The phytochemical resveratrol, which is found in grapes and wine, has been reported to have a variety of anti-inf lammatory, anti-platelet, and anticarcinogenic effects. Based on its structural similarity to diethylstilbestrol, a synthetic estrogen, we examined whether resveratrol might be a phytoestrogen. At concentrations (Ϸ3-10 M) comparable to those required for its other biological effects, resveratrol inhibited the binding of labeled estradiol to the estrogen receptor and it activated transcription of estrogen-responsive reporter genes transfected into human breast cancer cells. This transcriptional activation was estrogen receptor-dependent, required an estrogen response element in the reporter gene, and was inhibited by specific estrogen antagonists. In some cell types (e.g., MCF-7 cells), resveratrol functioned as a superagonist (i.e., produced a greater maximal transcriptional response than estradiol) whereas in others it produced activation equal to or less than that of estradiol. Resveratrol also increased the expression of native estrogen-regulated genes, and it stimulated the proliferation of estrogen-dependent T47D breast cancer cells. We conclude that resveratrol is a phytoestrogen and that it exhibits variable degrees of estrogen receptor agonism in different test systems. The estrogenic actions of resveratrol broaden the spectrum of its biological actions and may be relevant to the reported cardiovascular benefits of drinking wine.Resveratrol (trans-3, 4Ј, 5-trihydroxystilbene) occurs naturally in grapes and a variety of medicinal plants. In plants, resveratrol functions as a phytoalexin that protects against fungal infections (1). Because of its high concentration in grape skin, significant amounts of resveratrol are present in wine (2, 3), and it has been proposed to explain, at least in part, the apparent ability of moderate consumption of red wine to reduce the risk of cardiovascular disease (4 -7). Resveratrol also has been reported to have cancer chemopreventive activity (8). The similarity in structure between resveratrol and the synthetic estrogen diethylstilbestrol (DES; 4, 4Ј-dihydroxy-trans-␣, ␤-diethylstilbene) prompted us to investigate whether resveratrol might exhibit estrogenic activity, a property that is known to produce a cardioprotective benefit (9, 10).Estrogens, including phytoestrogens, act via the estrogen receptor, a member of the nuclear receptor superfamily. Estrogen binding to the receptor activates the transcription of estrogen-responsive target genes. We report here that resveratrol binds to and activates transcription by the estrogen receptor at concentrations that are comparable to those required for its other biological effects. EXPERIMENTAL PROCEDURESCell Culture. MCF-7 cells, subclone WS8 (estrogen receptor-positive), MDA-MB-231 cells, subclone 10A (estrogen receptor-negative), and T47D cells, subclone A18 (estrogen receptor-positive) are derived from human breast adenocarcinomas and were provided by V. Craig Jordan (Northwestern University Medical Sch...

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Ahch (also known as Dax1) encodes a transcription factor that has been implicated in sex determination and gonadal differentiation. Mutations in human AHC cause X-linked, adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HH). Duplication of the Xp21 dosage-sensitive sex reversal (DSS) region, which contains the Ahch locus, and transgenic overexpression of Ahch cause male-to-female sex reversal. Using Cre-mediated disruption of Ahch, we have generated a mouse model of AHC-HH that allows the function of Ahch to be examined in both males and females. Although Ahch has been postulated to function as an ovarian determination gene, the loss of Ahch function in females does not affect ovarian development or fertility. Ahch is instead essential for the maintenance of spermatogenesis. Lack of Ahch causes progressive degeneration of the testicular germinal epithelium independent of abnormalities in gonadotropin and testosterone production and results in male sterility. Ahch is thus not an ovarian determining gene, but rather has a critical role in spermatogenesis.

Context-Steroidogenic factor 1 (SF1/AdBP4/FTZF1, NR5A1) is a nuclear receptor transcription factor that plays a key role in regulating adrenal and gonadal development, steroidogenesis, and reproduction. Targeted deletion of Nr5a1 (Sf1) in the mouse results in adrenal and gonadal agenesis, XY sex-reversal, and persistent Müllerian structures in males. Consistent with the murine phenotype, human mutations in SF1 were described initially in two 46,XY individuals with female external genitalia, Müllerian structures (uterus) and primary adrenal failure.Objective-Given recent case reports of haploinsufficiency of SF1 affecting testicular function in humans, we aimed to identify SF1 mutations in a cohort of individuals with a phenotypic spectrum of 46,XY gonadal dysgenesis/impaired androgenization (now termed 46,XY Disorders of Sex Development, DSD) with normal adrenal function.Methods & Patients-Mutational analysis of NR5A1 in 30 individuals with 46,XY DSD, followed by functional studies of SF1 activity.Results-Heterozygous missense mutations in NR5A1 were found in four individuals (4/30, 13%) with this phenotype. These mutations (V15M, M78I, G91S, L437Q) were shown to impair Publisher's Disclaimer: "This is an un-copyedited author manuscript copyrighted by The Endocrine Society. This may not be duplicated or reproduced, other than for personal use or within the rule of "Fair Use of Copyrighted Materials" (section 107, Title 17, U.S. Code) without permission of the copyright owner, The Endocrine Society. From the time of acceptance following peer review, the full text of this manuscript is made freely available by The Endocrine Society at http://www.endojournals.org/. The final copy edited article can be found at http://www.endojournals.org/. The Endocrine Society disclaims any responsibility or liability for errors or omissions in this version of the manuscript or in any version derived from it by the National Institutes of Health or other parties. The citation of this article must include the following information: author(s), article title, journal title, year of publication and DOI." UKPMC Funders Group UKPMC Funders Group Author Manuscript UKPMC Funders Group Author Manuscripttranscriptional activation through abnormal DNA binding (V15M, M78I, G91S), altered sub-nuclear localization (V15M, M78I), or through disruption of the putative ligand-binding pocket (L437Q). Two mutations appeared to be de novo or germline changes. The other two mutations appeared to be inherited in a sex-limited dominant manner, as the mother is heterozygous for the change.Conclusions-These studies demonstrate that SF1 mutations are more frequent than previously suspected causes of impaired fetal and postnatal testicular function in 46,XY individuals.

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Ovarian follicles undergo exponential growth in response to follicle-stimulating hormone (FSH), largely as a result of the proliferation of granulosa cells (GCs). In vitro under serum-free conditions, rat GCs differentiate in response to FSH but do not proliferate unless activin is also present. In the presence of FSH plus activin, GCs exhibit enhanced expression of cyclin D2 as well as inhibin-alpha, aromatase, steroidogenic factor-1 (SF-1), cholesterol side chain (SCC), and epiregulin. In this report we sought to identify the signaling pathways by which FSH and activin promote GC proliferation and differentiation. Our results show that these responses are associated with prolonged Akt phosphorylation relative to time-matched controls and are dependent on phosphatidylinositol 3-kinase (PI 3-kinase) and Smad2/3 signaling, based on the ability of the PI 3-kinase inhibitor LY294002 or infection with adenoviral dominant negative Smad3 (DN-Smad3) mutant to attenuate induction of cyclin D2, inhibin-alpha, aromatase, SCC, SF-1, and epiregulin. The DN-Smad3 mutant also abolished prolonged Akt phosphorylation stimulated by FSH plus activin 24 h post-treatment. Infection with the adenoviral constitutively active forkhead box-containing protein, O subfamily (FOXO)1 mutant suppressed induction of cyclin D2, aromatase, inhibin-alpha, SF-1, and epiregulin. Transient transfections of GCs with constitutively active FOXO1 mutant also suppressed cyclin D2, inhibin-alpha, and epiregulin promoter-reporter activities. Chromatin immunoprecipitation results demonstrate in vivo the association of FOXO1 with the cyclin D2 promoter in untreated GCs and release of FOXO1 from the cyclin D2 promoter upon addition of FSH plus activin. These results suggest that proliferation and differentiation of GCs in response to FSH plus activin requires both removal of FOXO1-dependent repression and positive signaling from Smad2/3.

We created a nonclassical estrogen receptor (ER) knock-in mouse model by introducing a mutation that selectively eliminates classical ER signaling through estrogen response elements, while preserving the nonclassical ER pathway. Heterozygous nonclassical ER knock-in (NERKI) females are infertile. Their ovaries contain no corpora lutea, reflecting a defect in ovulation, and the stromal cells contain lipid droplets, suggesting altered steroidogenesis. The uteri are enlarged with evidence of cystic endometrial hyperplasia, and the mammary glands are hypoplastic. These phenotypic features indicate differential ER effects on growth and development in various estrogen-responsive tissues. These findings suggest that nonclassical ER signaling pathways play an important physiological role in the development and function of the reproductive system.

DAX1 mutations are a relatively frequent cause of adrenal failure in this group of boys. SF1 mutations causing adrenal failure in humans are rare and are more likely to be associated with significant underandrogenization and gonadal dysfunction in 46,XY individuals.