A set of biochemical and histological responses was measured in wild gudgeon collected upstream and downstream of urban and pharmaceutical manufacture effluents. These individual end-points were associated to fish assemblage characterisation. Responses of biotransformation enzymes, neurotoxicity and endocrine disruption biomarkers revealed contamination of investigated stream by a mixture of pollutants. Fish from sampled sites downstream of the industrial effluent exhibited also strong signs of endocrine disruption including vitellogenin induction, intersex and male-biased sex-ratio. These individual effects were associated to a decrease of density and a lack of sensitive fish species. This evidence supports the hypothesis that pharmaceutical compounds discharged in stream are involved in recorded endocrine disruption effects and fish population disturbances and threaten disappearance of resident fish species. Overall, this study gives argument for the utilisation of an effect-based monitoring approach to assess impacts of pharmaceutical manufacture discharges on wild fish populations.

Aromatase, a key steroidogenic enzyme that catalyses the conversion of androgens to estrogens, represent a target for endocrine disrupting chemicals. However, little is known about the effect of pollutants on aromatase enzymes in fish. In this study, we first optimized a rainbow trout (Oncorhynchus mykiss) microsomal aromatase assay to measure the effects of 43 substances belonging to diverse chemical classes (steroidal and non steroidal aromatase inhibitors, pesticides, heavy metals, organotin compounds, dioxins, polycyclic aromatic hydrocarbons) on brain and ovarian aromatase activities in vitro. Our results showed that 12 compounds were able to inhibit brain and ovarian aromatase activities in a dose-dependent manner with IC50 values ranging from the low nM to the high microM range depending on the substance: steroidal and non steroidal inhibitors of aromatase (4-hydroxyandrostenedione, androstatrienedione, aminogluthethimide), imidazole fungicides (clotrimazole, imazalil, prochloraz), triazole fungicides (difenoconazole, fenbuconazole, propiconazole, triadimenol), the pyrimidine fungicide fenarimol and methylmercury. Overall, this study demonstrates that rainbow trout brain and ovarian microsomal aromatase assay is suitable for evaluating potential aromatase inhibitors in vitro notably with respect to environmental screening. The results highlight that methylmercury and some pesticides that are currently used throughout the world, have the potential to interfere with the biosynthesis of endogenous estrogens in fish.

Androgens can induce complete spermatogenesis in immature or prepubertal teleost fish. However, many aspects of the role of androgens in adult teleost spermatogenesis have remained elusive. Since oestrogens inhibit androgen synthesis, we used an oestrogen-induced androgen depletion model to identify androgen-dependent stages during adult zebrafish spermatogenesis. Exposure to 10 nM 17b-oestradiol (E 2 ) in vivo at least halved the mass of differentiating germ cells (from type B spermatogonia to spermatids), while type A spermatogonia accumulated. Studies on the cellular dynamics revealed that a reduction of spermatogonial proliferation together with an inhibition of their differentiation to type B spermatogonia were the basis for the oestrogen-mediated disturbance of spermatogenesis. The capacity of the zebrafish testis to produce 11-ketotestosterone as well as the expression of steroidogenesis-related genes was markedly decreased after in vivo oestrogen exposure. Moreover, the androgen-release response to recombinant zebrafish Lh was lost after oestrogen exposure. We conclude that oestrogen exposure caused a state of androgen insufficiency in adult male zebrafish. Since the downregulation of the steroidogenic system as well as the disturbance of spermatogenesis in testicular explants exposed to E 2 ex vivo was much less severe than after in vivo exposure, the main inhibitory effect appears to be exerted via feedback inhibition of gonadotropin release. This experimental set-up helped to identify spermatogonial proliferation and their differentiation as androgen targets in adult zebrafish spermatogenesis.

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Many chemicals in the aquatic environment are able to adversely affect in vitro brain and ovarian aromatase expression/activity. However, it remains to be determined if these substances elicit in vivo effect in fish. With the view to further understanding possible effects of endocrine disrupting chemicals (EDCs) on aromatase function, we first developed methods to measure brain and ovarian aromatase expression/activity in a model species, the zebrafish, and assessed the effect of estradiol (E2) and androstatrienedione (ATD), a steroidal aromatase inhibitor. We showed that CYP19b gene was predominantly expressed in the brain whereas in the ovary CYP19a mRNA level was predominant. Moreover, aromatase activities (AA) were higher in brain than in ovary. In adult zebrafish, E2 treatment had no effect on aromatase expression/activity in brain, whereas at larval stage, E2 strongly triggered CYP19b expression. In the ovaries, E2 led to a complete inhibition of both CYP19a expression and AA. Exposure to ATD led to a total inhibition of both brain and ovarian AA but had no effect on CYP19 transcripts abundance. Together, these results provide relevant knowledge concerning the characterization of aromatase in the zebrafish, and reinforce the idea that brain and ovarian aromatase are promising markers of EDCs in fish and deserve further in vivo studies.

Many endocrine-disrupting chemicals act via estrogen receptor (ER) or aryl hydrocarbon receptor (AhR). To investigate the interference between ER and AhR, we studied the effects of 17beta-estradiol (E2) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the expression of zebra fish cyp19a (zfcyp19a) and cyp19b (zfcyp19b) genes, encoding aromatase P450, an important steroidogenic enzyme. In vivo (mRNA quantification in exposed zebra fish larvae) and in vitro (activity of zfcyp19-luciferase reporter genes in cell cultures in response to chemicals and zebra fish transcription factors) assays were used. None of the treatments affected zfcyp19a, excluding the slight upregulation by E2 observed in vitro. Strong upregulation of zfcyp19b by E2 in both assays was downregulated by TCDD. This effect could be rescued by the addition of an AhR antagonist. Antiestrogenic effect of TCDD on the zfcyp19b expression in the brain was also observed on the protein level, assessed by immunohistochemistry. TCDD alone did not affect zfcyp19b expression in vivo or promoter activity in the presence of zebra fish AhR2 and AhR nuclear translocator 2b (ARNT2b) in vitro. However, in the presence of zebra fish ERalpha, AhR2, and ARNT2b, TCDD led to a slight upregulation of promoter activity, which was eliminated by either an ER or AhR antagonist. Studies with mutated reporter gene constructs indicated that both mechanisms of TCDD action in vitro were independent of dioxin-responsive elements (DREs) predicted in the promoter. This study shows the usefulness of in vivo zebra fish larvae and in vitro zfcyp19b reporter gene assays for evaluation of estrogenic chemical actions, provides data on the functionality of DREs predicted in zfcyp19 promoters and shows the effects of cross talk between ER and AhR on zfcyp19b expression. The antiestrogenic effect of TCDD demonstrated raises further concerns about the neuroendocrine effects of AhR ligands.

International audienceOestrogens 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 (17 beta-oestradiol (E-2), 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, E-2 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

The effects of parasite infection by the cestode Ligula intestinalis on the reproductive function and endocrine system of wild roach Rutilus rutilus were evaluated. Gonad maturation, plasma vitellogenin, plasma steroid concentrations (i.e. progesterone, 11-keto-testosterone and 17-beta-estradiol) and brain aromatase activity were investigated in relation with parasitization. A low prevalence (8%) of ligulosed roach and a moderate impact of parasitization (mean parasitization index of 8.8%) were found in the studied population. Inhibition of gonad maturation generally resulted from infestation but 5% of the ligulosed roach nevertheless reached maturity. Main sex steroid plasma content was depleted in both genders. Male 11-keto-testosterone, female 17-beta-estradiol and progesterone plasma concentrations of both genders were, respectively, 27, 5 and 3 times lower in ligulosed fish when compared to their non-infected counterparts. Progesterone levels were negatively correlated with the parasitization index in females. Brain aromatase activity of infected roach was reduced to 50% of that of the non-infected fish. These results demonstrate significant negative effects on the reproductive function of wild roach infected by the tapeworm L. intestinalis collected from a site with low contamination.