title: Disruption of apoptosis pathways involved in zebrafish gonad differentiation by 17α-ethinylestradiol and fadrozole exposures.
abstract: Zebrafish (Danio rerio) sex determination seems to involve genetic factors (GSD) but also environmental factors (ESD), such as endocrine disrupting chemicals (EDCs) that are known to mimic endogenous hormones and disrupt gonad differentiation. Apoptosis has also been proposed to play a crucial role in zebrafish gonad differentiation. Nevertheless, the interactions between EDCs and apoptosis have received little attention. Thus, this study aimed to assess if and which apoptotic pathways are involved in zebrafish gonad differentiation and how EDCs may interfere with this process. With these purposes, zebrafish were exposed to 17α-ethinylestradiol (EE2, 4ng/L) and fadrozole (Fad, 50μg/L) from 2h to 35days post-fertilization (dpf). Afterwards, a gene expression analysis by qRT-PCR and a stereological analysis, based on systematic sampling and protein immunohistochemistry, were performed. The death receptors (FAS; TRADD), anti-apoptotic (BCL-2; MDM2), pro-apoptotic (CASP-2 and -6) and cell proliferation (BIRC5/survivin; JUN) genes and proteins were evaluated. In general, apoptosis was inhibited in females through the involvement of anti-apoptotic pathways, while in males apoptosis seemed to be crucial to the failure of the "juvenile ovary" development and the induction of testes transformation. The JUN protein was shown to be necessary in juvenile ovaries, while the BIRC5 protein seemed to be involved in zebrafish spermatogenesis. Both EDCs, EE2 and Fad, increased the apoptosis stimulus in zebrafish gonad. It was noticed that the few females that were resistant to Fad-induced sex reversal had increased anti-apoptotic factor levels, while males exposed to EE2 showed increased pro-apoptotic genes/proteins and were more advanced in gonad differentiation. Overall, our findings show that apoptosis pathways are involved in zebrafish gonad differentiation and that EDCs can disrupt this process.
authors.affiliation: Centre for the Research and Technology of Agro-Environmental and Biological Sciences, CITAB, Departamento de Biologia e Ambiente (DeBA), University of Trás-os-Montes and Alto Douro, UTAD, Quinta de Prados, Vila Real, 5000-801, Portugal; Life Sciences and Environment School, University of Trás-os-Montes and Alto Douro, UTAD, Quinta de Prados, Vila Real, 5000-801, Portugal. Electronic address: aluzio@utad.pt.
authors.forename: Ana
authors.initials: A
authors.lastname: Luzio
authors.affiliation: University of Lisbon, Faculty of Sciences, BioISI- Biosystems & Integrative Sciences Institute, Campo Grande, 1749-016 Lisbon, Portugal; Department of Genetics and Biotechnology, Life Sciences and Environment School (ECVA), University of Trás-os-Montes and Alto Douro, UTAD, Quinta de Prados, Vila Real, 5000-801, Portugal.
authors.forename: Manuela
authors.initials: M
authors.lastname: Matos
authors.affiliation: Life Sciences and Environment School, University of Trás-os-Montes and Alto Douro, UTAD, Quinta de Prados, Vila Real, 5000-801, Portugal.
authors.forename: Dércia
authors.initials: D
authors.lastname: Santos
authors.affiliation: Centre for the Research and Technology of Agro-Environmental and Biological Sciences, CITAB, Departamento de Biologia e Ambiente (DeBA), University of Trás-os-Montes and Alto Douro, UTAD, Quinta de Prados, Vila Real, 5000-801, Portugal; Life Sciences and Environment School, University of Trás-os-Montes and Alto Douro, UTAD, Quinta de Prados, Vila Real, 5000-801, Portugal.
authors.forename: António A
authors.initials: AA
authors.lastname: Fontaínhas-Fernandes
authors.affiliation: Centre for the Research and Technology of Agro-Environmental and Biological Sciences, CITAB, Departamento de Biologia e Ambiente (DeBA), University of Trás-os-Montes and Alto Douro, UTAD, Quinta de Prados, Vila Real, 5000-801, Portugal; Life Sciences and Environment School, University of Trás-os-Montes and Alto Douro, UTAD, Quinta de Prados, Vila Real, 5000-801, Portugal.
authors.forename: Sandra M
authors.initials: SM
authors.lastname: Monteiro
authors.affiliation: Centre for the Research and Technology of Agro-Environmental and Biological Sciences, CITAB, Departamento de Biologia e Ambiente (DeBA), University of Trás-os-Montes and Alto Douro, UTAD, Quinta de Prados, Vila Real, 5000-801, Portugal; Life Sciences and Environment School, University of Trás-os-Montes and Alto Douro, UTAD, Quinta de Prados, Vila Real, 5000-801, Portugal. Electronic address: acoimbra@utad.pt.
authors.forename: Ana M
authors.initials: AM
authors.lastname: Coimbra
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D052244:Endocrine Disruptors / Q000633:toxicity*; D004997:Ethinyl Estradiol / Q000633:toxicity*; D017316:Fadrozole / Q000633:toxicity*; D005260:Female; D008297:Male; D010053:Ovary / Q000187:drug effects; D011897:Random Allocation; D012733:Sex Differentiation / Q000187:drug effects* / Q000502:physiology; D013737:Testis / Q000187:drug effects; D018675:Toxicity Tests; D014874:Water Pollutants, Chemical / Q000633:toxicity*; D015027:Zebrafish / Q000502:physiology*
chemical_list: D052244:Endocrine Disruptors; D014874:Water Pollutants, Chemical; D004997:Ethinyl Estradiol; D017316:Fadrozole
keywords: Aromatase inhibitor; BCL-2; BIRC5; CASP-6; Immunohistochemistry; Xenoestrogen; qRT-PCR