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haracterization of vtg-1 mRNA expression during ontogeny in Oreochromis ossambicus ( PETERS )

The yolk-precursor lipoprotein, vitellogenin (VTG) has been widely recognized as a biomarker for the detection of estrogenic activity in water-borne chemical pollutants. We characterized the expression status of this important constituent of reproduction in the Mozambique tilapia (Oreochromis mossambicus), a tilapiine freshwater fish species indigenous to Southern Africa, and investigated its utility in detection of exposure to estrogen using a quantitative real-time polymerase chain reaction (QPCR) assay. We initially isolated a 3 kb upstream promoter region of the vtg gene and identified putative binding sites for several regulatory factors including estrogen receptor (ESR). Evidence for the expression of several splice-site vtg mRNA variants was found in a number of tissue types. A quantitative real-time polymerase chain reaction (QPCR) assay was subsequently developed based upon a specific primer pair (OMV6/9) that selectively amplified the liver-enriched transcript. The level of this transcript in liver tissue was high in females and lower, but detectable, in males and was significantly increased in male fish following laboratory exposure to 17 -estradiol (E2). This study further established that juvenile whole body homogenates (WBHs) contained extremely low levels of liver-specific vtg mRNA between 5 and 110 days post-fertilization (dpf) compared to adult male liver. Subsequent exposure of 20 dpf juveniles to E2 showed a substantial increase in this transcript within hours, and when compared to classic male model under same conditions, the juveniles were remarkably more sensitive. We therefore conclude that the quantification, using QPCR methodology, of vtg mRNA expression in 20 dpf O. mossambicus juveniles has promise for assessing aqua estrogenic EDC activity in