1. Dynamic and differential expression of the gonadal aromatase during the process of sexual differentiation in a novel transgenic cyp19a1a -eGFP zebrafish line
- Author
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Jean-Marc Porcher, Nathalie Hinfray, Edith Chadili, Morgane Caulier, Yann Guiguen, Benjamin Piccini, Frédéric Sohm, Camille Torchy, François Brion, Institut National de l'Environnement Industriel et des Risques (INERIS), Plateforme d'ingénierie génétique des animaux modèles (AMAGEN), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Physiologie et Génomique des Poissons (LPGP), Institut National de la Recherche Agronomique (INRA)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Agence Nationale de la Recherche, Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique )-Institut National de la Recherche Agronomique (INRA), This work was supported by the French National Ministry of Ecology and Sustainable Development [Program 190 ECOT AP 2013-2016, NEMO project and Program 181-DRC 50] and by the French National Research Program for Environmental and Occupational Health of Anses [AIDEZ project n° EST-2016/1/189]. AMAGEN received the support of the French National Research Agency (ANR) 'Investissement d’Avenir' program [TEFOR ANR-II-INSB-0014]., and ANR-11-INBS-0014,TEFOR,Transgenèse pour les Etudes Fonctionnelles sur les Organismes modèles(2011)
- Subjects
Male ,0301 basic medicine ,green fluorescent protein ,Embryo, Nonmammalian ,Sex Differentiation ,[SDV]Life Sciences [q-bio] ,sexual differentiation ,poisson zèbre ,Green fluorescent protein ,gonade ,Animals, Genetically Modified ,Endocrinology ,poisson ,cyprinidae ,Testis ,poisson transgenique ,Aromatase ,cyp19a1a ,Zebrafish ,protéine fluorescente verte ,endocrine disruptor ,perturbateur endocrinien ,Gene Expression Regulation, Developmental ,gonadal aromatase ,Cell biology ,medicine.anatomical_structure ,danio rerio ,lignée transgénique ,Female ,Development of the gonads ,danio devario ,différenciation sexuelle ,expression des gènes ,aromatase ,medicine.medical_specialty ,Gonad ,Transgene ,Green Fluorescent Proteins ,Biology ,gonad ,Gene Expression Regulation, Enzymologic ,reproduction ,03 medical and health sciences ,Internal medicine ,medicine ,Animals ,14. Life underwater ,transgenic zebrafish ,Gonads ,fish ,Sexual differentiation ,fungi ,ovaire ,Zebrafish Proteins ,biology.organism_classification ,Sexual dimorphism ,Germ Cells ,030104 developmental biology ,biology.protein ,Animal Science and Zoology ,ovary - Abstract
Accepted manuscript; In zebrafish, there exists a clear need for new tools to study sex differentiation dynamic and its perturbation by endocrine disrupting chemicals. In this context, we developed and characterized a novel transgenic zebrafish line expressing green fluorescent protein (GFP) under the control of the zebrafish cyp19a1a (gonadal aromatase) promoter. In most gonochoristic fish species including zebrafish, cyp19a1a, the enzyme responsible for the synthesis of estrogens, has been shown to play a critical role in the processes of reproduction and sexual differentiation. This novel cyp19a1a-eGFP transgenic line allowed a deeper characterization of expression and localization of cyp19a1a gene in zebrafish gonads both at the adult stage and during development. At the adult stage, GFP expression was higher in ovaries than in testis. We showed a perfect co-expression of GFP and endogenous Cyp19a1a protein in gonads that was mainly localized in the cytoplasm of peri-follicular cells in the ovary and of Leydig and germ cells in the testis. During development, GFP was expressed in all immature gonads of 20 dpf-old zebrafish. Then, GFP expression increased in early differentiated female at 30 and 35 dpf to reach a high GFP intensity in well-differentiated ovaries at 40 dpf. On the contrary, males consistently displayed low GFP expression as compared to female whatever their stage of development, resulting in a clear dimorphic expression between both sexes. Interestingly, fish that undergoes ovary-to-testis transition (35 and 40 dpf) presented GFP levels similar to males or intermediate between females and males. In this transgenic line our results confirm that cyp19a1a is expressed early during development, before the histological differentiation of the gonads, and that the down-regulation of cyp19a1a expression is likely responsible for the testicular differentiation. Moreover, we show that although cyp19a1a expression exhibits a clear dimorphic expression pattern in gonads during sexual differentiation, its expression persists whatever the sex suggesting that estradiol synthesis is important for gonadal development of both sexes. Monitoring the expression of GFP in control and exposed-fish will help determine the sensitivity of this transgenic line to EDCs and to refine mechanistic based-assays for the study of EDCs. In fine, this transgenic zebrafish line will be a useful tool to study physiological processes such as reproduction and sexual differentiation, and their perturbations by EDCs.
- Published
- 2018