Sex-related changes in estrogen receptors and aromatase gene expression and enzymatic activity during early development and sex differentiation in the European sea bass (Dicentrarchus labrax) Mercedes Blázquez a, * , Alicia González a,1 , Maria Papadaki b , Constantinos Mylonas b , Francesc Piferrer a a Institut de Ciències del Mar, Consejo Superior de Investigaciones Científicas (CSIC), Passeig Marítim, 37-49, 08003 Barcelona, Spain b Hellenic Center for Marine Research, Institute of Aquaculture, P.O. Box. 2214, Iraklion, Crete 71003, Greece article info Article history: Received 19 February 2008 Revised 15 May 2008 Accepted 3 June 2008 Available online 8 June 2008 Keywords: Aromatase cyp19 Estrogens Estrogen receptors Sex differentiation Sea bass Dicentrarchus labrax abstract The present study addresses the role of aromatase and estrogen receptors in sex differentiation and development. With this purpose, a sea bass female- and a male-dominant group were obtained by suc- cessive size gradings since in this species females are already larger than males at the time of sex differ- entiation. Changes in cyp19a and cyp19b gene expression and enzymatic activity were monitored by a validated real-time PCR and a tritiated water assay, respectively, during early development and sex dif- ferentiation. Changes in mRNA expression of estrogen receptors, both erb1 and erb2, were also assessed during this period. Results show clear sex-related differences in cyp19a gene expression and enzymatic activity in gonads, with females exhibiting significantly higher levels than males at 150 days post hatch- ing (DPH), when histological signs of sex differentiation were evident. cyp19b gene expression and activ- ity in brain were detectable during early ontogenesis at 50 DPH but no clear sex-related differences were observed. Both erb1 and erb2 showed higher gene expression levels in testis than in ovaries around 200– 250 DPH, corresponding with the time of testicular differentiation and precocious male maturation, but no sex-related differences were found in the brain. Together these results indicate that in the European sea bass high expression levels of cyp19a are associated with ovarian differentiation and thus cyp19a can be considered as a suitable molecular marker of ovarian differentiation. However, the involvement of cyp19b in sex differentiation cannot be concluded. In addition, the higher levels of erb1 and erb2 in males versus females during sex differentiation, coinciding with precocious male maturation in the sea bass, suggest an important role for these receptors in testicular development and maturation. Ó 2008 Elsevier Inc. All rights reserved. 1. Introduction Sex steroids are implicated in fish gonadal differentiation and sexual behaviour (Devlin and Nagahama, 2002). Androgen admin- istration has generally resulted in masculinization, whereas estro- gens are essential for female differentiation (Piferrer, 2001; Devlin and Nagahama, 2002). P450 aromatase is the key enzyme respon- sible for the conversion of androgens into estrogens (Simpson et al., 2002). Two isoforms of cyp19, the gene that encodes P450 aromatase, termed cyp19a and cyp19b (also named cyp19A1 and cyp19A2), preferentially expressed in gonads and brain, respec- tively, are present in teleost fish. Treatment of sexually undifferen- tiated genotypic females with aromatase inhibitors resulted in the development of phenotypic males in several fish species (Piferrer et al., 1994; Guiguen et al., 1999; Kwon et al., 2000; Kitano et al., 2000). Moreover, in the Nile tilapia, high cyp19a expression and therefore high estrogen levels are required for ovarian differentia- tion (D’Cotta et al., 2001; Kwon et al., 2001). The classical mecha- nism of estrogen actions include binding specifically to their receptors (Bain et al., 2007). In this regard, three estrogen recep- tors, era, erb1 and erb2 (also named esr1, esr2b and esr2a, respec- tively) were isolated from fish, with different binding capacities, tissue-distribution, regulation and different expression patterns (Hawkins et al., 2000; Menuet et al., 2002; Hawkins and Thomas, 2004; Halm et al., 2004; Filby and Tyler, 2005; Greytak and Callard, 2007; Nagler et al., 2007). Detailed studies on the temporal pattern of expression of estro- gen receptors and/or aromatase during fish sex differentiation are scarce and mainly concentrated on their transcriptional regulation at the gonadal level, hence not considering the contribution of the brain (Guiguen et al., 1999; Kitano et al., 2000). Moreover, in those studies in which the brain was considered, the actual aromatase enzymatic activity that accounts for possible post-transcriptional 0016-6480/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2008.06.001 * Corresponding author. Fax: +34 932309555. E-mail address: blazquez@icm.csic.es (M. Blázquez). 1 Present address: Departamento de Fisiología y Farmacología, Facultad de Medicina, Universidad de Cantabria, Avda. Herrera Oria s/n 39011 Santander, Spain. General and Comparative Endocrinology 158 (2008) 95–101 Contents lists available at ScienceDirect General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen