Aromatase cytochrome P450:: Cloning, intron variation, and ontogeny of gene expression in southern flounder (Paralichthys lethostigma)

Aromatase cytochrome P450 (P450(arom)) is the enzyme complex responsible for conversion of androgens to estrogens in vertebrates. Consequently, in some fishes its activity appears critical to ovarian differentiation. Southern flounder (Paralichthys lethostigma) is a commercially important flatfish in which females grow larger than males and sex determination is temperature sensitive. Through cloning of the P450(arom), gene in ovary and quantitative reverse transcription-polymerase chain reaction, we developed a biomarker for early female differentiation in southern flounder. The deduced amino acid sequence for southern flounder P450(arm), is similar to other teleosts. Comparison of P450(arom) intron sequences from fish of different populations revealed substantial inter-individual variation. Adult ovary and spleen exhibited high levels of P450(arom) mRNA, while P450(arom) mRNA was only weakly detected in testes. Brain, liver, intestine, kidney, gill, muscle, and heart showed little or no P450(arom) mRNA expression. Gonads of wild and hatchery-produced juvenile flounder of sizes spanning the period of sex differentiation initially exhibited low levels of P450arom mRNA followed by increases in some individuals and bifurcation into two clearly segregated groups (i.e., putative males and females) beginning at similar to 65 mm in total length. Gonadal histology confirmed predictions of sex based on P450(arom) expression in juvenile flounder, demonstrating that the patterns of P450(arom) expression observed relate to sex-specific differentiation. This research represents a unique approach to assessing sex differentiation in a natural population, and a powerful technique for better understanding mechanisms of flounder sex determination and rapidly defining conditions for controlling sex for aquaculture.