Reproductive maturation of the tropical clawed frog: Xenopus tropicalis.

The tropical clawed frog, Xenopus tropicalis, is a relatively new model species being used in developmental biology and amphibian toxicology studies. In order to increase our understanding of reproductive maturation and the role of steroid hormones in X. tropicalis, we collected baseline reproductive data in this species from metamorphosis to adulthood. One cohort of frogs was maintained for 42 weeks post-metamorphosis (PM) with endpoints representative of important reproductive parameters collected at 1- or 2-week intervals. These endpoints were then correlated to titers of either estradiol or testosterone. Male frogs exhibited nuptial pads, starting at 8 weeks (PM) when measureable concentrations of circulating testosterone (5.3 ng/mL plasma) first appeared. Testosterone concentrations remained above this level at all later time points, but were highly variable among individuals. Testes sizes in males reached their peak at 22 weeks PM (21 mg) with sperm counts peaking at the same time (25 million sperm/male). In females, estradiol becomes elevated in the blood at 16 weeks PM (1.5 ng/mL plasma) which corresponds with the presences of vitellogenin (4.4 mg/mL plasma), vitellogenic oocytes in the ovary, ovarian growth, and oviduct growth. Vitellogenic oocytes increased in number up to 15,000 per female at 30 weeks PM and accounted for 75% of the total number of oocytes present in the ovary. The ovary and oviducts continued to grow in mass until 30 weeks PM at which point they had reached sizes of 3.6g and 0.8 g, respectively. These data indicate that male and female X. tropicalis reach reproductive maturation at 22 and 30 weeks PM, respectively. Results from this study are valuable for the design of amphibian toxicology assays and increase our understanding of the reproductive biology of this relatively new model species.

Trenbolone causes mortality and altered sexual differentiation in Xenopus tropicalis during larval development.

Trenbolone is an androgen agonist used in cattle production and has been measured in aquatic systems associated with concentrated animal-feeding operations. In this study, the authors characterized the effects of aqueous exposure to 17ß-trenbolone during larval Xenopus tropicalis development. Trenbolone exposure resulted in increased mortality of post-Nieuwkoop-Faber stage 58 tadpoles at concentrations ≥100 ng/L. Morphological observations and the timing of this mortality are consistent with hypertrophy of the larynx. Development of nuptial pads, a male secondary sex characteristic, was induced in tadpoles of both sexes at 100 ng/L. Effects on time to complete metamorphosis or body sizes were not observed; however, grow-outs placed in clean media for six weeks were significantly smaller in body size at 78 ng/L. Effects on sex ratios were equivocal, with the first experiment showing a significant shift in sex ratio toward males at 78 ng/L. In the second experiment, no significant effects were observed up to 100 ng/L, although overall sex ratios were similar. Histological assessment of gonads at metamorphosis showed half with normal male phenotypes and half that possessed a mixed-sex phenotype at 100 ng/L. Hypertrophy of the Wolffian ducts was also observed at this concentration. These results indicate that larval 17ß-trenbolone exposure results in effects down to 78 ng/L, illustrating potential effects from exposure to androgenic compounds in anurans.

Sex reversal of the amphibian, Xenopus tropicalis, following larval exposure to an aromatase inhibitor.

Aromatase is a steroidogenic enzyme that catalyzes the conversion of androgens to estrogens in vertebrates. Modulation of this enzyme's activity by xenobiotic exposure has been shown to adversely affect gonad differentiation in a number of diverse species. We hypothesized that exposure to the aromatase inhibitor, fadrozole, during the larval development of the tropical clawed frog, Xenopus tropicalis, would result in masculinization of the developing female gonad. Tadpoles were exposed to fadrozole at nominal concentrations from 1 to 64 microg/L in a flow-through system from < 24 h post-fertilization (Nieuwkoop Faber (NF) stage 15-20) to metamorphosis (NF stage 66). At metamorphosis, morphologically examined gonads indicated complete masculinization of all tadpoles at concentrations of 16 microg/L and above and a significant bias in sex ratio towards males at concentrations of 1 microg/L and above. No effects on time to metamorphosis, body mass, or body length were observed. A random subsample of frogs was raised to reproductive maturity (39 weeks post-fertilization) in control water. All frogs exposed as tadpoles to 16 microg/L fadrozole or greater possessed testes at sexual maturity. Intersexed gonads characterized by the presence of both testicular and ovarian tissue were observed in 12% of frogs in the 4 microg/L treatment. No differences in estradiol, testosterone, or vitellogenin plasma concentrations were observed in exposed males or females compared to controls. Females in the 4 microg/L treatment possessed a significantly greater percentage of pre-vitellogenic oocytes than controls and were significantly smaller in body mass. No differences in sperm counts were observed in exposed males compared to controls. Results from this study demonstrate that larval exposure to an aromatase inhibitor can result in the complete masculinization of female gonads. These masculinized females are phenotypically indistinguishable from normal males at adulthood. Lower levels of aromatase inhibition resulted in intersexed gonads and possible female reproductive impairment at adulthood. These results indicate that exposure of amphibians to xenobiotics capable of inhibiting aromatase would result in adverse reproductive consequences.