Cloning Of Cyp19a1 Genes And Effects Of Endocrine Disrupting Chemicals On Cyp19a1 Of Rare Minnow

Cytochrome P450 aromatase encoded by cyp19a1 genes is an important steroidogenic enzyme responsible for conversion of androgens to estrogens at rate-limiting step. The regulation of cyp19a1 gene can alter the production of estrogen, disturbing the local and total level of estrogen. There are two subtypes of cyp19a1 gene, cyp19a1a and cyp19a1b. Cyp19a1a is predominantly expressed in ovary while cyp19a1b is mainly in brain. Cyp19a1 genes are involved in sex determination and differentiation.To elucidate the effects of endocrine disrupting chemicals (EDCs) on aromatase and then predict the adverse effects of EDCs on vertebrate and human, the rare minnow ovarian and brain P450 aromatase (cyp19a1a and cyp19a1b) cDNA were isolated and characterized using homology cloning and RACE strategy. The fragments for both transcripts were aligned to produce a 2239 bp cyp19a1a and a 2167 bp cyp19a1b full-length cDNA sequence. The coding region of cyp19a1a and cyp19a1b encoded a putative peptide of 520 and 507 amino acids, respectively. The similarity of amino acids between rare minnow and other vetebrates was above 90%. Several conserved functional domains were found in the amino acid sequence of the rare minnow. And they were I-helix (involved in steroid substrate binding), Ozol's peptide, aromatase-specific conserved region, and heme-binding region. Tissue distribution was analyzed with semi-quantitative RT-PCR. The results revealed that the rare minnow cyp19a1a mRNA was predominantly expressed in ovary while cyp19a1b was predominantly expressed in brain with low expression in other tissues.The gemomic DNA of rare minnow was extracted using phenol-chloroform method. The 5'flanking regions of cyp19a1 genes were isolated using genome walking method. Using TESS software, sequences for binding sites of steroidogenic factor-1, retinoic acid receptor, Wilm Tumor 1 (WT1-KTS), estrogen responsive element half-sites, glucocorticoid responsive element (GRE) half-sites, sex-determining region Y gene product (SRY) , CCAAT/enhancer binding protein, GATA transcription factor 1 (GATA-1) and activator protein 1 (AP-1) were identified on promoter regions of cyp19a1a gene. Besides SF-1, RAR, GR, C/EBP, GATA-1, sequences for binding sites of peroxisome proliferators-activated receptor, aryl hydrocarbon receptor, cAMP-responsive element binding protein and estrogen responsive element were identified on promoter regions of cyp19a1b gene.The rare minnow juveniles of 31 dpf were exposed to EDCs (clofibrate,EE2,NP,BPA). Each EDCs contains three concentrations and each concentration contains 6 random fish. After 3 d exposure, the total RNA was extracted from the whole fish. And then the influence of several EDCs on the transcript abundance of cyp19a1a and cyp19a1b was investigated in rare minnow juveniles. Clofibrate did not influence the expression of either cyp19a1 genes. Exposure to 1 nM ethinylestradiol (EE2) for 3 days significantly downregulated the expression of cyp19a1a gene, however 0.1 and 1 nM EE2 significantly increased the gene expression of cyp19a1b. Exposure to 0.1 and 1μM Nonylphenol (NP) significantly suppressed the cyp19a1a expression, but it had no effect on the expression of cyp19a1b gene.Bisphenol A (BPA) strongly suppressed the cyp19a1b gene expression from 0.1 to 10 nM and significantly suppressed the gene expression of cyp19a1a only at 10nM. These results indicate that EDCs may influence the expression of cyp19a1 genes through differential transcriptional modulation and the other adjusting modes in rare minnow juveniles. Cyp19a1 can be a target molecular for EDCs. And it also helps to predict the adverse effects of EDCs on vertebrate and human.