Intersex Occurrence in Rainbow Trout (Oncorhynchus mykiss) Male Fry Chronically Exposed to Ethynylestradiol Sophie Depiereux 1 *, Me ´ lanie Liagre 1 , Lorraine Danis 1 , Bertrand De Meulder 2 , Eric Depiereux 2 , Helmut Segner 3 , Patrick Kestemont 1 * 1 Unit of Research in Environmental and Evolutionary Biology (URBE-NARILIS), Laboratory of Ecophysiology and Ecotoxicology, University of Namur, Namur, Belgium, 2 Unit of Research in Molecular Biology (URBM-NARILIS), University of Namur, Namur, Belgium, 3 Centre for Fish and Wildlife Health, Vetsuisse Faculty, University of Bern, Bern, Switzerland Abstract This study aimed to investigate the male-to-female morphological and physiological transdifferentiation process in rainbow trout (Oncorhynchus mykiss) exposed to exogenous estrogens. The first objective was to elucidate whether trout develop intersex gonads under exposure to low levels of estrogen. To this end, the gonads of an all-male population of fry exposed chronically (from 60 to 136 days post fertilization – dpf) to several doses (from environmentally relevant 0.01 mg/L to supra- environmental levels: 0.1, 1 and 10 mg/L) of the potent synthetic estrogen ethynylestradiol (EE2) were examined histologically. The morphological evaluations were underpinned by the analysis of gonad steroid (testosterone, estradiol and 11-ketotestosterone) levels and of brain and gonad gene expression, including estrogen-responsive genes and genes involved in sex differentiation in (gonads: cyp19a1a, ER isoforms, vtg, dmrt1, sox9a2; sdY; cyp11b; brain: cyp19a1b, ER isoforms). Intersex gonads were observed from the first concentration used (0.01 mg EE2/L) and sexual inversion could be detected from 0.1 mg EE2/L. This was accompanied by a linear decrease in 11-KT levels, whereas no effect on E2 and T levels was observed. Q-PCR results from the gonads showed downregulation of testicular markers (dmrt1, sox9a2; sdY; cyp11b) with increasing EE2 exposure concentrations, and upregulation of the female vtg gene. No evidence was found for a direct involvement of aromatase in the sex conversion process. The results from this study provide evidence that gonads of male trout respond to estrogen exposure by intersex formation and, with increasing concentration, by morphological and physiological conversion to phenotypic ovaries. However, supra-environmental estrogen concentrations are needed to induce these changes. Citation: Depiereux S, Liagre M, Danis L, De Meulder B, Depiereux E, et al. (2014) Intersex Occurrence in Rainbow Trout (Oncorhynchus mykiss) Male Fry Chronically Exposed to Ethynylestradiol. PLoS ONE 9(7): e98531. doi:10.1371/journal.pone.0098531 Editor: Patrick Prunet, Institut National de la Recherche Agronomique (INRA), France Received April 19, 2013; Accepted May 5, 2014; Published July 17, 2014 Copyright: ß 2014 Depiereux et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Fonds de la Recherche Scientifique – FNRS, Belgium, grant number: FC 83738 (http://www.frs-fnrs.be/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: sophiedepiereux@gmail.com (SD); patrick.kestemont@unamur.be (PK) Introduction Gonads are unique among all organs given their bipotential nature: they can develop either into an ovary or a testis from a single primordium. The germinal epithelium is composed of germ cells which develop into sperm or eggs and somatic cells, involved in gamete maturation, which differentiate into Sertoli or follicular cells, depending on the destiny of the gonad. This leads to a plasticity of this developmental pathway. In mammals, the way by which cells choose between either fate is genetically controlled by the expression of a main male gene SRY that leads irrefutably to testis development [1]. Though these pathways are still not well understood in mammals, the situation is even more complex in fish, since they display a great variety of sex determination systems, and because sex differentiation can be subject to exogenous influences, which can override the presumptive developmental route (i.e. temperature in zebrafish) [2]. Sex steroids play a key role in these differentiation processes, acting as inducers of the developmental pathway and organizers of cellular differentiation [3]. Numerous studies in different fish species have demonstrated that the administration of exogenous hormones during the critical period of gonadal development can override endogenous sex determining/differentiating mechanisms in the developing fish, enabling the induction of phenotypic sex reversal. Given that one mating type can be more marketable, this practice is now commonly used in fish farming to obtain unisexual populations [4–6]. From an environmental perspective, this lability in gonad sexual differentiation leads to a sensitivity of fish to endocrine disruptors, which are molecules that mimic natural endogenous hormones or interfere with the endogenous synthesis of hormones. These molecules can reach surface waters through wastewaters if they are not properly removed by sewage treatment plants, and can locally attain harmful concentrations for wild fish [7–10]. The effective concentrations appear to be very low, being comprised within the ng to mg/L range, depending on the potency of the molecule and the duration of the exposure [10]. Nowadays there is considerable evidence that fish exposed to these chemicals display reproductive failures [11–13]. Among the endocrine disruptors, xenoestrogens such as the synthetic estrogen ethynylestradiol (EE2 - the main component of the contraceptive pill) in particular have attracted attention [14]. EE2 is widespread in surface waters with mean reported concentrations around 0.05 ng EE2/L but can attain PLOS ONE | www.plosone.org 1 July 2014 | Volume 9 | Issue 7 | e98531