[show abstract][hide abstract] ABSTRACT: 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.
Journal de la Société de Biologie 02/2009; 203(1):29-38.
[show abstract][hide abstract] 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.
[show abstract][hide abstract] ABSTRACT: 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.
Journal of Ethnopharmacology 03/2008; 116(1):152-60. · 2.76 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have characterized the intronic promoter of the rat estrogen receptor (ER) alpha gene, responsible for the lactotrope-specific truncated ER product (TERP)-1 isoform expression. Transcriptional regulation was investigated by transient transfections using 5'-deletion constructs. TERP promoter constructs were highly active in MMQ cells, a pure lactotrope cell line, whereas a low basal activity was detected in alphaT3-1 gonadotrope cells or in COS-7 monkey kidney cells. Serial deletion analysis revealed that 1) a minimal -693-bp region encompassing the TATA box is sufficient to allow lactotrope-specific expression; 2) the promoter contains strong positive cis-acting elements both in the distal and proximal regions, and 3) the region spanning the -1698/-1194 region includes repressor elements. Transient transfection studies, EMSAs, and gel shifts demonstrated that estrogen activates the TERP promoter via an estrogen-responsive element (ERE1) located within the proximal region. Mutation of ERE1 site completely abolishes the estradiol-dependent transcription, indicating that ERE1 site is sufficient to confer estrogen responsiveness to TERP promoter. In addition, ERalpha action was synergized by transfection of the pituitary-specific factor Pit-1. EMSAs showed that a single Pit-1 DNA binding element in the vicinity of the TATA box is sufficient to confer response by the TERP promoter. In conclusion, we demonstrated, for the first time, that TERP promoter regulation involves ERE and Pit-1 cis-elements and corresponding trans-acting factors, which could play a role in the physiological changes that occur in TERP-1 transcription in lactotrope cells.
[show abstract][hide abstract] ABSTRACT: To explain the effect of estrogen on skeletal muscle, the presence of estrogen receptor alpha mRNA (ERalpha mRNA) was investigated in human skeletal muscle.
The highly sensitive technique of nested reverse transcriptase-polymerase chain reaction (nested RT-PCR) was applied on a variety of tissue samples of both sexes: women (deltoid, pectoral, and uterus muscles) (N= 3) and men (deltoid muscle) (N= 3). The total ribonucleic acid was isolated from each tissue sample, reverse transcribed in a thermocycler, and nested PCR was then performed with specific primers. The by-products were analyzed by agarose gel electrophoresis. Internal standard 28S was simultaneously amplified. The ERalpha mRNA level was quantitated by using the ERalpha mRNA/28S mRNA ratio.
The expected 204-bp product corresponding to ERalpha was amplified in all tested tissue samples, i.e., deltoid, pectoral, and uterine muscles from women and deltoid muscle from men. The ERalpha mRNA/28S mRNA ratios indicating the receptor expression levels in deltoid muscle from men and women were 0.945 +/- 0.393 (mean +/- SD) (N= 3) and 0.973 +/- 0.136 (mean +/- SD) (N= 2), respectively.
In conclusion, the nested RT-PCR technique identified the presence of transcript encoding ERalpha mRNA in human skeletal muscles. Semi-quantification did not reveal gender difference.
Medicine & Science in Sports & Exercise 04/2003; 35(3):439-43. · 4.48 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have characterized the intronic promoter of the rat estro- gen receptor (ER) gene, responsible for the lactotrope- specific truncated ER product (TERP)-1 isoform expression. Transcriptional regulation was investigated by transient transfections using 5-deletion constructs. TERP promoter constructs were highly active in MMQ cells, a pure lactotrope cell line, whereas a low basal activity was detected in T3-1 gonadotrope cells or in COS-7 monkey kidney cells. Serial deletion analysis revealed that 1) a minimal 693-bp region encompassing the TATA box is sufficient to allow lacto- trope-specific expression; 2) the promoter contains strong positive cis-acting elements both in the distal and proximal regions, and 3) the region spanning the 1698/1194 region includes repressor elements. Transient transfection stud- ies, EMSAs, and gel shifts demonstrated that estrogen ac- tivates the TERP promoter via an estrogen-responsive ele- ment (ERE1) located within the proximal region. Mutation of ERE1 site completely abolishes the estradiol-dependent transcription, indicating that ERE1 site is sufficient to con- fer estrogen responsiveness to TERP promoter. In addition, ER action was synergized by transfection of the pituitary- specific factor Pit-1. EMSAs showed that a single Pit-1 DNA binding element in the vicinity of the TATA box is sufficient to confer response by the TERP promoter. In conclusion, we demonstrated, for the first time, that TERP promoter reg- ulation involves ERE and Pit-1 cis-elements and corre- sponding trans-acting factors, which could play a role in the physiological changes that occur in TERP-1 transcription in lactotrope cells. (Endocrinology 144: 2845-2855, 2003)
[show abstract][hide abstract] ABSTRACT: The aim of this study was to examine whether the expression levels of mRNA of the three estrogen receptor (ER) subtypes, ERalpha, ERbeta, and truncated ER product-1 (TERP-1) found in the rat pituitary gland were modified during gestation, lactation, and postlactation periods. By using relative quantitative RT-PCR, we found that ERalpha mRNA significantly peaked in midpregnancy. However, the ERalpha protein level remained constant. ERbeta gene expression did not change throughout pregnancy, suggesting that it was not related to estradiol levels during this reproductive period. In contrast, both TERP-1 mRNA and protein levels dramatically increased throughout the second half of gestation, being faintly detectable in early pregnancy. TERP-1 expression was rapidly reversed by lactation, whereas neither pituitary ERalpha nor ERbeta relative levels were significantly altered. In addition, pup removal for 24-96 h on d 9 postpartum significantly reduced the expression of both ERalpha and ERbeta mRNA compared with that in lactating animals, but the expression of TERP-1 mRNA was no longer detected. Collectively, our data indicate that 1) TERP-1, ERalpha, and ERbeta expression levels are differentially regulated in the pituitary; 2) TERP-1 is variably expressed depending on the hormonal environment related to the estrous cycle, pregnancy, and lactation; and 3) TERP-1/ERalpha ratios dramatically change depending on reproductive periods, suggesting a critical role for TERP-1 in reproductive events.
[show abstract][hide abstract] ABSTRACT: It is now widely accepted that radial glial cells play a fundamental role in the process of embryonic neurogenesis. In contrast to mammals, radial glial cells are maintained during adulthood in teleost fishes and this persistence is likely to be related to the intense neurogenic activity of the adult fish brain. We and others have recently shown that radial glial cells are progenitor cells capable of generating new neurons in adult zebrafish (Danio rerio). More precisely, we showed that aromatase B (CYP19B, AroB), the synthetic enzyme of estradiol, is strongly expressed in a subset of radial cells, essentially in the forebrain of adult males and females. We also demonstrated that many of these aromatasepositive radial cells have mitotic activity and generate neurons. The purpose of this work is to further characterize cells presenting proliferative activity by looking at other radial cells markers. Among potential markers, BLBP (Brain lipid binding protein) appeared pertinent as it is is highly expressed in the developing central nervous system of mammals and considered as a marker of radial glial cells and immature astrocytes. BLBP (Fabp7 or BFABP) is a brain specific member of the large family of hydrophobic ligand-binding protein (Fatty Acid Binding Proteins: FABPs). In the present study, we compare the expression of BLBP in radial glial cells of the zebrafish with that of AroB using immunohistochemistry. The data showed that BLBP is highly expressed in radial glial cells of the entire brain. In the regions where AroB is strongly expressed, there is a good correlation between the distribution of AroB-positive and BLBP-positive radial glial cells. Double staining for AroB and BLBP showed that most of radial glial cells of the forebrain coexpress the two markers; however, some express only one. We also performed double staining with BLBP and PCNA (Proliferating Cell Nuclear Antigen). Data showed that most BLBP-positive radial glial cells are also PCNA-positive cells, suggesting that the BLBP-positive cells are able to divide. The in vivo data presented here extend previously published data on radial glial markers in adult fish and demonstrate that BLBP is an excellent marker of radial glial cells of the zebrafish adult brain. Nevertheless, the present data suggest that heterogeneity occurs within the radial glial cell population. A more detailed study is thus needed to further characterize the phenotype of mitotically-active radial glial cells in adult fish.
11EME journée scientifique du réseau LARC Neurosciences.
[show abstract][hide abstract] ABSTRACT: 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.