Cloning and molecular characterization of an ovarian-derived (brain-like) P450 aromatase cDNA and development of a competitive RT-PCR assay to quantify its expression in the fathead minnow (Pimephales promelas)

In higher vertebrates, estrogens are key players in sexual differentiation and development and their production is essential for normal sexual development in both females and males. Cytochrome P450 aromatase (P450arom) is the steroidogenic enzyme responsible for the production of estrogens from aromatizable androgens. In this study P450arom cDNA was cloned in the fathead minnow (Pimephales promelas) and a RT-PCR assay developed to quantify its expression. By PCR strategy, we obtained a 2688 bp full-length P450arom cDNA from an ovary in mid-vitellogenic development, encoding a protein of 508 amino acids. The ovary-derived P450arom cDNA displayed highest amino acid identity with brain-type P450aroms (88.4% compared with goldfish brain P450arom). Northern blot analysis revealed a major transcript of 2.7 kb in female and male brain, but no signal was detected in either ovary or testis. A specific competitive RT-PCR assay was developed to quantify P450arom mRNA expression in brain, testis and ovary of fathead minnow. Target and competitor cRNA had similar amplification efficiencies in the linear range of PCR amplification. The sensitivity of the assay (36 fM competitor RNA) allowed us to quantify mRNA expression in tissues from individual fish. Using the competitive RT-PCR assay, it was established that the cloned P450arom was expressed in adult fathead minnow brain at levels between 10 and 100- fold higher than in ovary and testis. Expression levels of the P450arom in the ovary and testis samples were similar.