Dynamic and differential expression of the gonadal aromatase during the process of sexual differentiation in a novel transgenic cyp19a1a-GFP zebrafish line

In most gonochoristic fish species, aromatase, the enzyme responsible for the synthesis of estrogens, has been shown to play a critical role in the process of sexual differentiation (SD). In zebrafish, that undergoes a juvenile intersex stage, there is still a lack of knowledge regarding the precise localization and pattern of expression of gonadal aromatase (Cyp19a1a) impeding the formal characterization of the role of cyp19a1a in this process. To fulfill this gap, the spatio-temporal expression of cyp19a1a was analyzed in a novel transgenic zebrafish model expressing Green Fluorescent Protein (GFP) under the control of the zebrafish cyp19a1a gene promoter. First, we showed a perfect co-expression of GFP and endogenous Cyp19a1a protein in adult gonads that was localized in the cytoplasm of oogonia, young oocytes and peri-follicular cells of the ovary and in the cytoplasm of Leydig and germ cells in the testis. Then, the spatio-temporal expression pattern of cyp19a1a was studied during SD using GFP fluorescence imaging in gonads at 20, 30, 35 and 40 day post-fertilisation (dpf). Our data showed that GFP was expressed in all undifferentiated gonads of 20 dpf-old zebrafish. At later stages of development, GFP expression increased in early differentiated female at 30 and 35 dpf to reach high GFP intensity in well-differentiated ovaries at 40 dpf. This pattern of gonadal GFP expression markedly differed from that of males which consistently displayed low GFP expression as compared to female whatever their stage of development, resulting in a clear dimorphic expression between males and females. Interestingly, fish that undergoes ovary-to-testis transition (35 and 40 dpf) presented an intermediary GFP level as compared to those measured in males and females. Our results suggest that (1) cyp19a1a is expressed early during gonadal development before the histological process of SD, (2) the down-regulation of cyp19a1a expression is critical for the testicular differentiation, (3) although cyp19a1a expression exhibit a clear dimorphic expression in gonads during SD, its expression persists whatever the sex suggesting that estradiol synthesis is important for gonadal development in both male and female. Monitoring the expression of the GFP in control and exposed-fish will help to identify the interest of this model to study the mechanisms of action of endocrine disrupting chemicals on critical physiological processes such as SD.