Scientific RepoRts | 6:23825 | DOI: 10.1038/srep23825
Sex reversal assessments reveal
dierent vulnerability to endocrine
disruption between deeply
diverged anuran lineages
Stephanie Tamschick1, Beata Rozenblut-Kościsty2, Maria Ogielska2, Andreas Lehmann3,
Petros Lymberakis4, Frauke Homann1, Ilka Lutz1, Werner Kloas1 & Matthias Stöck1
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 aect the sexual development
of vertebrates including amphibians. We test the susceptibility to an environmentally relevant
contraceptive, the articial 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 ow-through-system and pioneers
genetic and histological sexing of metamorphs in non-model anurans for EDC-studies. This novel
methodology reveals striking quantitative dierences in genetic-male-to-phenotypic-female sex
reversal in non-model vs. model species. Our ndings qualify molecular sexing in EDC-analyses as
requirement to identify sex reversals and state-of-the-art approaches as mandatory to detect species-
specic vulnerabilities to EDCs in amphibians.
Amphibians face a global ongoing decline1,2. Anthropogenic causes such as industrial agriculture3, habitat
destruction4,5, invasive species6, climate change7, land use8 and infectious diseases9, including several forms of
chytridiomycosis10,11, are among the major threats. However, the sum of multiple stressors1,7, some of which
poorly known, is considered to be the true reason for the massive population declines. One potential cause rep-
resents endocrine disrupting compounds (EDCs)12. Besides pesticides, EDCs comprise either natural products
or synthetic chemicals that mimic, enhance (an agonist), or inhibit (an antagonist) the action of hormones and
in this way interfere with the synthesis, secretion, transport, binding, action, or elimination of natural hor-
mones, which are responsible for the maintenance of homeostasis, reproduction, development, and/or behav-
ior13. Considerable amounts of EDCs are globally found in waste and surface waters14,15 and can easily enter the
body of aquatic organisms and impair their natural hormonal pathways. EDCs are well known for their negative
impacts on the sexual development of aquatic organisms such as sh16,17 and are suspected to cause fertility
problems in humans18,19. However, their impact to non-model amphibians with aquatic larvae is not well studied,
despite recent evidence for high EDC-relevance to suburban frog populations20. One globally relevant EDC is
17α -ethinylestradiol (EE2), a synthetically stabilized estrogen and main ingredient of many female contraceptive
pills. e inert EE2 is then excreted and insuciently eliminated by sewage plants and hence reaches aquatic
ecosystems14. It is a main hormonal pollutant, resistant to degradation, that accumulates in sediments and biota14.
Concentrations from 24 to 831 ng/L have been detected in European and American surface waters21–23. Such con-
centrations have been shown to alter behavior and somatic and sexual development in sh and amphibians12,14,15.
Due to their semi-aquatic life cycle, oen aquatic reproduction and a highly permeable skin, amphibians are
1Leibniz-Institute of Freshwater Ecology and Inland Fisheries (IGB), Müggelseedamm 301 & 310, D-12587 Berlin,
Germany. 2Department of Evolutionary Biology and Conservation of Vertebrates, Wroclaw University, Sienkiewicza
21, 50-335 Wroclaw, Poland. 3Federal Institute for Materials Research and Testing (BAM), Richard-Willstätter-Str. 11,
D-12489 Berlin, Germany. 4Natural History Museum of Crete, University of Crete, Knossou Ave., 71409 Heraklion,
Crete, Greece. Correspondence and requests for materials should be addressed to M.S. (email: matthias.stoeck@
Received: 09 December 2015
Accepted: 15 March 2016
Published: 31 March 2016
Scientific RepoRts | 6:23825 | DOI: 10.1038/srep23825
especially sensitive to EDCs. Effects on development and reproduction are best examined in clawed frogs,
Xenopus laevis and X. tropicalis. In these amphibian models, EE2-concentrations as low as 0.3 ng/L have been
shown to aect calling behavior and mating success24. Higher but still environmentally relevant amounts of EE2
(29 to 840 ng/L) have been shown to aect body morphology, metamorphosis and hemoglobin catabolism25,26.
Importantly, EE2 can lead to impaired sexual development as mirrored by gonad histomorphology, demonstrat-
ing that male clawed frogs (X. laevis) develop mixed sex (= ‘intersex’, see below) gonads or even show complete
phenotypic sex reversal26–29. e undierentiated anuran gonad is bipotential and can develop into either ovary
or testis30. erefore, exogenous hormones can override the primary genetic sex determination signal and lead to
developmental disturbances, mixed sexes or complete sex reversal. One major obstacle of studying EDC-eects
in amphibians has been the mostly inaccessible information about genetic sex. In most previous EDC-studies,
sex reversal had to be inferred by comparing sex ratios of control and exposed frogs, assuming a normal 1:1 pro-
portion, which may have easily led to wrong conclusions about EDC-impacts on sex ratios. While all amphibian
species investigated show genetic sex determination31, exhibiting either male (XX/XY) or female (ZZ/ZW) heter-
ogamety, an extrapolated 96% of all species have microscopically indistinguishable sex chromosomes32, requiring
molecular sexing methods. Although EDC-studies with molecular sexing were applied to the model Xenopus26,33,
sex markers have become only recently available for some non-model anurans32,34–38, and have not been used in
Using a high-standard ow-through-system and the rst direct experimental approach of its kind, we simul-
taneously exposed European tree frogs (H. arborea), green toads (B. viridis) and the well investigated but deeply
diverged model-species X. laevis to EE2, applied molecular sexing followed by histological analysis and compared
impacts on their sexual development. We found striking dierences in the susceptibility to sex reversal between
model and non-model species, showing that state-of-the-art approaches are an important prerequisite to detect
species-specic vulnerabilities to EDCs in amphibians.
Phenotypic sex reversal of genetic males. Among all three anuran species, simultaneous exposure to
three EE2-concentrations under ow-through-conditions resulted in dierent proportions of male-to-female sex
reversal, ranging from 15 to 100% (Table1 and Fig.1), which was solely revealed when comparing genetic and
phenotypic sex of experimental animals. Importantly, no sex reversal occurred in control groups. While sex rever-
sal (Figs1 and 2) was generally correlated to EE2-concentration, interspecies dierences (p ≤ 0.010) between
clawed frogs (X. laevis) and tree frogs (H. arborea) were found at all concentrations, and between clawed frogs
and green toads (B. viridis) at the highest concentration (5,000 ng/L; p ≤ 0.001). While EE2-treatment produced
similar percentages of sex-reversed tree frogs and green toads (15 to 36%), clawed frogs appeared most suscepti-
ble (up to 100%). At the lowest concentration (50 ng/L) 31.3% of genetically male clawed frogs developed female
phenotypes, i.e. ovaries, while no sex reversal occurred in the non-model species (H. arborea, B. viridis). As
expected for a feminizing EDC, sex reversal occurred always from genetic male to phenotypic female. According
to gross morphological observation and histological evidence, sex-reversed genetic male frogs and toads devel-
oped ovaries that showed no dierence to those of genetic control and untreated39 females.
Mixed sex gonads. In addition to sex reversals, EE2-treatment provoked the development of various per-
centages of mixed sex40 gonads (equivalent to ‘intersexes’ of some authors41–43) that were histologically recorded
in all three species (Fig.3 and Table1). Such altered gonads are characterized by the presence of ovarian within
testicular tissue in genetic males and were never found in control groups. In contrast to the sex reversal analy-
ses, X. laevis formed fewer mixed sex gonads than B. viridis (p ≤ 0.026). No signicant susceptibility dierences
between H. arborea and the model species were found. Both non-model species also diered in their susceptibil-
ity at the lowest concentration (50 ng/L; p ≤ 0.015).
Using a new combination of experimental features, we provide evidence for different quantities of
genetic-male-to-phenotypic-female sex reversal in three amphibian species, diverged between 78 million years44
(Hyla, Bufo) and 206 My (Xenopus), under exposure to the estrogen EE2. is synthetic substance is globally of
high relevance for EDC-pollution of aquatic ecosystems14,15. Our new approach combined simultaneous exposure
of tadpoles to three EE2-concentrations in a ow-through-system and genetic sexing of metamorphs of model
and non-model experimental anurans. We applied environmentally (pollution) and physiologically (expected
eects in X. laevis) relevant concentrations of EE2. Genetic sexing of metamorphosed tree frogs and green toads
revealed these two non-model species to have similar susceptibilities to sex reversal among each other, while
both signicantly diered from X. laevis. is model-species, in which genetic sex is governed by a female heter-
ogametic (ZZ/ZW) chromosome system45, proved to be more sensitive to EE2 with a lower dose provoking sex
reversals and more aected animals (Table1). On the other hand, B. viridis and H. arborea, both diverged 206 My
from X. laevis and possessing male heterogametic (XX/XY) sex chromosomes32,35, showed higher percentages of
mixed sex individuals than X. laevis.
All of this suggests that species-specic developmental stages, sex determination systems or endocrine path-
ways, shaped by long separate evolutionary histories, were dierently aected by EE2, and such a wide spectrum
of eects can be generally expected also for other EDCs among diverged anuran lineages.
e occurrence of more than 50% of genetic females among the randomly chosen hatchlings in several of
our test tanks underlines the importance of genetic sexing. Unavailability of genetic sexing, as in many previous
studies, could easily lead to wrong conclusions about the strength of feminizing (or masculinizing) eects of
Scientific RepoRts | 6:23825 | DOI: 10.1038/srep23825
EDCs when determining “no observed eect concentration” (NOEC) and “lowest observed eect concentration”
(LOEC) for endocrine active substances.
Different estrogenic compounds with concentrations reaching from the low nanogram- to the high
microgram-per-liter range have been shown to provoke phenotypic male-to-female sex reversals in X. laevis46–48
models. To our knowledge, only one previous study26 has examined sex reversals aer EE2-exposure using molec-
ular sexing in X. laevis, examining a similar range of concentrations (90, 840, 8,810 ng/L). In contrast to our study,
male-to-female sex reversals were not detected under the 90 ng/L treatment, and at the higher concentrations
with only 7 and 17%, respectively. However, these authors used a static and not a ow-through-system, which
may explain the deviating results to our study, as EE2-concentrations may stronger uctuate due to eects of met-
abolic activity of microorganisms in tanks49,50, due to greater biomass sorption of EE251, or due to simple adsorp-
tion to surfaces of exposure tanks. Beyond the synthetic EE2, on which we focused due to its high environmental
relevance, previous sex reversal estimates in X. laevis, evaluating only sex ratios, involved the natural, ephemeral
sexed Females Males Females Males Sex-reversed Mixed sex
N N N % % N % N %
Control 35 24 11 68.6 31.4 0 0 0 0
50 ng/L 38 22 16 57.9 42.1 5*31.3*0x0.0x
500 ng/L 37 20 17 54.1 45.9 13*,# 76.5*,# 1 12.5
5,000 ng/L 38 21 17 55.3 44.7 17*,x, # 100*,x, # 0x0.0x
Control 36 13 23 36.1 63.9 0 0 0 0
50 ng/L 36 15 21 41.7 58.3 0*0.0*0° 0.0°
500 ng/L 41 22 19 53.7 46.3 6*,# 31.6*,# 3 30
5,000 ng/L 37 17 20 45.9 54.1 3*15.0*3 27.3
Control 25 13 12 52 48 0 0 0 0
50 ng/L 24 11 13 45.8 54.2 0 0 4x,° 57.1x,°
500 ng/L 27 15 12 55.6 44.4 4 36.4 4#80.0#
5,000 ng/L 27 12 15 44.4 55.6 5x33.3x9x,# 69.2x,#
Table 1. Eects of three 17α-ethinylestradiol (EE2) concentrations (50, 500 and 5,000 ng/L) on the sexual
development of model and non-model amphibian species. Species comprised African clawed frogs (Xenopus
laevis), European tree frogs (Hyla arborea), and European green toads (Bufo viridis); numbers and percentages
of genetically sexed individuals, sex-reversed males and mixed sex individuals. Signicant inter-species
susceptibility dierences occurred at all concentrations, resulting in genetic-male-to-phenotypic-female sex
reversal and development of mixed sex individuals. *Signicant dierence between clawed frogs and tree frogs;
xbetween clawed frogs and green toads; °between tree frogs and green toads; #signicant dierence between
treatment and control groups within the same species.
Figure 1. Quantities of sex reversal (contradiction between genetic and phenotypic sex) under the
inuence 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 dierences in genetic-male-to-phenotypic-female sex reversal occurred at all concentrations:
(*) signicant dierences between clawed frogs and tree frogs (p ≤ 0.010); (x) signicant dierences 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).
Scientific RepoRts | 6:23825 | DOI: 10.1038/srep23825
17β -estradiol (E2). Such E2-treatments provoked skewed sex ratios40,48,52–54 or complete feminization46,53,55,56. In
H. arborea and B. viridis eects have only been studied56 at the very high 100,000 ng/L E2-concentration. In both
species, no female-biased sex ratios but a high percentage (59.3%) of undierentiated gonads in B. viridis were
found. Since gonad dierentiation in bufonid toads is slower compared to the other species at this developmental
stage39, we assume that the time of dissection at metamorphosis may have inuenced these results.
Several inconsistent outcomes in the literature may be explicable by the potentially wrong assumption of
initial 1:1 sex ratios of experimental amphibians. Based on our data, we strongly recommend genetic sexing,
whenever available, as a hallmark of appropriate evaluation of EDC-eects in amphibians. is demand can be
extended to other vertebrates and generalized to EDC-research in organisms with homomorphic sex chromo-
somes, including invertebrates57. Otherwise, as shown here, complete sex reversal as a very profound EDC-eect,
occurring at low concentrations, may be completely overlooked. Furthermore, deep phylogenetic dierences may
result in strong susceptibility dierences towards EDCs. ough we do not advocate the extensive use of endan-
gered amphibians, we conclude that results gained from earlier studies in X. laevis in general and without genetic
sex information specically should not be uncritically extrapolated to other anuran species.
Figure 2. Histological sections of three anuran species under the inuence 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); – fat body; o – ovary; t – testis; arrows
indicate seminiferous tubules; *ovarian cavity; arrowheads – diplotene oocytes. Scale bars are 100 micrometers.
Scientific RepoRts | 6:23825 | DOI: 10.1038/srep23825
Animals. is experiment was approved by the German State Oce of Health and Social Aairs (LaGeSo,
Berlin, Germany; G0359/12); all methods were carried out in accordance with approved guidelines. Xenopus
laevis tadpoles were obtained from the stock at the Leibniz-Institute for Freshwater Ecology and Inland Fisheries.
Induction of spawning and tadpole husbandry followed standard methods58. Parental animals of B. viridis and H.
arborea were caught at several localities in Greece (Supplementary Table 1), and non-invasively DNA-sampled59.
Parts of their clutches were transferred to IGB (permit 115790/229) and acclimated at 22 ± 1 °C in 10 L Milli-Q
grade water, supplemented with 2.5 g marine salt (Tagis, Germany).
Hormone exposure and experimental conditions. 17α -Ethinylestradiol (Sigma-Aldrich, Germany),
dissolved in dimethyl sulfoxide (DMSO; Roth, Germany), was applied in nominal concentrations of 50, 500 and
5,000 ng/L (Supplementary Fig. 1 and Supplementary Table 2, for measurements during the experiment); control
animals received 0.00001% DMSO. EE2-concentrations in test tanks were checked weekly by high performance
liquid chromatography/mass spectrometry (HPLC-MS/MS), and adjusted if required. In order to minimize
adsorption or release of EDCs, we used glass tanks and all connections of the ow through system consisted of
inert materials involving mainly PTFE (Polytetrauoroethylene, “Teon”)-coating or Platinum-cured Silicon tub-
ing (Cole-Parmer). Exposure of tadpoles started at Gosner60-stage 22–23 in B. viridis and H. arborea, equivalent
to Nieuwkoop-Faber61 stage 42–44 in X. laevis, distinctly prior to the sensitive phase of sex determination in all
species30,62. Twenty randomly chosen individuals per species and treatment were transferred into each test tank
in a high-standard ow-through-system (details52). Two replicates per exposure group (including control) com-
prised in total 160 tadpoles per species. Stock solutions and water were piped via a peristaltic pump into a mixing
chamber, mixed to nal EE2-concentrations, and supplied to a cluster of three test tanks each. Concentrations
were thus identical for all three species in each treatment group. Tadpoles were reared in a 12/12 h light/dark cycle
at constantly 22 ± 1 °C in suciently aerated and regularly cleaned tanks. Weekly monitored water parameters
comprised: dissolved oxygen, nitrate, ammonium, pH, conductivity, and hardness; values were adequate as in
previous studies involving the same equipment40. Tadpoles were fed SeraMicron (Sera, Germany), H. arborea and
B. viridis were additionally supplied with TetraMin (Tetra, Germany). To imitate natural conditions under which
H. arborea and B. viridis leave water at metamorphosis, animals were transferred to glass terraria at Gosner stage
46. Xenopus laevis were dissected at equivalent Nieuwkoop-Faber stage 66; hylids and bufonids aer sucient
Phenotypic sexing based on gonad gross morphology and histology. Animals were anesthetized
by immersion in tricaine methanesulfonate (MS 222; Sigma-Aldrich), decapitated and dissected under a binoc-
ular microscope (Olympus SZX7). Gonadal anatomy served for preliminary phenotypic sexing and detection of
underdeveloped gonads. To improve visualization, a drop of Bouin’s solution (Sigma-Aldrich) was added; and
in situ anatomical photographs were taken (Olympus DT5 camera). For histology, gonads were carefully dissected,
separated from adjacent tissue, xed in Bouin (24 h) and subsequently rinsed several rounds in 70% ethanol.
Histological sections were prepared for 50% of study animals (from each tank 10 randomly chosen individuals,
i.e. 20 per treatment group). Analyses were performed according to established protocols30,39,63. Using Stemi SV11
(Zeiss) microscope and camera, separated gonads were photographed, embedded in paraplast, sectioned into
7 m longitudinal slices, stained with Mallory’s trichrome, and examined using Zeiss Axioskop 20 microscope.
Images were acquired by a cooled Carl Zeiss AxioCam HRc CCD camera. Histological sections were screened
slide by slide to establish phenotypic sex. Ovaries were recognized by the presence of ovarian cavities, early mei-
otic oocytes and/or diplotenes, and testes by spermatogonia, spermatocytes and/or seminiferous cords or tubules.
Figure 3. Histological sections of mixed sex gonads of three anuran species under the inuence 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,
specic of bufonid toads’ gonads; – 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.
Scientific RepoRts | 6:23825 | DOI: 10.1038/srep23825
In the case of B. viridis, the most anterior part of both male and female gonads is Bidder’s organ, an ovary-like
structure, characteristic of bufonids64. In B. viridis mixed sex was dened when ovarian meiocytes were found
inside male testicular tissue behind the physiological transition region between Bidder’s organ and the actual
gonad. All phenotypic sexing was performed without prior knowledge about genetic sex of animals.
Genotypic sex determination. DNA extraction involved the BioSprint robotic workstation with
its 96 DNA Plant Kit (Qiagen, Germany) according to the manufacturer’s protocol. To establish genetic sex,
species-specic polymerase chain reactions (PCRs) were conducted on Eppendorf Mastercyclers (Ep Gradient S).
For X. laevis, two genes were amplied45,65: DMRT1 and the female-specic DM-W (Supplementary Table 3).
Genetic sexing of non-model species involved microsatellites WHa5–201 and Ha-H10834,36 (H. arborea) and
C20132,66, HNRNPD and CHD167 (B. viridis; Supplementary Table 3). Genotypes were analyzed on a sequencer
(3500 × L Genetic Analyzer, Applied Biosystems) and G v. 4.0 was used for visualization of peaks.
DNA quality issues (four H. arborea) and homomorphy of microsatellites (33 B. viridis) prevented genetic sexing
in these individuals that were excluded from sex reversal analyses.
Detection of complete sex reversal and mixed sex. Phenotypic sexing of all animals was based on
gross morphology and histology of gonads39,52. Complete sex reversal was stated if genetic males showed a phe-
notypically female gonad, irrespective of the degree of its dierentiation and not diering from those of control
females. Mixed sex gonads were detected by the presence of ovarian and testicular tissue in the same gonad.
Statistics. All data were analyzed with SPSS Statistics 22 (IBM, Armonk, NY). Intra- and inter-specic dif-
ferences in EE2-susceptibility were examined. For evaluations of sex reversal and mixed sex, we rst compared
both replicates per species and parameter using Fisher’s exact test. If no dierences (exact p ≥ 0.05) were found,
both replicates per treatment were pooled in order to compare control and exposure groups within and between
species using cross-tabulations with 2-sided Chi square tests (α = 0.05). Post-hoc Fisher’s exact tests (2-sided)
were applied for pairwise comparisons including False Discovery Rate corrections68.
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Scientific RepoRts | 6:23825 | DOI: 10.1038/srep23825
This work was supported by the German Science Foundation (DFG; grant Sto 493/3-1) and a Heisenberg-
Fellowship (Sto 493/2-1, Sto 493/2-2) to MS. We thank the Ministry of the Environment of Greece that provided
the permit (115790/229) to collect the specimens. We thank M. Papadimitrakis for help during the eld work, W.
Kleiner, J. Garmshausen, M. Brehm, and A. Weißhuhn for animal care and/or laboratory assistance, J.F. Gerchen
for unpublished primers, M. Kazmierczak for help with microphotography, E. Serwa for excellent histology, I.
Haufe for detailed statistical advice, J. Plötner for access to the DNA extraction work station and M. Monaghan
and K. Preuss for access to the IGB genotyping facility. e publication of this article was funded by the Open
Access Fund of the Leibniz Association.
M.S. and W.K. conceived and designed the research. M.S. and P.L. did field work; S.T., M.S., F.H. and I.L.
conducted the experiments. S.T. performed genotyping and, supported by F.H. statistical analyses. S.T. and M.S.
wrote the paper; B.R.K. and M.O. performed histology and evaluated, supported by I.L. gonadal dierentiation.
A.L. analyzed water samples. All authors contributed to the nal manuscript.
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing nancial interests: e authors declare no competing nancial interests.
How to cite this article: Tamschick, S. et al. Sex reversal assessments reveal dierent vulnerability to endocrine
disruption between deeply diverged anuran lineages. Sci. Rep. 6, 23825; doi: 10.1038/srep23825 (2016).
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