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Oestrogens can affect expression of genes encoding steroidogenic enzymes in fish gonads. However, little information is available on their effects at the protein level. In this context, we first analysed the expression of key steroidogenic enzyme genes and proteins in zebrafish testis, paying attention also to other cell types than Leydig cells. Gene expression was analysed by quantitative PCR on fluorescence-activated cell-sorting fractions coupled or not to differential plating, while protein synthesis was studied by immunohistochemistry using specific antibodies against zebrafish Cyp17a1, Cyp19a1a and Cyp19a1b. Furthermore, we have evaluated the effect of oestrogen treatment (17β-oestradiol (E2), 10 nM) on the localization of these enzymes after 7 and 14 days of in vivo exposure in order to study how oestrogen-mediated modulation of their expression is linked to oestrogen effects on spermatogenesis. The major outcomes of this study are that Leydig cells express Cyp17a1 and Cyp19a1a, while testicular germ cells express Cyp17a1 and both, Cyp19a1a and Cyp19a1b. As regards Cyp17a1, both protein and mRNA seem to be quantitatively dominating in Leydig cells. Moreover, E2 exposure specifically affects only Leydig cell Cyp17a1 synthesis, preceding the disruption of spermatogenesis. The oestrogen-induced suppression of the androgen production capacity in Leydig cells is a major event in altering spermatogenesis, while germ cell steroidogenesis may have to be fuelled by precursors from Leydig cells. Further studies are needed to elucidate the functionality of steroidogenic enzymes in germ cells and their potential role in testicular physiology.

International audience; In freshwater ecosystems, a large number of chemical substances are able to disturb homeostasis of fish by modulating one or more physiological functions including the immune system. The aim of this study was to assess multi-biomarker responses including immunotoxicity induced by urban and agricultural pressure in European bullheads living in a small French river basin. For this purpose, a set of biochemical, immunological, physiological and histological parameters was measured in wild bullheads from five locations characterized by various environmental pressures. Moreover, to address effects of physiological status and contamination level variation on biomarker responses, fish were sampled during three periods (April, July and October). Results revealed a clear impact of environmental pressure on fish health and particularly on immunological status. An increase of EROD activity was recorded between upstream and downstream sites. Upstream sites were also characterized by neurotoxicological effects. Fish exhibited up stream/downstream variations of immunological status but strong differences were observed according to sampling season. Conversely, regarding biochemical and immunological effects, no significant response of physiological indexes was recorded related to environmental pressures. According to these results, the European bullhead appears as a valuable model fish species to assess adverse effects in wildlife due to urban and agricultural pressures.

International audience; Clotrimazole is a pharmaceutical fungicide known to inhibit several cytochrome P450 enzyme activities, including several steroidogenic enzymes. This study aimed to assess short-term in vivo effects of clotrimazole exposure on blood 11-ketotestosterone (11-KT) levels and on the transcriptional activity of genes in pituitary and testis tissue that are functionally relevant for androgen production with the view to further characterize the mode of action of clotrimazole on the hypothalamus-pituitary-gonad axis in zebrafish, a model vertebrate in toxicology. Adult male zebrafish were exposed to measured concentrations in water of 71, 159 and 258 micro g/L of clotrimazole for 7 days. Expression of pituitary gonadotropins ß subunit (lhb, fshb), testicular gonadotropins receptors (lhcgr, fshr) and testicular steroidogenesisrelated genes (e.g., star, cyp17a1, cyp11c1) were assessed. Blood concentrations of 11-KT were measured. Short-term exposure to clotrimazole induced a concentration-dependent increase of star, cyp17a1, and cyp11c1 gene expression and Cyp17a1 and Cy11c1 protein synthesis in Leydig cells, but androgen levels in blood remained unchanged. fshb, but not lhb mRNA levels in the pituitary tended to increase in clotrimazole-exposed zebrafish. Testicular expression of the Fsh receptor gene was significantly upregulated following exposure, when expression of this receptor was significantly correlated to the expression of steroidogenesis-related genes. Moreover, the Fsh-regulated insulin-like growth factor 3 (igf3) gene, a fish-specific Igf peptide expressed in Sertoli cells, was induced in testes. By using a network of genes functioning in pituitary and testis tissue, our study demonstrated that clotrimazole induced a cascade of molecular and cellular events which are in agreement with a role for Fsh in stimulating Leydig cell steroidogenesis to compensate the inhibitory action of clotrimazole on 11-KT synthesis and in inducing the expression of Fsh-regulated igf3 in Sertoli cells.