Real-time PCR analysis of cytochrome P450 aromatase expression in zebrafish: gene specific tissue distribution, sex differences, developmental programming, and estrogen regulation.

In teleost fish, the predominant brain form of cytochrome P450 aromatase (P450aromB) is a neural marker of estrogen effect, and an entry point for studying the role of hormonal and environmental estrogens on neurodevelopment and neuroplasticity. As part of a project using zebrafish to investigate these issues, we developed and validated a rapid, sensitive, and reproducible real-time polymerase chain reaction (PCR) assay for quantifying and comparing P450aromB and P450aromA expression in unfertilized eggs, embryos/larvae, and dissected tissues of adult fish. Results confirm that P450aromB and -A predominate in brain and ovary, respectively, and further show that the degree of overlapping expression (ratio, B:A) is 100:1 in brain, 1:50 in ovary, 1:1 in eye, and 2:1 in testis. Sex differences were observed in eye only (female>male). When compared to whole ovaries, unfertilized eggs had similar levels of P450aromA but enrichment of P450aromB, which suggests preferential synthesis or accumulation in mature oocytes. Both of the maternally derived aromatase isoforms were rapidly degraded post-fertilization, but the onset of embryonic P450aromB expression (5 hpf) was much earlier than P450aromA (48 hpf), and reached higher maximum levels (e.g., 10-fold at 72 hpf). Consistent with earlier reports, P450aromB but not -A was estrogen-inducible, but the estrogen response system in embryos was far more robust than in adults (>100- vs. <4-fold maximal induction, respectively). Application of this real-time PCR assay to measurement of P450aromB and -A in zebrafish embryos has utility for routine screening of chemicals and environmental samples for estrogen-like bioactivity and neural effects.