Localisation et expression d'enzymes de la stéroïdogenèse et de régulateurs transcriptionnels dans les gonades du poisson zèbre

: intérêt d'une approche basée sur l'imagerie en microscopie

Abstract

Endocrine disrupting chemicals (EDCs) are known to interfere with the endocrine system leading to adverse effects on the reproductive health of organisms. During fish reproductive cycle , steroidogenesis plays an important role since it allows the biosynthesis of steroid hormones (including androgens and estrogens). This physiological process is extremely sensitive to EDCs. In zebrafish, a model species widely used in developmental biology and ecotoxicology, there is still a lack of knowledge regarding the gonadal steroidogenesis. This lack of knowledge clearly impedes the study of the mechanisms of action of EDCs on physiological processes related to steroidogenesis such as sexual differentiation.
In order to improve this knowledge, we developed new tools to study the expression of steroidogenic enzymes (Star, Cyp11a1, Hsd3b1, Cyp17a1, Cyp11c1, Cyp19a1a, Cyp19a1b) and some of their regulators (Foxl2a, Fsta, Fstb) in zebrafish, in a tissue and individual-specific way : in toto hybridization, immunofluorescence and zebrafish transgenic lines (Cyp19a1b-GFP, Cyp19a1a-GFP and Cyp11c1-GFP). Using these techniques, we characterized the spatial and temporal expression profiles of these targets in differentiated and differentiating gonads in this species.
We identified all the sites of expression of steroidogenic enzymes in adult gonads of zebrafish. Their localization in both germ cells and somatic cells confirmed the results obtained in other fish species. However, we characterized for the first time the germinal expression of Cyp19a1b in zebrafish gonads. We also showed that Cyp19a1a, Cyp19a1b and Foxl2a are more highly expressed in the ovary while Cyp11c1 is more highly expressed in the testis. Dimorphic expression patterns of these enzymes in differentiated gonads are consistent with what is known about circulating steroids levels in males and females. The immunohistochemical approach used showed its interest for the study of enzyme endocrinology and their regulation.
During gonadal development, a dimorphic expression of Cyp19a1a was highlighted in differentiating gonads, confirming the important role of aromatase A in the process of sexual differentiation. In zebrafish, the inhibition of Cyp19a1a expression in differentiating gonads seemed to induce testicular differentiation.
This work has led to a better characterization of steroidogenesis in zebrafish gonads. The expression profiles of steroidogenic enzymes and some of their regulators described in this work will allow a better understanding of the modes of actions of EDCs on these targets as well as on sexual differentiation and reproductive processes.

Date of Award	13 May 2015
Original language	French
Awarding Institution	University of Namur
Supervisor	Patrick Kestemont (Supervisor), Frederic Silvestre (President), Xavier Cousin (Jury), François Brion (Jury), Nathalie Hinfray (Jury) & Yann Guiguen (Jury)