. MER-25, at the same dose (50 mug), had no estrogenic or antiestrogenic activity. Both CI and UA rapidly deplete the cytoplasmic estrogen binding capacity, reducing it to barely detectable levels for 24-42 h. Although during this period, no IP or uterine wet weight response can be elicited by estradiol, administration of saturating levels of [3H]estradiol in vivo or in vitro results in the appearance of considerable [3H]estradiol in the nucleus, bound to a macromolecule sedimenting identically with that of the nuclear receptor-estradiol complex (5.5S) formed in the absence of prior antiestrogen exposure. Hence, the estradiol which becomes bound in the nucleus after antiestrogen is biologically ineffective. The return of IP responsiveness after antiestrogen correlates well with the level of cytoplasmic receptor capable of translocation to the nucleus, and not with the nuclear estradiol uptake capacity, Presumably, then, the antiestrogenic action of CI and UA results from their depletion of cytoplasmic receptor sites and not from their ability to block specific estradiol-nuclear receptor binding per se. These studies indicate that one should be cautious in assuming that the magnitude of an estrogen response is necessarily related to the level of estrogen receptor complex in the nucleus.

Animals↗
PubMed1976

Synthesis of 17beta-estradiol by isolated ovarian tissues of the pregnant rat: aromatization in the corpus luteum.

Corpora lutea from pregnant rats were incubated to determine their ability to produce 17beta-estradiol and to aromatize testosterone in vitro. Corpora lutea and non-luteal ovarian tissues were removed from rats on days 7, 15, and 22 of pregnancy, and these tissues were immediately frozen or incubated separately in medium 199 at 37 C in an atmosphere of 95% O2-5% CO2 for 4 h. 17Beta-Estradiol in tissue and medium were quantified by a highly specific radioimmunoassay. The estradiol content ivnariably increased in non-luteal tissues during incubation, while it decreased or remained the same in incubated corpora lutea. The synthesis in non-luteal tissues, which was 18 to 400-fold greter. The incubation of corpora lutea (5 to 25 mg of tissue) with testosterone (200 ng) on days 7, 15, and 22 of pregnancy resulted in a mean accumulation of 17beta-estradiol in medium of 2.5 x 103 pg/mg tissue, compared with a mean value of 6 pg/mg for luteal tissue removed from the same ovaries and incubated without testosterone. The incubation of corpora lutea from 15-day pregnant rats with (7alpha-3H)-testosterone resulted in 15% conversion to presumptive (7alpha-3H)17beta-estradiol, which was isolated identically to estradiol isolated for radioimmunoassay. Recrystallization to constant specific activity revealed a high degree of radiochemical purity (75%) of the isolated (3H)estradiol. Rat diaphragm muscle and rabbit corpora lutea did not aromatize testosterone to 17beta-estradiol in amounts detectable by radioirpora lutea in vitro is virtually diol by non-luteal ovarian tissues. However,the corpora lutea show a striking capacity to aromatize testosterone, which might explain the high estradiol content of the rat corpora lutea during pregnancy. The physiological significance of this aromatizing system and of 17beta-estradiol in the corpus luteum is unknown but may be related to the luteotropic action of estradiol in the pregnant rat.

Animals↗
PubMed1992

Effects of rat alpha-fetoprotein administration on estradiol free fraction, the onset of puberty, and neural and uterine nuclear estrogen receptors.

The cause of the onset of puberty in the rat or any other mammalian species is unknown. According to one theory, puberty is initiated through switching of the brain "gonadostat." It is hypothesized here that puberty in the rat is the consequence of the appearance of free, and therefore physiologically active, estrogen in the circulation. To test this, the unbound fraction of estradiol in serum of immature female rats was measured in relation to the nuclear receptor occupancy of estradiol in the hypothalamus, preoptic area, and uterus at various times after birth. In addition, an attempt was made to alter the free fraction of estradiol by injection of the estradiol-binding protein alpha-fetoprotein (AFP) into immature female rats. The free fraction of estradiol was low (less than 1%), but began rising at about 20 days of age, and a significant increase in nuclear bound estradiol was observed in 23-day-old rats (P less than 0.001). By day 30, unbound levels attained adult values (3.99 +/- 0.15%). At this time, nuclear bound estradiol in all tissues examined fell (P less than 0.01), but by day 40, these were greatly increased in rats in estrus (P less than 0.001), being trebled in the preoptic areas and doubled in the hypothalamus. Injection of AFP into immature female rats extended the period of low free estradiol (1.22 +/- 0.08%), while in albumin-injected rats, the free fraction was 4.44 +/- 0.1%. Injection of AFP resulted in levels of nuclear-bound estradiol that were less than half those measured in nuclei from AFP-injected animals (P less than 0.001), and AFP delayed puberty. The affinity of the reaction between estradiol and nuclear receptors in brains of immature and mature rats was not significantly different; the Kd fell within the range of 0.05-0.08 nM. It is suggested that in the rat, puberty is the result of the appearance in the circulation of physiologically active estradiol after day 20.

Animals↗
PubMed1998

Evidence that the mediobasal hypothalamus is the primary site of action of estradiol in inducing the preovulatory gonadotropin releasing hormone surge in the ewe.

Although a neural site of action for estradiol in inducing a LH surge via a surge of GnRH is now well established in sheep, the precise target(s) for estrogen within the brain is unknown. To address this issue, two experiments were conducted during the breeding season using an artificial model of the follicular phase. In the first experiment, bilateral 17beta-estradiol microimplants were positioned in either the medial preoptic area (MPOA) or the mediobasal hypothalamus (MBH), and LH secretion was monitored. An initial negative feedback inhibition of LH secretion was observed in ewes that had estradiol microimplants located in the MPOA (6 of 6 ewes) or caudal MBH in the vicinity of the arcuate nucleus (4 of 4). In contrast, a normal LH surge was only found in animals bearing estradiol microimplants in the MBH (5 of 10). Detailed analysis of estradiol microimplant location with respect to the estrogen receptor-alpha-immunoreactive cells of the hypothalamus revealed that 4 of the 5 ewes exhibiting a LH surge had microimplants located bilaterally within or adjacent to the area of estrogen receptor-expressing cells of the ventromedial nucleus. Two of these ewes exhibited a LH surge without showing any form of estrogen negative feedback. In the second experiment, we used the technique of hypophyseal portal blood collection to monitor GnRH secretion directly at the time of the LH surge induced by estradiol delivered either centrally or peripherally. Central estradiol implants induced the GnRH surge. The duration and mean plasma concentration of GnRH during the surge were not different between animals given peripheral or central MBH estradiol implants. Cholesterol-filled MBH microimplants did not evoke a GnRH surge. We conclude that the ventromedial nucleus is the primary site of action for estradiol in stimulating the preovulatory GnRH surge of the ewe, whereas the MPOA and possibly the caudal MBH are sites at which estrogen can act to inhibit LH secretion. These data provide evidence for the sites within the ovine hypothalamus responsible for mediating the bimodal influence of estradiol on GnRH secretion and suggest that different, and possibly independent, neuronal cell populations are responsible for the negative and positive feedback actions of estradiol.

Animals↗
PubMed2000

Responsiveness of the ovine gonadotropin-releasing hormone receptor gene to estradiol and gonadotropin-releasing hormone is not detectable in vitro but is revealed in transgenic mice.

Although the ability of estradiol to enhance pituitary sensitivity to GnRH is established, the underlying mechanism(s) remain undefined. Herein, we find that approximately 9,100 bp of 5' flanking region from the ovine GnRH receptor (oGnRHR) gene is devoid of transcriptional activity in gonadotrope-derived cell lines and is not responsive to either estradiol or GnRH. In stark contrast, this same 9,100 bp promoter fragment directed tissue-specific expression of luciferase in multiple lines of transgenic mice. To test for hormonal regulation of the 9,100-bp promoter, ovariectomized transgenic females were treated with a GnRH antiserum alone or in combination with estradiol. Treatment with antiserum alone reduced pituitary expression of luciferase by 80%. Pituitary expression of luciferase in animals receiving both antiserum and estradiol was approximately 50-fold higher than animals receiving antiserum alone. The estradiol response of the -9,100-bp promoter was equally demonstrable in males. In addition, a GnRH analog (D-Ala-6-GnRH) that does not cross-react with the GnRH antiserum restored pituitary expression of luciferase in males passively immunized against GnRH to levels not different from castrate controls. Finally, treatment with both estradiol and D-Ala-6-GnRH increased pituitary expression of luciferase to a level greater than the sum of the individual treatments suggesting synergistic activation of the transgene by these two hormones. Thus, despite the complete absence of transcriptional activity and hormonal responsiveness in vitro, 9,100 bp of proximal promoter from the oGnRHR gene is capable of directing tissue-specific expression and is robustly responsive to both GnRH and estradiol in transgenic mice. To begin to refine the functional boundaries of the critical cis-acting elements, we next constructed transgenic mice harboring a transgene consisting of 2,700 bp of 5' flanking region from the oGnRHR gene fused to luciferase. As with the -9,100 bp promoter, expression of luciferase in the -2,700 lines was primarily confined to the pituitary gland, brain and testes. Furthermore, the passive immunization-hormonal replacement paradigms described above revealed both GnRH and estradiol responsiveness of the -2,700-bp promoter. Thus, 2,700 bp of proximal promoter from the oGnRHR gene is sufficient for tissue-specific expression as well as GnRH and estradiol responsiveness. Given the inability to recapitulate estradiol regulation of GnRHR gene expression in vitro, transgenic mice may represent one of the few viable avenues for ultimately defining the molecular mechanisms underlying estradiol regulation of GnRHR gene expression.

Animals↗
PubMed2002

Estradiol feedback alters potassium currents and firing properties of gonadotropin-releasing hormone neurons.

GnRH neurons are regulated by estradiol feedback through unknown mechanisms. Voltage-gated potassium channels determine the pattern of activity and response to synaptic inputs in many neurons. We used whole-cell patch-clamp to test whether estradiol feedback altered potassium currents in GnRH neurons. Adult mice were ovariectomized and some treated with estradiol implants to suppress reproductive neuroendocrine function; 1 wk later, brain slices were prepared for recording. Estradiol affected the amplitude, decay time, and the voltage dependence of both inactivation and activation of A-type potassium currents in these cells. Estradiol also altered a slowly inactivating current, I(K.) The estradiol-induced changes in I(A) contributed to marked changes in action potential properties. Estradiol increased excitability in GnRH neurons, decreasing both threshold and latency for action potential generation. To test whether estradiol altered phosphorylation of the channels or associated proteins, the broad-spectrum kinase inhibitor H7 was included in the recording pipette. H7 acutely reversed some but not all effects of estradiol on potassium currents. Estradiol did not affect I(A) or I(K) in paraventricular neurosecretory neurons, demonstrating a degree of specificity in these effects. Potassium channels are thus one target for estradiol regulation of GnRH neurons; this regulation involves changes in phosphorylation of potassium channel components.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗