Analysis of the goldfish (Carassius auratus) aromatase (P450arom) gene promoters by green fluorescent protein (GFP) expression in living zebrafish (Danio rerio) embryos

It is well established that estrogen has permanent, organizational effects on CNS development. Although estrogen is generally regarded as a circulating hormone derived from the gonads, access of estrogen to neural targets is limited by high levels of plasma binding proteins or neural metabolizing enzymes. Thus, developmental effects are dependent on estrogen formed from circulating androgen at sites of action within the brain itself. Aromatization of androgen to estrogen is catalyzed by cytochrome P450 aromatase (P450arom), a product of the CYP19 gene, and this transformation regulates the quantity of ligand available for binding to estrogen receptors. Teleosts are unique in having exceptionally high brain levels of P450arom when compared to the ovaries of the same fish (300-fold higher) or to the brains of other vertebrates (100- to 1000-fold higher). This makes teleosts a good model to study the synthesis and action of estrogen in the brain. We previously reported that the goldfish has two P450arom isoforms which are encoded by separate and unique gene loci, CYP19A and CYP19B. Neural tissues express high levels of P450aromB and much lower levels of P450aromA, whereas P450aromA is the only isoform expressed in the ovary where levels are very low. Recently we isolated and cloned the 5'-flanking regions of the two CYP19 genes. Sequence analyses showed a low percent identity between the promoter regions of CYP19A and B, suggesting that they differ with respect to critical cis-acting regulatory elements. Analysis of fish promoters is hampered by the lack of suitable fish cell lines and their resistance to transfection. Given the close taxonomic relationship of goldfish and zebrafish, the goal of the present study was to test the utility of the zebrafish embryo as an in vivo whole animal system for functional analysis of CYP19 gene transcription.