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Sci Rep
2016 Mar 31;6:23825. doi: 10.1038/srep23825.
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Sex reversal assessments reveal different vulnerability to endocrine disruption between deeply diverged anuran lineages.
Tamschick S, Rozenblut-Kościsty B, Ogielska M, Lehmann A, Lymberakis P, Hoffmann F, Lutz I, Kloas W, Stöck M.
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Multiple anthropogenic stressors cause worldwide amphibian declines. Among several poorly investigated causes is global pollution of aquatic ecosystems with endocrine disrupting compounds (EDCs). These substances interfere with the endocrine system and can affect the sexual development of vertebrates including amphibians. We test the susceptibility to an environmentally relevant contraceptive, the artificial estrogen 17α-ethinylestradiol (EE2), simultaneously in three deeply divergent systematic anuran families, a model-species, Xenopus laevis (Pipidae), and two non-models, Hyla arborea (Hylidae) and Bufo viridis (Bufonidae). Our new approach combines synchronized tadpole exposure to three EE2-concentrations (50, 500, 5,000 ng/L) in a flow-through-system and pioneers genetic and histological sexing of metamorphs in non-model anurans for EDC-studies. This novel methodology reveals striking quantitative differences in genetic-male-to-phenotypic-female sex reversal in non-model vs. model species. Our findings qualify molecular sexing in EDC-analyses as requirement to identify sex reversals and state-of-the-art approaches as mandatory to detect species-specific vulnerabilities to EDCs in amphibians.
Figure 1. Quantities of sex reversal (contradiction between genetic and phenotypic sex) under the influence of 17α-ethinylestradiol (EE2) in three deeply diverged anuran amphibians.Percentages of genetic-male-to-phenotypic-female sex reversal in African clawed frogs (Xenopus laevis, red), European tree frogs (Hyla arborea, green), and European green toads (Bufo viridis, blue) exposed to three concentrations of EE2 and in control animals; pooled data from two replicate experiments for each treatment or control. Susceptibility differences in genetic-male-to-phenotypic-female sex reversal occurred at all concentrations: (*) significant differences between clawed frogs and tree frogs (p ≤ 0.010); (x) significant differences between clawed frogs and green toads (p ≤ 0.001). Statistical analyses were conducted using cross-tabulation, Chi square and Fisher´s exact tests (α = 0.05).
Figure 2. Histological sections of three anuran species under the influence of 17α-ethinylestradiol (EE2).(a–c) Normal male, normal female and phenotypically sex-reversed gonad of African clawed frog (Xenopus laevis). (d–f) Normal male, normal female and phenotypically sex-reversed gonad of European green toad (Bufo viridis). (g–i) Normal male, normal female and phenotypically sex-reversed gonad of European tree frog (Hyla arborea). Bo – Bidder’s organ, characteristic of bufonid gonads (for details: Methods); fb – fat body; o – ovary; t – testis; arrows indicate seminiferous tubules; *ovarian cavity; arrowheads – diplotene oocytes. Scale bars are 100 micrometers.
Figure 3. Histological sections of mixed sex gonads of three anuran species under the influence of 17α-ethinylestradiol (EE2).(a) African clawed frog (Xenopus laevis), (b) European green toad (Bufo viridis), (c) European tree frog (Hyla arborea); Fig. 2 for control and sex-reversed individuals. Bo – Bidder’s organ, specific of bufonid toads’ gonads; fb – fat body; m – meiocytes; o – ovary; st – seminiferous tubules; t – testis; *a cavity separating testicular and ovarian parts of the mixed sex gonad; white arrow indicates ovarian cavity in the ovarian portion of the mixed gonad; white arrowheads show diplotene oocytes; yellow dotted lines separate testicular and ovarian parts of the mixed sex gonads. Scale bars represent 100 micrometers.
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