Other isotype-selective estrogen receptor (ER) agonists, the selective ER␣ agonist 3,17-dihydroxy-19-nor-17␣-pregna-1,3,5 (10)-triene-21,16␣-lactone and the selective ER␤ agonist 8-vinylestra-1,3,5 (10)-triene-3,17␤-diol, were used in hypophysectomized rats, gonadotropin-releasing hormone antagonist-treated mice, as well as intact rats to elucidate the effects of isotype-selective estrogens on the physiology of folliculogenesis and ovulation. In hypophysectomized rats and gonadotropin-releasing hormone antagonisttreated mice, the ER␤ agonist caused stimulation of early folliculogenesis, a decrease in follicular atresia, induction of ovarian gene expression, and stimulation of late follicular growth, accompanied by an increase in the number of ovulated oocytes similar to 17␤-estradiol (E2). In contrast, the ER␣ agonist had little or no effect on these parameters, implying that direct estrogen effects on ovarian follicular development are mediated by ER␤. In intact rats, E2 and the ER␣ agonist dose-dependently inhibited ovulation, in contrast to the ER␤ agonist. On the other hand, the ER␤ agonist did not stimulate uterine weight in intact rats, in contrast to E2 and the ER␣ agonist. This finding is in line with the assumption that estrogen mediated ovulation inhibition and stimulation of uterine growth are mediated by ER␣ but not by ER␤.pharmacology ͉ folliculogenesis ͉ endocrinology E strogens play a central role in the development and maintenance of female reproductive organs, mammary glands, and sexual behavior. In addition, estrogen's involvement in the function of a number of other tissues such as the bone, the cardiovascular, and the CNS has also been recognized (1, 2).Estrogens exhibit most of their physiological effects through nuclear receptor proteins, the estrogen receptors (ERs). Until recently, it had been assumed that there is only one ER. In 1996, a second ER was detected (3-5), which shares a high degree of homology with the ''classic'' counterpart, specifically in the DNA-and ligand-binding domains. The newly detected receptor is called ER␤, the classic counterpart being renamed as ER␣. The expression pattern of the two ER isotypes is different: ER␤ is predominantly expressed in the ovary and prostate but also other organs not subserving reproduction-related functions. In contrast, its expression is low in the pituitary, the thymus, the uterus, and the liver; these organs express ER␣ at high levels (6, 7). A distinct tissue distribution of ER␣ and ER␤ suggests specific biological functions of ER␣ and ER␤.ER␤ is the predominant ER in ovarian follicles in rodents and nonrodents (8-10). The hypothesis that ER␤ plays an important role in the control of ovarian function is supported by data from various lines of ER␤ knockout mice showing different degrees of female subfertility due to reduced follicular maturation and ovulation rate (11).The stimulatory activity of estrogens on ovarian cell growth, especially folliculogenesis, has been demonstrated in rodent models (12,13). It is suggested that estroge...

Defining an appropriate and efficient assessment of drug‐induced corrected QT interval (QTc) prolongation (a surrogate marker of torsades de pointes arrhythmia) remains a concern of drug developers and regulators worldwide. In use for over 15 years, the nonclinical International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) S7B and clinical ICH E14 guidances describe three core assays (S7B: in vitro hERG current & in vivo QTc studies; E14: thorough QT study) that are used to assess the potential of drugs to cause delayed ventricular repolarization. Incorporating these assays during nonclinical or human testing of novel compounds has led to a low prevalence of QTc‐prolonging drugs in clinical trials and no new drugs having been removed from the marketplace due to unexpected QTc prolongation. Despite this success, nonclinical evaluations of delayed repolarization still minimally influence ICH E14‐based strategies for assessing clinical QTc prolongation and defining proarrhythmic risk. In particular, the value of ICH S7B‐based “double‐negative” nonclinical findings (low risk for hERG block and in vivo QTc prolongation at relevant clinical exposures) is underappreciated. These nonclinical data have additional value in assessing the risk of clinical QTc prolongation when clinical evaluations are limited by heart rate changes, low drug exposures, or high‐dose safety considerations. The time has come to meaningfully merge nonclinical and clinical data to enable a more comprehensive, but flexible, clinical risk assessment strategy for QTc monitoring discussed in updated ICH E14 Questions and Answers. Implementing a fully integrated nonclinical/clinical risk assessment for compounds with double‐negative nonclinical findings in the context of a low prevalence of clinical QTc prolongation would relieve the burden of unnecessary clinical QTc studies and streamline drug development.

The sterol 4,4-dimethyl-5alpha-cholesta-8,14,24-trien-3beta-ol (FF-MAS [follicular-fluid meiosis-activating sterol]) from human follicular fluid has recently been identified as a compound that induces the resumption of meiosis. FF-MAS and various oxysterols have been reported to transactivate the orphan receptor LXRalpha. The objective was to determine the biological activity of synthetic FF-MAS on the resumption of meiosis and final maturation of mouse oocytes in vitro. In order to evaluate whether LXRalpha might mediate FF-MAS action on the oocyte, we compared the capability of various compounds to activate LXRalpha-dependent transcription and to induce resumption of meiosis in the oocyte assay. Ovaries were isolated from immature mice primed with FSH 48 h before collection. Naked oocytes (NkO) and cumulus enclosed oocytes (CEO) were isolated from follicles. The oocytes were cultured in two groups, NkO and CEO, respectively, in media containing either 3 mM hypoxanthine, 5 microM IBMX, or 0.100 mM dbcAMP to maintain the oocytes in the germinal vesicle stage. The resumption of meiosis was assessed by the frequency of germinal vesicle breakdown (GVBD) after 24 h of in vitro culture. FF-MAS overcame the meiotic inhibition by hypoxanthine in both the NkO group and CEO group in a dose-dependent manner within the concentration range 0.07-7 microM. FF-MAS displayed similar potency in all inhibitory agents used. Also, FF-MAS significantly increased the formation of polar bodies in both the CEO and NkO group. The oxysterols 22(R)-hydroxycholesterol (a potent ligand for the LXRalpha receptor), 16-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol, as well as cholesterol, were tested without any significant effect on maturation compared to that of controls. Oxysterols and FF-MAS were observed to activate LXRalpha. In conclusion, the results reported here clearly demonstrate that synthetic FF-MAS exclusively is capable of mediating resumption of meiosis in vitro in both NkO and CEO irrespective of the inhibitory substance used. In contrast, the oxysterols and cholesterol had no significant biological activity on this oocyte function, and consequently we found no correlation between LXRalpha activation and meiosis stimulation.

It has been suggested that the collagenolytic enzymes released from white blood cells which infiltrate the pregnant human uterine cervix at term are responsible for connective tissue changes which take place during the ripening process. Similarly, an infiltration of inflammatory cells occurs in pregnant guinea-pigs either spontaneously at term or at preterm after treatment with the antiprogestin onapristone. The objective of this study was to evaluate the effects of the inflammatory cytokines interleukin 8 (IL-8), interleukin 1 beta (IL-1 beta), tumour necrosis factor alpha (TNF-alpha) and a combination of IL-1 beta and TNF-alpha on cervical ripening in guinea-pigs during advanced pregnancy. The cytokines were applied locally (intracervically) in a gel for 2 days and the effects were assessed on the third day by both extensibility measurements and morphological evaluation. IL-8 treatment on days 42 and 43 post coitum (p.c) and on days 48 and 49 p.c. (term: day 67 +/- 3 p.c.) significantly (P < 0.05) increased cervical extensibility at both stages of pregnancy. Although IL-1 beta treatment (days 42 and 43 p.c.) led to a slight increase in cervical extensibility, this effect was not statistically significant. An electron microscope study performed on days 48 and 49 p.c. revealed a pronounced cervical ripening accompanied by the dissolution of collagen fibres, stromal oedema and the infiltration of polymorphonuclear leukocytes in all cytokine-treated groups. The morphological effects of IL-8 and IL-1 beta were indistinguishable from those observed during normal cervical ripening at term.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract-Both known estrogen receptors, ER␣ and ER␤, are expressed in blood vessels. To gain further insight into the role of ER␣ in a functional setting, we investigated the effect of the novel highly selective ER␣ agonist Cpd1471 on vascular reactivity in ovariectomized spontaneously hypertensive rats (SHR). After ovariectomy or sham operation, 12-week-old female SHR received either 17␤-estradiol (E2, 2 g/kg body wt per day), the selective ER␣ agonist Cpd1471 (30 g/kg body wt per day), or placebo. Acetylcholine-induced endothelium-dependent vasorelaxation was significantly blunted in aortas from ovariectomized rats (R max , 53%Ϯ3% versus sham, 79%Ϯ2%; PϽ0.001). Treatment with E2 or Cpd1471 significantly augmented acetylcholine-induced relaxation in ovariectomized rats (R max , 70%Ϯ2%; resp, 73%Ϯ2%). Endothelium-independent relaxation induced by sodium nitroprusside was not different among the four groups. The contractile response induced by the nitric oxide (NO) synthase inhibitor N-nitro-L-arginine, an index of basal NO formation, was significantly lower in ovariectomized rats compared with sham-operated animals (53Ϯ2% versus 77%Ϯ5%; PϽ0.01) and was normalized by both E2 (70%Ϯ2%) and Cpd1471 (70%Ϯ3%). Aortic endothelial NO synthase (eNOS) expression and phosphorylation of the vasodilator-stimulated phosphoprotein, an index of NO/cGMP-signaling, was reduced in ovariectomized SHR and normalized by E2 and Cpd1471. In SHR after ovariectomy, endothelium-dependent NO-mediated vasorelaxation and eNOS expression are attenuated. The novel selective ER␣ agonist Cpd1471 prevented these pathophysiological changes to a similar extent as E2. Thus, the pharmacological principle of selective ER␣ activation mediates positive vascular effects. Key Words: estrogen Ⅲ endothelium Ⅲ nitric oxide Ⅲ nitric oxide synthase Ⅲ rats, spontaneously hypertensive G ender differences in the risk for cardiovascular diseases are well recognized, with premenopausal women exhibiting a lower risk than age-matched men. The advantage of women over men in cardiovascular morbidity disappears after menopause, suggesting that estrogen plays an important role in cardiovascular health. 1 Estrogens are known to exert beneficial effects on the vascular wall. Long-term estrogen treatment improves endothelial dysfunction, a major contributor to the pathophysiology of cardiovascular disease, through upregulation of endothelial cell genes, such as endothelial nitric oxide synthase (eNOS). 2-4 Furthermore, estrogen has rapid nongenomic effects on the vascular endothelium, including activation of nitric oxide (NO) synthesis. 5,6 However, despite the positive effects on vascular function in animal models 7-9 and humans, 10 -14 estrogen replacement therapy with 17␤-estradiol or mixtures of equine estrogens as in the Heart and Estrogen/progestin Replacement Study (HERS) has failed to protect from cardiovascular diseases in large controlled clinical trials. [15][16][17][18] Therefore, in recent years, research has focused on selective estrogen receptor modulation as...

These results support the hypothesis that activation of ERalpha favorably affects cardiac hypertrophy, myocardial contractility, and gene expression in ovariectomized SHR. Further studies are required to determine whether activation ERbeta mediates redundant or divergent effects.

Abstract-Experimental and population-based studies indicate that female gender and estrogens protect the cardiovascular system against aldosterone-induced injury. Understanding the function of estrogens in heart disease requires more precise information on the role of both estrogen receptor (ER) subtypes, ER␣ and ER␤. Therefore, we determined whether selective activation of ER␣ or of ER␤ would confer redundant, specific, or opposing effects on cardiovascular remodeling in aldosterone salt-treated rats. The ER␣ agonist 16␣-LE2, the ER␤ agonist 8␤-VE2, and the nonselective estrogen receptor agonist 17␤-estradiol lowered elevated blood pressure, cardiac mass, and cardiac myocyte cross-sectional areas, as well as increased perivascular collagen accumulation and vascular osteopontin expression in ovariectomized rats receiving chronic aldosterone infusion plus a high-salt diet for 8 weeks. Uterus atrophy was prevented by 16␣-LE2 and 17␤-estradiol but not by 8␤-VE2. Cardiac proteome analyses by 2D gel electrophoresis, mass spectrometry, and peptide sequencing identified specific subsets of proteins involved in cardiac contractility, energy metabolism, cellular stress response and extracellular matrix formation that were regulated in opposite directions by aldosterone salt treatment and by different estrogen receptor agonists. We conclude that activation of either ER␣ or ER␤ protects the cardiovascular system against the detrimental effects of aldosterone salt treatment and confers redundant, as well as specific, effects on cardiac protein expression. Nonfeminizing ER␤ agonists such as 8␤-VE2 have a therapeutic potential in the treatment of hypertensive heart disease. (Hypertension. 2007;50:432-438.)

Ligand-dependent activation of ERbeta confers blood pressure lowering effects in SHR that are superior to those of 17beta-estradiol or the ERalpha agonist 16alpha-LE2 and attenuates cardiac hypertrophy primarily by a reduction of cardiac afterload without promoting uterine growth.