Communication in Genomics and Proteomics Cloning and developmental expression of the cytochrome P450 aromatase gene (CYP19) in the European eel (Anguilla anguilla) Itai Tzchori a,b, * , Gad Degani b,c , Avshalom Hurvitz b , Boaz Moav a a Department of Zoology, Tel Aviv University, Tel Aviv 69978, Israel b MIGAL, Galilee Technological Center, Kiryat Shmona 10200, Israel c School of Science and Technology, Academic Tel-Hai College, Israel Received 23 February 2004; revised 7 June 2004; accepted 21 June 2004 Available online 13 August 2004 Abstract To characterize the involvement of the aromatase gene during the process of sex determination in the European eel (Anguilla anguilla), the expression of its gonadal form was determined during various developmental stages. The cloned cDNA from the Euro- pean eel gonad (EeCYP19) contains an open reading frame of 1539 bp, encoding a deduced protein of 513 residues. The predicted amino acid sequence shows 97% identity with that of the Japanese eel, and 59–69% of identity with those of the CYP19 gonadal and brain forms of other teleost fish. Two potential initiation sites (ATG) were found downstream of the first ATG codon. A fluorescent- based method of real-time PCR was developed to quantify EeCYP19 expression. The expression levels of EeCYP19 in the gonads of adult males were approximately 12- and 30-fold lower than the levels in adult females and juvenile eels previously treated with E2, respectively. Expression of aromatase was found only in a single specimen in the control group. In contrast, no difference was found among sexes in the aromatase expression in the brain. Treatment with aromatase inhibitor (AI) of juvenile eel resulted in the total loss of aromatase expression in the gonads and brains. The results of this work revealed that AI treatment not only reduces the synthesis of estradiol, but reduces the expression levels of EeCYP19 as well. No evidence for the presence of a distinct extra-gonadal (brain) form of aromatase in the European eel could be provided. Ó 2004 Elsevier Inc. All rights reserved. Keywords: European eel; Sex determination; Estradiol-17b; CYP19; Expression; Aromatase inhibitor; Real-time PCR 1. Introduction Steroids play an important role in gonadal differenti- ation in lower vertebrates (Bogart, 1987; Devlin and Nagahama, 2002; Yamamoto, 1969). Treating fish with exogenous sex steroid hormones during early develop- mental stages may cause sex-inversion in spite of the genotypic sex. It has been demonstrated in many fish species that exogenous androgens generally have a masculinizing effect, while exogenous estrogens have feminizing effects (Yamazaki, 1983). However, the mechanism of action of these steroids on sex differentia- tion is not well understood. Estrogens are C-18 steroids indispensable for normal sexual development of the female, but have been associ- ated with sexual function in male too (Devlin and Naga- hama, 2002). The synthesis of estrogens is dependent on the activity of the steroidogenic enzyme P450 aromatase (CYP19). CYP19 catalyses aromatization of the A ring of C19 steroids to form the phenolic ring characteristic of estrogens, with concomitant loss of the C19 angular methyl group as formic acid. CYP19 activity is an important modulator of E2 concentration and is critical to the regulation processes controlled by E2 (Brodie, 1991). 0016-6480/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2004.06.007 * Corresponding author. Present address: Bldg 6B, LMGD/ NICHD, National Institutes of Health, Bethesda, MD, USA. Fax: +301-402-0543. E-mail address: tzchorii@mail.nih.gov (I. Tzchori). www.elsevier.com/locate/ygcen General and Comparative Endocrinology 138 (2004) 271–280 GENERAL AND COMPARATIVE ENDOCRINOLOGY