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Development, standardization and refinement of procedures for evaluating effects of endocrine active compounds on development and sexual differentiation of Xenopus laevis.
Lutz I, Kloas W, Springer TA, Holden LR, Wolf JC, Krueger HO, Hosmer AJ.
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Xenopus laevis has been introduced as a model to study effects of endocrine-active compounds (EAC) on development and sexual differentiation. However, variable and inconsistent data have raised questions about the reliability of the test methods applied. The current study was conducted in two laboratories to develop, refine, and standardize procedures and protocols. Larvae were exposed in flow-through systems to 17beta-estradiol (E2), at concentrations from 0.2 to 6.0 microg E2 L(-1) in Experiment 1A, and 0.015 to 2.0 microg E2 L(-1) in Experiment 1B. In both studies survival (92%, 99%) and percentage of animals that completed metamorphosis (97%, 99%) indicated reproducible biological performance. Furthermore, minor variations in husbandry led to significant differences in snout-to-vent length, weight, and gonad size. In Experiment 1A, almost complete feminization occurred in all E2 treatment groups whereas a concentration response was observed in Experiment 1B resulting in an EC(50) of 0.12 microg E2 L(-1). The final verified protocol is suitable for determining effects of EAC on development and sexual differentiation in X. laevis.
Fig. 1. Comparison of measured mean E2 concentrations in water samples of E2 treatments in the flow-through system. Mean values represent the concentrations of E2 of weekly sampling intervals from dpf 6 or 8 through the last sampling interval on termination of the experiments on dpf 82. a, E2 concentrations of Experiment 1A; b, E2 concentrations of Experiment 1B. The limit of quantification was 0.00500 μg E2 L−1, calculated as the product of the concentration of the lowest calibration standard (0.100 μg E2 L−1) and the dilution factor of the matrix blank samples (0.0500 μg E2 L−1)
Fig. 2. Body weight (means ± SD) of a, females, and b, males, after exposure to E2 from dpf 6 or 8 in a flow-through system until completion of metamorphosis or day 82 post-fertilization. 0.015 μg E2 L−1 was not used in Experiment 1A and 6.0 μg E2 L−1 was not used in Experiment 1B. Neg Ctrl, negative control group
Fig. 3. Cumulative portion of X. laevis frogs completing metamorphosis after exposure to different concentrations of E2 from day 6 or 8 post-fertilization in a flow-through system until completion of metamorphosis or day 82 post-fertilization. The number of individuals per treatment group (n) is given in the legend. a, portion of females in Experiment 1A; b, portion of females in Experiment 1B; c, portion of males in Experiment 1A (just one male was observed in the 6.0 μg E2 L−1 group); d, portion of males in Experiment 1B. Neg Ctrl, negative control group
Fig. 4. Mean age at completion of metamorphosis (mean ± SD) by treatment group for female, a, and male, b, frogs at completion of metamorphosis or on day 82 post-fertilization. 0.015 μg E2 L−1 was not used in Experiment 1A and 6.0 μg E2 L−1 was not used in Experiment 1B. Only one male frog was observed in the 6.0 μg E2 L−1 group in Experiment 1A. Neg Ctrl, negative control group. For Experiment 1B significant differences are marked by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001; two-sided Wilcoxon–Mann–Whitney test of equality with negative control group). A Kruskal–Wallis test of overall group differences was significant (females p < 0.0001, males p = 0.0009) and a Jonckheere–Terpstra test of trend was significant (females p < 0.0001, males p = 0.0062)
Fig. 5. Individual bars represent frequencies of sex of X. laevis frogs among all animals according to treatment group based on gross morphological evaluation; the total number of frogs (n) that completed metamorphosis, is given on top of the bars. Tadpoles were exposed to E2, in a flow-through system, from day 6 or 8 post-fertilization through completion of metamorphosis or day 82 post-fertilization. The EC50 was calculated from the results of Experiment 1B and was 0.12 μg E2. The numbers of frogs in the negative control in Experiment 1A was higher for evaluation of gender because the animals which were excluded from calculation of weight and length were included in calculation of the portion of males, females, and mixed sex. Neg Ctrl, negative control group. For Experiment 1B asterisks denote significant differences (**p < 0.01, ***p < 0.001; two-sided Fisher’s exact test for comparison to negative control). Both an overall exact chi-square test of equality of all experimental groups and a Cochran–Armitage trend test were statistically significant (p < 0.05) in every case
Fig. 6. a. Ovary of X. laevis revealing segmented, lobular structure and clearly visible internal melanocytes in the tissue; the length of the ovary is slightly shorter than the length of the kidney. b. In contrast, a testis is shorter relative to the length of the kidney (half of the kidney), straight shaped, and does not contain internal melanocytes. In general testis tissue appears much denser than ovary tissue. c. Mixed sex gonads were defined as the co-occurrence of both ovarian and testicular tissue in a single gonad. The ovary tissue in the presented gonad shows a reduced lobular structure, just a few melanophores, whereas the testis tissue appears bulbous. Arrows indicate the different gonad structures, f, ovary-like tissue, and m, male like tissue. d. Pearling (p) was characterized by the presence of multiple, prominent segmental enlargements and/or attenuations along the length of one or both gonads. e. Segmental aplasia (segA) was classified as nodular islands of testicular or ovarian tissue, with either intervening membranous connections or no connections. The kidney and the attached gonad tissue were fixed with Bouin’s solution
Fig. 7. Correlation of changes in E2 concentration and the biomass of growing tadpoles during the exposure period in Experiments 1A and 1B
Fig. 8. Proportion of male and females frogs (n) of the negative control groups completing metamorphosis during the course of the studies, based on the total number of animals surviving in Experiments 1A and 1B
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