Phthalates Impair Germ Cell Number in the Mouse Fetal Testis by an Androgen- and Estrogen-Independent Mechanism

Data from experiments conducted almost exclusively in the rat have established that some phthalates have deleterious effects on the fetal testis probably due to their antiandrogenic and/or estrogenic effects, but their mechanisms of action remain unknown. A recent study reported that phthalates also have deleterious effects on human fetal testis with germ cell number, but not steroidogenesis altered. Therefore, we used organ culture of fetal testes at different stages of development to analyze the direct effects of phthalates on both steroidogenesis and gonocyte development and to determine if the effects of MEHP on these functions reported in the rat can be extended to other mammalian species. We defined specific periods of sensitivity of the fetal mouse testis to MEHP for these two functions and showed that the effects of phthalates on steroidogenesis vary with the developmental stage. Conversely, the strong deleterious effects of phthalates on germ cells were constantly present during the active phases of gonocyte development and thus share no relationship with the steroidogenic status. Moreover, all the effects of phthalates were unchanged in testes from mice deficient for estrogen (ERαKO or ERβKO) or androgen (Tfm) receptors. In conclusion, our results demonstrate that phthalates impair mouse fetal germ cell number similarly to other mammalian species, but are neither estrogenic nor antiandrogenic molecules because their effects do not involve, directly or indirectly, ER or AR

The role of activation functions 1 and 2 of estrogen receptor-α for the effects of estradiol and selective estrogen receptor modulators in male mice

Estradiol (E2) is important for male skeletal health and the effect of E2 is mediated via estrogen receptor (ER)-α. This was demonstrated by the findings that men with an inactivating mutation in aromatase or a non-functional ERα had osteopenia and continued longitudinal growth after sexual maturation. The aim of the present study was to evaluate the role of different domains of ERα for the effects of E2 and SERMs on bone mass in males. Three mouse models lacking either ERαAF-1 (ERαAF-1(0)), ERαAF-2 (ERαAF-2(0)) or the total ERα (ERα(−/−)) were orchidectomized (orx) and treated with E2 or placebo. E2 treatment increased the trabecular and cortical bone mass and bone strength, while it reduced the thymus weight and bone marrow cellularity in orx wild type (WT) mice. These parameters did not respond to E2 treatment in orx ERα(−/−) or ERαAF-2(0) mice. However, the effects of E2 in orx ERαAF-1(0) mice were tissue-dependent, with a clear response in cortical bone parameters and bone marrow cellularity, but no response in trabecular bone. To determine the role of ERαAF-1 for the effects of SERMs, we treated orx WT and ERαAF-1(0) mice with Raloxifene (Ral), Lasofoxifene (Las), Bazedoxifene (Bza) or vehicle. These SERMs increased total body areal bone mineral density (BMD) and trabecular volumetric BMD to a similar extent in orx WT mice. Furthermore, only Las increased cortical thickness significantly and only Bza increased bone strength significantly. However, all SERMs showed a tendency towards increased cortical bone parameters. Importantly, all SERM-effects were absent in the orx ERαAF-1(0) mice. In conclusion, ERαAF-2 is required for the estrogenic effects on all evaluated parameters, while the role of ERαAF-1 is tissue specific. All evaluated effects of Ral, Las and Bza are dependent on a functional ERαAF-1. Our findings might contribute to the development of bone specific SERMs in males

Profil d'expression de gènes contrôlés par l'oestradiol et/ou par les récepteurs nucléaires des oestrogènes ERa et ERb

Mon projet a eu pour ambition de progresser dans la compréhension des mécanismes cellulaires et moléculaires qui sous-tendent les multiples effets médiés par l oestradiol et les récepteurs des oestrogènes. La dissection des mécanismes de signalisation a passé par l étude de l expression génique à grande échelle (microarray) dans certains modèles de souris que nous avons développés. Nous disposions plus particulièrement de modèles de souris:1- porteuses de mutations germinales pour ERa et ERb (ERKOs); 2- porteuses de la mutation germinale pour l aromatase (ArKO). Notre projet devait nous permettre i) - d identifier des gènes dont l expression est controlée par l oestradiol et/ou par les récepteurs nucléaires des oestrogènes ii) - d évaluer les effets oestrogène-dépendants mais oestrogènes récepteurs indépendants (récepteurs membranaires) d une part et de les distinguer des évènements oestrogène-indépendants mais oestrogènes récepteurs dépendants d autre part. Les étapes de l approche sont résumées comme suit : valorisation des résultats [détection du signal, analyse des données brutes (expression différentielle, normalisation, filtrage, statistiques, regroupement)], vérification et confirmation des données issues de la puce à ADN par PCR, interprétation biologique des données. En conclusion : les informations obtenues démontrent que les résultats des expériences de microarrays permettent de sélectionner des gènes de manière reproductible et non-ambigüe. Dans les tissus étudiés (prostate et tissu adipeux blanc) les analyses du profil d expression de ces gènes révèlent que les effets sont principalement médiés par le récepteur alpha des oetrogènes et de manière indépendante de l hormone.The definition of the role of estrogen is a long-date scientific preoccupation. On the basis of the studies carried out in the last twenty years, it is now well accepted that estradiol and its cognate receptors are relevant transcription regulators in reproductive as well as non-reproductive tissues. Models of estrogen (E2) insufficiency (ArKO) and estrogen receptors (ER) dysfunction (ERKOs) have revealed new and unexpected roles of estradiol and its receptors both in female and male. The purpose of this study is to use mouse models of estrogen insufficiency (ArKO) and estrogen receptors dysfunction (ERKOs) to provide a genomic insight in the multiple and complex mechanisms defining estrogenic signaling to help understanding its role in physiological and pathological conditions. In particular the objective was to identify the genes responsive to estrogen signaling according to various possible mechanisms: 1) estrogen and estrogen receptordependent actions; 2) estrogen-independent and estrogen-receptor-dependent actions; 3) estrogen receptor-independent estrogen-dependent actions. To reach this aim, estrogen and estrogen receptors dependent genes expression profiling were performed by microarray analysis in ventral and dorso-lateral prostate and gonadal white adipose tissue from mouse models of impaired estrogen synthesis (ArKO) and ER action (ERKOs). The experimental and biological reproducibility of microarray data was first verified and confirmed providing a correlation between real-time PCR and microarrays in fold change measurements and in expression profiles across all tissues. The results obtained from the analysis of the expression profiles indicate that the classical and the non-genomic actions of estrogen are not to represent the main mechanisms of estrogenic signaling in prostate and adipose tissue. Conversely it appears that the estrogenic signaling in these tissues is exerted via estrogen receptors with an estrogen-independent mechanism of action. ERa appear to be the main mediator of the observed estrogenic effects, with mechanisms that differ according to the specific tissue. In ventral and dorso-lateral prostates, ERa seems to have inhibitory effects on transcription of target genes, supporting the hypothesis of its implication as a tumor suppressor in the prostate gland. Additional studies need to be performed to permit the identification of genes whose regulation can be directly modulated by ERs.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Analyse du rôle des récepteurs des oestrogènes a et b chez la souris

STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Paracrine signaling through the epithelial estrogen receptor alpha is required for proliferation and morphogenesis in the mammary gland.

International audienceEstradiol is a major regulator of postnatal mammary gland development and thought to exert its effects through estrogen receptor alpha (ERalpha) expressed in the mammary gland stroma and epithelium. Previous studies, however, were confounded by the use of an ERalpha mutant strain that retains some of the protein with transactivation activity. Here, we use an ERalpha-/- mouse strain in which no ERalpha transcript can be detected to analyze mammary gland development in the complete absence of ERalpha signaling. The ERalpha-/- females show no development beyond a rudimentary ductal system. By grafting ERalpha-/- epithelium or stroma in combination with ERalpha WT stroma or epithelium, we show that the primary target for estradiol is the mammary epithelium, whereas a direct response of the mammary stroma is not required for mammary gland development to proceed normally. Mammary glands reconstituted with ERalpha-/- mammary epithelium exposed to pregnancy hormones show increased transcription of milk protein genes, indicating that ERalpha signaling is not an absolute requirement for a transcriptional response to pregnancy hormones. When ERalpha-/- mammary epithelial cells are in close vicinity to ERalpha WT cells, they proliferate and contribute to all aspects of mammary gland development, indicating that estradiol, like progesterone, orchestrates proliferation and morphogenesis by a paracrine mechanism, affecting nearby cells in the mammary epithelium

Retinoic acid-induced glandular metaplasia in mouse skin is linked to the dermal expression of retinoic acid receptor β mRNA

The distribution of transcripts of nuclear (RARα, RARβ, and RARγ) and cytosolic (CRABP) retinoic acid receptors was analyzed in 13.5-day mouse embryo upper-lip skin, cultured in vitro for 48 hr with or without added retinoic acid. The results show a significant up-regulation of the transcription of the RARβ gene concomitant with the initiation of an alteration of hair vibrissae follicle development, leading, after transfer for 8 days to the chick embryo chorioallantoic membrane, to an exocrine-type gland morphogenesis

Estrogen Receptor α Signaling in Inflammatory Leukocytes Is Dispensable for 17β-Estradiol-Mediated Inhibition of Experimental Autoimmune Encephalomyelitis

Estrogen treatment has been shown to exert a protective effect on experimental autoimmune encephalomyelitis (EAE), and is under clinical trial for multiple sclerosis. Although it is commonly assumed that estrogens exert their effect by modulating immune functions, we show in this study that 17beta-estradiol (E2) treatment can inhibit mouse EAE without affecting autoantigen-specific T cell responsiveness and type 1 cytokine production. Using mutant mice in which estrogen receptor alpha (ERalpha) has been unambiguously inactivated, we found that ERalpha was responsible for the E2-mediated inhibition of EAE. We next generated irradiation bone marrow chimeras in which ERalpha expression was selectively impaired in inflammatory T lymphocytes or was limited to the radiosensitive hemopoietic compartment. Our data show that the protective effect of E2 on clinical EAE and CNS inflammation was not dependent on ERalpha signaling in inflammatory T cells. Likewise, EAE development was not prevented by E2 treatment in chimeric mice that selectively expressed ERalpha in the systemic immune compartment. In conclusion, our data demonstrate that the beneficial effect of E2 on this autoimmune disease does not involve ERalpha signaling in blood-derived inflammatory cells, and indicate that ERalpha expressed in other tissues, such as CNS-resident microglia or endothelial cells, mediates this effect

Identification of a second human retinoic acid receptor

International audienceWe have previously described a human complementary DNA that encodes a novel protein which is homologous to members of the steroid/thyroid nuclear receptor multigene family. This novel protein (hap for hepatoma) exhibits strong homology with the human retinoic acid receptor (RAR) which has been recently characterized. To test the possibility that the hap protein might also be a retinoid receptor, a chimaeric receptor was created by replacing the putative DNA binding domain of hap with that of the human oestrogen receptor (ER). The resulting hap-ER chimaera was then tested for its ability to trans-activate an oestrogen-responsive reporter gene (vit-tk-CAT) in the presence of possible receptor ligands. Here we show that retinoic acid (RA) at physiological concentrations is effective in inducing the expression of this reporter gene by the hap-ER chimaeric receptor. This demonstrates the existence of two human retinoic acid receptors designated RAR-alpha and RAR-beta