Marie-Lise Thieulant

Androgens Upregulate cyp19a1b (Aromatase B) Gene Expression in the Brain of Zebrafish (Danio rerio) Through Estrogen Receptors

Abstract The brain of teleosts is known for its strong aromatase expression, exhibiting unique features compared with other vertebrates. Among these features is the high sensitivity of aromatase B (the product of cyp19a1b) to estrogens. This effect involves the binding of estrogen receptors on an estrogen-responsive element (ERE) of the cyp19a1b promoter. Given the presence of potential androgen-responsive elements (AREs) on this promoter, in vivo and in vitro effects of androgens were studied. Using immunohistochemistry and quantitative PCR on zebrafish embryos, we found that cyp19a1b is upregulated by testosterone, an aromatizable androgen, and by 5alpha-dihydrotestosterone (DHT), a nonaromatizable androgen, suggesting a potential androgenic regulation of cyp19a1b through androgen receptors (ARs). To assess a putative direct regulation of the cyp19a1b gene by ARs, we transfected U251MG cells with zebrafish AR together with a luciferase reporter gene driven by 3000 bp of the proximal cyp19a1b promoter containing the ERE and potential AREs. Interestingly, although zebrafish AR activated luciferase reporter genes controlled by AREs, they failed to induce the cyp19a1b-luciferase construct. These data suggest that the androgenic regulation of cyp19a1b does not involve AR. We further showed that regulation of the cyp19a1b gene by testosterone is, in fact, due to aromatization, whereas the effect of DHT involves conversion into 5alpha-androstane-3beta,17beta-diol (betadiol), a metabolite of DHT with known estrogenic activity. The blockage of the androgen regulation of cyp19a1b expression using antiestrogens further confirmed the involvement of estrogen receptors in mediating these effects.

Synthesis of estrogens in progenitor cells of adult fish brain : Evolutive novelty or exaggeration of a more general mechanism implicating estrogens in neurogenesis?

In contrast to other vertebrates, in which the adult brain shows limited adult neurogenesis, teleost fishes exhibit an unparalleled capacity to generate new neurons as adults, suggesting that their brains present a highly permissive environment for the maintenance and proliferation of adult progenitors. Here, we examine the hypothesis that one of the factors permitting establishment of this favourable environment is estradiol. Indeed, recent data showed that radial glial cells strongly expressed one of two aromatase duplicated genes. Aromatase is the estrogen-synthesizing enzyme and this observation is of great interest, given that radial glial cells are progenitor cells capable of generating new neurons. Given the well-documented roles of estrogens on cell fate, and notably on cell proliferation, these data suggest that estradiol could be involved in maintaining and/or activating these progenitors. Examination of recent data in birds and mammals suggests that the situation in fish could well be an exaggeration of a more general mechanism implicating estrogens in neurogenesis. Indeed, there is accumulating evidence that estrogens are involved in embryonic, adult or reparative neurogenesis in other vertebrates, notably in mammals.

Testosterone “receptors” in purified nuclei of rat anterior hypophysis

Abstract In this work, the presence of two macromolecular associations of [ 3 H]-testosterone in purified nuclei of rat anterior hypophysis was demonstrated. After incubation of slices of hypophyseal tissue with [ 3 H]-testosterone purified nuclei were obtained free from cytoplasmic contamination. After extraction with M NaCl and gel filtration on Sephadex G 200, two peaks of bound radioactivity were obtained. The first, with a concentration of 0·6 × 10 −13 mol by mg of proteins is excluded from gel Sephadex G 200, the second with a concentration of 2·1 × 10 −13 mol by mg proteins is retained on this gel. There is no binding with histones. The radioactive material bound to the nuclear “receptors” is mainly testosterone. The amount of dihydrotestosterone is small. Again, we have noted the presence of a non polar steroid. They represent respectively 66,7 and 7% of the radioactivity of the chromatogram.

Binding and metabolism of [3H]-testosterone in the nuclei of rat pituitary in vivo

Bound radioactivity in the nuclei of the rat anterior hypophysis reached a maximum 60 min after i.p. injection of (3H)-testosterone. At this time, the level of nuclear radioactivity was 7 times greater than that of the cytoplasmic fraction. The nuclear radioactivity consisted of bound (61%) and unbound radioactivity. In the nuclei of the pituitary gland, [3H]-testosterone represents 63 % of the total radioactivity and dihydrotestosterone 21%.

Œstrogènes et neurogenèse : de nouvelles fonctions pour une vieille hormone. Leçons tirées du poisson zèbre

In contrast to other vertebrates, in which the adult brain shows limited adult neurogenesis, teleost fish exhibit an unparalleled capacity to generate new neurons as adults, suggesting that their brains present a highly permissive environment for the maintenance and proliferation of adult progenitors. Here, we examine the hypothesis that one of the factors permitting establishment of this favourable environment is estradiol. Indeed, recent data showed that radial glial cells strongly expressed one of two aromatase duplicated genes. Aromatase is the estrogen-synthesizing enzyme and this observation is of great interest, given that radial glial cells are progenitor cells capable of generating new neurons. Given the well documented roles of estrogens on cell fate, and notably on cell proliferation, these data suggest that estradiol could be involved in maintaining and/or activating these progenitors. Examination of recent data in birds and mammals suggests that the situation in fish could well be an exaggeration of a more general mechanism implicating estrogens in neurogenesis. Indeed, there is accumulating evidence that estrogens are involved in embryonic, adult or reparative neurogenesis in other vertebrates, notably in mammals.

BINDING AND EFFECTS OF 5α-ANDROSTANE-3β, 17β-DIOL IN THE MALE RAT PITUITARY

Summary 5α-androstane-3β,17β-diol (Adiol) binds to cytosol proteins from male rat pituitary with a relatively high affinity (KD = 15 ± 6 nM) and a low capacity (n = 92 ± 8 fmol/mg protein). These saturable proteins which bind Adiol are characterized as estrogen receptor. This conclusion was based on the binding characteristics, the binding stereospecificity and the sedimentation coefficient in sucrose linear gradients. Moreover, Adiol induces, in vivo, the nuclear translocation of estrogen receptor and some effects of estrogen action. It is efficient to induce progesterone receptor and to increase pituitary protein content but inefficient to increase DNA synthesis. Results suggest a mechanism of Adiol action in the male rat pituitary similar to that observed with androgens in other target tissues. Moreover, the study of Adiol and 17β-estradiol binding suggests two forms of estrogen receptor in the cytosol from male rat pituitary. The maximal concentration of binding sites was observed at 22–30 days of age for E2 and at 37–42 days of age for Adiol. On the other hand, the nuclear ontogenic pattern suggested a single class of binding sites for E2 and Adiol in the pituitary nuclei.

Estrogen effects of Daldinia concentrica and Psathyrella efflorescens extracts in vitro

Daldinia concentrica and Psathyrella efflorescens are two fungi used in African traditional medicine. In the present study, their extracts were evaluated for their steroid activities in estrogen- or androgen-dependent cell lines using as endpoints steroid-dependent transcriptional activity and cell proliferation. Treatment of human breast cancer MCF-7 cells with 15 or 30 microg/ml of Daldinia concentrica or Psathyrellaefflorescens extracts in the absence of 17beta-estradiol (E2) significantly increased the transcriptional activity of an estrogen receptor (ER)-dependent reporter gene, in the same range as E2. Similar data were obtained in gonadotrope cell line alpha-T3-1. All the effects were prevented by the pure estrogen antagonist, ICI 182,780. In the absence of steroid addition, the two extracts induced cell proliferation of ER-dependent MCF-7 and Ishikawa Var-I cell lines by approximately 100% of the E2 response. Combination treatments with E2 showed no competitive or additive effects in the two latter cell lines. Interestingly, the extracts had no androgen-like response in androgen receptor (AR)-positive and ER-negative MDA-MB231 cells, suggesting that fungi effects are estrogen specific and extracts are not toxic at used concentrations. Results provided evidence that Daldinia concentrica or Psathyrellaefflorescens extracts induce estrogen-like effects in ER-positive cell lines, which could be responsible of the effects observed in vivo.

Effets œstrogène-like des extraits de Dadinia concentrica et de Psathyrella efflorescens au niveau moléculaire

RésuméLes extraits de Daldinia concentrica et de Psathyrella efflorescens ont été évalués pour leurs activités œstrogène-like sur des cellules œstrogènes ou androgènes dépendantes. Les extraits de D. concentrica ou de P. efflorescens augmentent significativement l’activité transcriptionnelle des gènes dépendant des récepteurs œstrogènes. Ces effets sont inhibés par un antagoniste pur de l’œstrogène, l’ICI 182,780. Les deux extraits induisent une prolifération des cellules de la lignée ER-dépendant MCF-7 et-Ishikawa-Var-1 avec approximativement 100 % de la réponse de E2. Une combinaison de traitements avec E2 ne montre aucun effet compétitif ou additif dans les deux dernières lignées cellulaires. Les extraits n’ont aucune réponse androgène-like dans les cellules AR-positif et ER-négatif MDA-MB231, suggérant que les effets des champignons sont spécifiquement œstrogéniques. Ces résultats prouvent que ces extraits induisent un effet œstrogène-like dans les lignées cellulaires ER-positif qui pourraient être responsables des effets observés in vivo.AbstractExtracts of Daldinia concentrica and Psathyrella efflorescens were evaluated for their estrogen-like activities in estrogen-or androgen-dependent cell lines. D. concentrica or P. efflorescens extracts significantly increased the transcriptional activity of estrogen receptor (ER)-dependent reporter genes. All the effects were prevented by the pure estrogen receptor antagonist, ICI 182,780. The two extracts induced cell proliferation of ER-dependent MCF-7 and-Ishikawa-Var-1 cell lines by approximately 100 % of the E2 response. Combination treatments with E2 showed no competitive or additive effects in the two latter cell lines. Interestingly, the extracts had no androgen-like response in androgen receptor (AR)-positive and ER-negative MDA-MB231 cells, suggesting that fungi effects are estrogen specific. These results provide evidence that the extracts induce estrogen-like effects in ER positive cell lines, which could be responsible for the effects observed in vivo.

5α-DHT metabolism in monolayer cultures of distinct pituitary cell populations

Abstract 5α-Dihydrotestosterone (DHT) metabolism into 5α-androstane-3α, 17β-diol (α-diol) and 5α-androstane-3β, 17β-diol (β-diol) was studied in monolayer cultures of distinct cell populations from prepubertal male rats pituitaries. Cells were characterized through immunocytochemistry with the various antihormone antisera. Centrifugal elutriation was used to prepare a gonadotrope-enriched population “G” and a gonadotrope-depleted population “L”, containing most lactotropes and somatotropes. Using centrifugation on Percoll gradient, two sub-populations, P1 and P2, were prepared by further fractionation of the “L” population. Cells were incubated for 48 h with [3H]DHT (1 μM, sp. act. 0.9 Ci/mmol) and metabolites extracted from the whole cell and medium. DHT was metabolized to about the same extent (30–40%) in all cell fractions. Compared with unfractionated population, the conversion of DHT into a-diol increased significantly in the P1 fraction, consisting of lactotropes, somatotropes and highly depleted in gonadotropes. This increase was lower in the somatotrope-enriched P2 fraction in which the amount of lactotropes was similar to P1 but that of gonadotropes slightly higher. In contrast, the conversion of DHT into α-diol decreased significantly in the “G” population compared with total or “L” fractions, whereas androstanedione formation, low in every population, increased significantly. The increase in a-diol formation could be related either to the decrease of gonadotropes or to a role of non-immunoreactive cells. As the β-diol formation was constant in all cell types, the β-diol/α-diol amount increased significantly in gonadotropes. Then, β-diol and DHT could be both active steroids in gonadotrope regulation inasmuch as specific binding sites were identified for these two steroids. It can be concluded that DHT action at the pituitary level is subject to complex control mechanisms involving a specific balance of its metabolites in each particular cell type.

Antiestrogen binding within different pituitary cell populations. Comparison with androgen and estrogen receptors

Gonadotrope-enriched populations were prepared from 42-day old male rats by centrifugal elutriation. They contained 4.8 +/- 0.7% of the cells, 51 +/- 10% of the LH and less than 3% of the PRL (n = 4). Gonadotrope-depleted fractions were also obtained that contained most of PRL cells. Specific antiestrogen binding sites (AEBS) were quantitated in these populations after destruction of estrogen receptor. Results showed the presence of a distinct, specific high affinity binding site for antiestrogen in dispersed pituitary cells and in enriched fractions. However, AEBS are not specific of a pituitary cell type. Thus, AEBS appear different from estrogen receptors in pituitary gland: by the thermal stability of AEBS, by the localization of AEBS in particulate material, by the uniform distribution of AEBS in different populations which differ markedly for E2 binding sites. Whereas the ratio of binding AE/E2 averaged 11.4 in the initial cell suspension it reached only 2.9 in the gonadotropes. The dissociation constants for AEBS were in the same range (1.16 - 2.27 X 10(-9) M) for the different populations.