Targeted Pathway-based In Vivo Testing Using Thyroperoxidase Inhibition to Evaluate Plasma Thyroxine as a Surrogate Metric of Metamorphic Success in Model Amphibian Xenopus laevis.

Chemical safety evaluation is in the midst of a transition from traditional whole-animal toxicity testing to molecular pathway-based in vitro assays and in silico modeling. However, to facilitate the shift in reliance on apical effects for risk assessment to predictive surrogate metrics having characterized linkages to chemical mechanisms of action, targeted in vivo testing is necessary to establish these predictive relationships. In this study, we demonstrate a means to predict thyroid-related metamorphic success in the model amphibian Xenopus laevis using relevant biochemical measurements during early prometamorphosis. The adverse outcome pathway for thyroperoxidase inhibition leading to altered amphibian metamorphosis was used to inform a pathway-based in vivo study design that generated response-response relationships. These causal relationships were used to develop Bayesian probabilistic network models that mathematically determine conditional dependencies between biochemical nodes and support the predictive capability of the biochemical profiles. Plasma thyroxine concentrations were the most predictive of metamorphic success with improved predictivity when thyroid gland sodium-iodide symporter gene expression levels (a compensatory response) were used in conjunction with plasma thyroxine as an additional regressor. Although thyroid-mediated amphibian metamorphosis has been studied for decades, this is the first time a predictive relationship has been characterized between plasma thyroxine and metamorphic success. Linking these types of biochemical surrogate metrics to apical outcomes is vital to facilitate the transition to the new paradigm of chemical safety assessments.

Flow cytometric analysis of Xenopus laevis and X. tropicalis blood cells using acridine orange.

Automated blood cell counters can distinguish cells based on their size and the presence or absence of a nucleus. However, most vertebrates have nucleated blood cells that cannot be counted automatically. We established an alternative automatic method for counting peripheral blood cells by staining cells with the fluorescent dye acridine orange (AO) and analysing cell populations using flow cytometry (FCM). As promising new animal models, we chose Xenopus laevis and three inbred strains of X. tropicalis. We compared the haematological phenotypes, including blood cell types, cell sizes, cellular structure, and erythrocyte lifespans/turnover rate among X. laevis and the three inbred strains of X. tropicalis. Each cell type from X. laevis was sorted according to six parameters: forward- and side-scattered light emission, AO red and green fluorescence intensity, and cellular red and green fluorescence. Remarkably, the erythrocyte count was the highest in the Golden line, suggesting that genetic factors were associated with the blood cells. Furthermore, immature erythrocytes in anaemic X. laevis could be separated from normal blood cells based on red fluorescence intensity. These results show that FCM with AO staining allows for an accurate analysis of peripheral blood cells from various species.

Effects of multiple life stage exposure to the fungicide prochloraz in Xenopus laevis: Manifestations of antiandrogenic and other modes of toxicity.

The Larval Amphibian Growth and Development Assay (LAGDA) is an internationally harmonized testing guideline for evaluating effects of chronic chemical exposure in amphibians. In order to evaluate the effects of chronic exposure to an antiandrogenic chemical in an amphibian model, prochloraz was tested using a variation of the LAGDA design. Exposure was initiated with <1d post-fertilization embryos at nominal concentrations of 0, 6.7, 20, 60 and 180 µg/L (0, 18, 53, 159, 478 nM) and continued in flow-through conditions until two months following the median time that controls completed metamorphosis. Growth, developmental rate, circulating thyroid hormone and thyroid gland histopathology were evaluated in a subsample at completion of metamorphosis. There were no effects on growth or development at this stage, but circulating thyroid hormone was elevated in the 20, 60 and 180 µg/L treatments and minimal to mild thyroid follicular cell hypertrophy was observed histologically in the 180 µg/L treatment. Growth, overt toxicity, and reproductive development were evaluated at test termination. There were no effects on growth in either gender, but livers and kidneys exhibited treatment-related pathologies consistent with organ toxicity related to metabolism and presumably impaired excretion of prochloraz metabolites. Histological assessments of female ovaries resulted in minimal pathologies only in the 180 µg/L treatment while male testes exhibited numerous treatment-related pathologies that are consistent with previously reported antiandrogenic effects of prochloraz in other species. The most severe testis pathologies occurred in the 180 µg/L treatment; however, incidences of treatment-related pathologies occurred in all prochloraz treatments. Müllerian duct regression in males was inhibited by prochloraz exposure while Müllerian duct maturation in females was accelerated, characteristic of a feminizing effect. Gene expression levels of potential biomarkers of testis function were also measured. Relative abundance of cyp17a1 transcripts was generally unaffected by prochloraz exposure whereas the Insl3 orthologue, rflcii, was elevated by 3 and >5-fold in the 60 and 180 µg/L treatments, respectively, indicating impaired Leydig cell maturation and testosterone signaling. Overall, prochloraz exposure caused effects characteristic of an antiandrogenic mode of action, which is consistent with previously reported results in other species and supports the utility of the LAGDA design for chemical testing.

Caspase-9 has a nonapoptotic function in Xenopus embryonic primitive blood formation.

Caspases constitute a family of cysteine proteases centrally involved in programmed cell death, which is an integral part of normal embryonic and fetal development. However, it has become clear that specific caspases also have functions independent of cell death. In order to identify novel apoptotic and nonapoptotic developmental caspase functions, we designed and transgenically integrated novel fluorescent caspase reporter constructs in developing Xenopus embryos and tadpoles. This model organism has an external development, allowing direct and continuous monitoring. These studies uncovered a nonapoptotic role for the initiator caspase-9 in primitive blood formation. Functional experiments further corroborated that caspase-9, but possibly not the executioners caspase-3 and caspase-7, are required for primitive erythropoiesis in the early embryo. These data reveal a novel nonapoptotic function for the initiator caspase-9 and, for the first time, implicate nonapoptotic caspase activity in primitive blood formation.

Acute and Chronic Outcomes of Gas-Bubble Disease in a Colony of African Clawed Frogs (Xenopus laevis).

Gas-bubble disease occurs in aquatic species that are exposed to water that is supersaturated with gases. In February 2007, municipal water supersaturated with gas was inadvertently pumped into the vivarium's aquatic housing systems and affected approximately 450 adult female Xenopus laevis. The inflow of supersaturated water was stopped immediately, the holding tanks aggressively aerated, and all experimental manipulations and feeding ceased. Within the first 6 h after the event, morbidity approached 90%, and mortality reached 3.5%. Acutely affected frogs showed clinical signs of gas-bubble disease: buoyancy problems, micro- and macroscopic bubbles in the foot webbing, hyperemia in foot webbing and leg skin, and loss of the mucous slime coat. All of the frogs that died or were euthanized had areas of mesenteric infarction, which resulted in intestinal epithelial necrosis and degeneration of the muscular tunic. Over the subsequent 2 wk, as gas saturation levels returned to normal, the clinical symptoms resolved completely in the remaining frogs. However, 3 mo later, 85% of them failed to lay eggs or produce oocytes, and the remaining 15% produced oocytes of low number and poor quality, yielding cytosolic extracts with poor to no enzymatic activity. Histology of the egg mass from a single 2- to 3-y-old frog at 3 mo after disease resolution revealed irregularly shaped oocytes, few large mature oocytes, and numerous small, degenerating oocytes. At 6 mo after the incident, the remaining frogs continued to fail to produce eggs of sufficient quantity or quality after hormonal priming. The researchers consequently opted to cull the remainder of the colony and repopulate with new frogs.

Quantification of X. laevis vitellogenin by liquid chromatography tandem mass spectrometry.

Over the last several decades, there has been an increase in public awareness and regulatory activity in regard to the presence of emerging contaminants in the environment that may have the potential to interact with the endocrine system of exposed wildlife. Alterations in vitellogenin (VTG), a high density yolk precursor protein, can indicate endocrine activity in oviparous species, including many fish and amphibians. While various methodologies and experiments have been performed to characterize baseline VTG concentrations among commonly studied fish species, fewer methodologies for accurately quantifying amphibian VTG are available. Since there is relatively little information available on background VTG levels in male and female frogs, the present investigation set out to quantify baseline levels of VTG in juvenile as well as adult male and female African clawed frogs (Xenopus laevis) using a newly developed liquid chromatography tandem mass spectrometry method. This new methodology for quantifying VTG in X. laevis frog blood plasma can be applied in mechanistic and toxicity studies with X. laevis to better characterize potential endocrine modes of action.

Biochemical and Hematologic Reference Intervals for Aged Xenopus laevis in a Research Colony.

Xenopus laevis, the African clawed frog, is commonly used in developmental and toxicology research studies. Little information is available on aged X. laevis; however, with the complete mapping of the genome and the availability of transgenic animal models, the number of aged animals in research colonies is increasing. The goals of this study were to obtain biochemical and hematologic parameters to establish reference intervals for aged X. laevis and to compare results with those from young adult X. laevis. Blood samples were collected from laboratory reared, female frogs (n = 52) between the ages of 10 and 14 y. Reference intervals were generated for 30 biochemistry analytes and full hematologic analysis; these data were compared with prior results for young X. laevis from the same vendor. Parameters that were significantly higher in aged compared with young frogs included calcium, calcium:phosphorus ratio, total protein, albumin, HDL, amylase, potassium, CO2, and uric acid. Parameters found to be significantly lower in aged frogs included glucose, AST, ALT, cholesterol, BUN, BUN:creatinine ratio, phosphorus, triglycerides, LDL, lipase, sodium, chloride, sodium:potassium ratio, and anion gap. Hematology data did not differ between young and old frogs. These findings indicate that chemistry reference intervals for young X. laevis may be inappropriate for use with aged frogs.

17α-Ethinylestradiol can disrupt hemoglobin catabolism in amphibians.

Different chemical substances, which enter the environment due to anthropogenic influences, can affect the endocrine system and influence development and physiology of aquatic animals. One of these endocrine disrupting chemicals is the synthetic estrogen, 17α-ethinylestradiol (EE2), which is a main component of various oral contraceptives and demonstrably affects many different aquatic vertebrates at extremely low concentrations by feminization phenomena. The aim of the present study was to investigate whether a four week exposure to three different concentrations of EE2 (0.3 ng/L, 29.6 ng/L and 2960 ng/L) affects the catabolism of hemoglobin of the amphibian Xenopus laevis. The results of this study demonstrate for the first time that beside an increase of the hepatic vitellogenin gene expression, exposure to EE2 also decreases the gene expression of the hepatic heme oxygenase 1 and 2 (HO1, HO2), degrading heme of different heme proteins to biliverdin, as well as of the biliverdin reductase A (BLVRA), which converts biliverdin to bilirubin. The results further suggest that EE2 already at the environmentally relevant concentration of (29.6 ng/L) can disrupt hemoglobin catabolism, indicated by decreased gene expression of HO2, which becomes evident at the highest EE2 concentration that led to a severe increase of biliverdin in plasma.

VEGFA-dependent and -independent pathways synergise to drive Scl expression and initiate programming of the blood stem cell lineage in Xenopus.

The first haematopoietic stem cells share a common origin with the dorsal aorta and derive from putative adult haemangioblasts in the dorsal lateral plate (DLP) mesoderm. Here we show that the transcription factor (TF) stem cell leukaemia (Scl/Tal1) is crucial for development of these adult haemangioblasts in Xenopus and establish the regulatory cascade controlling its expression. We show that VEGFA produced in the somites is required to initiate adult haemangioblast programming in the adjacent DLP by establishing endogenous VEGFA signalling. This response depends on expression of the VEGF receptor Flk1, driven by Fli1 and Gata2. Scl activation requires synergy between this VEGFA-controlled pathway and a VEGFA-independent pathway controlled by Fli1, Gata2 and Etv2/Etsrp/ER71, which also drives expression of the Scl partner Lmo2. Thus, the two ETS factors Fli1 and Etv6, which drives the VEGFA expression in both somites and the DLP, sit at the top of the adult haemangioblast gene regulatory network (GRN). Furthermore, Gata2 is initially activated by Fli1 but later maintained by another ETS factor, Etv2. We also establish that Flk1 and Etv2 act independently in the two pathways to Scl activation. Thus, detailed temporal, epistatic measurements of key TFs and VEGFA plus its receptor have enabled us to build a Xenopus adult haemangioblast GRN.

Cytochemical and immunocytochemical characterization of blood cells and immunohistochemical analysis of spleen cells from 2 species of frog, Rana (Aquarana) catesbeiana and Xenopus laevis.

BACKGROUND: Mechanisms of amphibian diseases are not characterized as well as those in domestic mammalian species. Antemortem laboratory testing is limited in frogs, presenting a diagnostic challenge to zoos, laboratories, and exotic veterinarians. OBJECTIVE: This study aimed to characterize blood cells and splenic cells from 2 anuran species based on characteristics identified by Wright staining, cytochemical staining, and immunochemical analysis and on histologic examination of spleens. METHODS: Blood specimens and spleens were obtained from 2 species of frog, the American bullfrog (Rana [Aquarana] catesbeiana) and the African clawed frog (Xenopus laevis). Blood smears were evaluated after Wright staining and cytochemical staining for α-naphthyl butyrate esterase (NBE), chloroacetate esterase (CAE), myeloperoxidase (PER), Sudan black B (SBB), and leukocyte alkaline phosphatase (LAP) reactions and for immunoreactivity for antibodies against CD3ε, CD79a, and BLA.36 antigens. Histologic sections of spleen were evaluated after staining with H&E and for immunoreactivity for CD3ε, CD79a, and BLA.36 antigens. RESULTS: In bullfrogs, neutrophils, eosinophils, and monocytes were positive for some or all of the following: NBE, CAE, PER, and SBB; lymphocytes occasionally were positive for CAE. In clawed frogs, neutrophils, basophils, and monocytes were positive for some or all of the following: NBE, CAE, PER, and SBB; eosinophils occasionally were positive for CAE and PER, and lymphocytes were negative for all cytochemical stains. LAP was not a useful marker for any leukocyte type. In both species, peripheral blood lymphocytes were strongly immunoreactive for CD3ε, CD79a, and BLA.36. In splenic tissue, histologic patterns varied and there was diffuse immunoreactivity for CD79a and BLA.36 with focal reactivity for CD3ε, but with different distribution patterns in each species. CONCLUSION: Cytochemical and immunochemical analysis of cells may be helpful in identification and characterization of amphibian blood cells and splenic cells for evaluation of the health of these animals.

Friend of GATA (FOG) interacts with the nucleosome remodeling and deacetylase complex (NuRD) to support primitive erythropoiesis in Xenopus laevis.

Friend of GATA (FOG) plays many diverse roles in adult and embryonic hematopoiesis, however the mechanisms by which it functions and the roles of potential interaction partners are not completely understood. Previous work has shown that overexpression of FOG in Xenopus laevis causes loss of blood suggesting that in contrast to its role in mammals, FOG might normally function to repress erythropoiesis in this species. Using loss-of-function analysis, we demonstrate that FOG is essential to support primitive red blood cell (RBC) development in Xenopus. Moreover, we show that it is specifically required to prevent excess apoptosis of circulating primitive RBCs and that in the absence of FOG, the pro-apoptotic gene Bim-1 is strongly upregulated. To identify domains of FOG that are essential for blood development and, conversely, to begin to understand the mechanism by which overexpressed FOG represses primitive erythropoiesis, we asked whether FOG mutants that are unable to interact with known co-factors retain their ability to rescue blood formation in FOG morphants and whether they repress erythropoiesis when overexpressed in wild type embryos. We find that interaction of FOG with the Nucleosome Remodeling and Deacetylase complex (NuRD), but not with C-terminal Binding Protein, is essential for normal primitive RBC development. In contrast, overexpression of all mutant and wild type constructs causes a comparable repression of primitive erythropoiesis. Together, our data suggest that a requirement for FOG and its interaction with NuRD during primitive erythropoiesis are conserved in Xenopus and that loss of blood upon FOG overexpression is due to a dominant-interfering effect.

Thyroid-stimulating hormone (TSH): measurement of intracellular, secreted, and circulating hormone in Xenopus laevis and Xenopus tropicalis.

Thyroid-stimulating hormone (TSH) is an important regulator of the hypothalamic-pituitary-thyroid (HPT) axis in Xenopus laevis. To evaluate the role of this hormone on developing tadpoles, immunologically-based Western blots and sandwich ELISAs were developed for measuring intracellular (within pituitaries), secreted (ex vivo pituitary culture), and circulating (serum) amounts. Despite the small size of the tadpoles, these methods were able to easily measure intracellular and secreted TSH, and circulating TSH was measurable in situations where high levels were induced. The method was validated after obtaining a highly purified and enriched TSH sample using anti-TSH-ß antibodies conjugated to magnetic beads. Subsequent mass-spectrometric analysis of the bands from SDS-PAGE and Western procedures identified the presence of amino acid sequences corresponding to TSH subunits. The purified sample was also used to prepare standard curves for quantitative analysis. The Western and ELISA methods had limits of detection in the low nanogram range. While the majority of the developmental work for these methods was done with X. laevis, the methods also detected TSH in Xenopus tropicalis. To our knowledge this is the first report of a specific detection method for TSH in these species, and the first to measure circulating TSH in amphibians. Examples of the utility of the methods include measuring a gradual increase in pituitary TSH at key stages of development, peaking at stages 58-62; the suppression of TSH secretion from cultured pituitaries in the presence of thyroid hormone (T4); and increases in serum TSH following thyroidectomy.

Identification of erythroid progenitors induced by erythropoietic activity in Xenopus laevis.

Oxygen is essential for the survival of animals. Red blood cells in the circulation, i.e. peripheral erythrocytes, are responsible for transporting oxygen to tissues. The regulation of erythropoiesis in vertebrates other than mammals is yet to be elucidated. Recently we identified erythropoietin, a primary regulator of erythropoiesis, in Xenopus laevis, which should enable us to identify target cells, including erythroid progenitors, and to investigate the production and development of erythroid cells in amphibians. Here, we established a semi-solid colony-forming assay in Xenopus laevis to clarify the existence of colony-forming unit-erythroid cells, the functional erythroid progenitors identified in vitro. Using this assay, we showed that recombinant xlEPO induces erythroid colony formation in vitro and detected an increased level of erythropoietin activity in blood serum during acute anemic stress. In addition, our study demonstrated the possible presence of multiple, non-xlEPO, factors in anemic serum supportive of erythroid colony formation. These results indicate that erythropoiesis mediated by erythropoietin is present in amphibian species and, furthermore, that the regulatory mechanisms controlling peripheral erythrocyte number may vary among vertebrates.

Serum clinical biochemical and hematologic reference ranges of laboratory-reared and wild-caught Xenopus laevis.

The South African clawed frogs Xenopus laevis and X. tropicalis are fully aquatic amphibians and well-established animal models. Because genetically engineered laboratory Xenopus are now being produced, the establishment of normal reference ranges for serum biochemical and hematologic parameters is essential for phenotyping and as a diagnostic aide. We determined normal reference ranges for hematologic values from 3 populations of X. laevis: wild-caught frogs (n = 43) and frogs from 2 commercial sources (A, n = 166; B, n = 109). For serum biochemistry, we determined normal reference ranges for frogs from source A and wild-caught frogs divided by sex and season. Significant differences across populations were found in WBC and RBC counts, hemoglobin concentration, hematocrit, mean corpuscular hemoglobin concentration, and mean corpuscular volume. Among serum biochemical analytes, significant differences were found for albumin:globulin ratio, anion gap, and concentrations of albumin, globulin, total protein, lipase, alanine transaminase, γ-glutamyl transpeptidase; creatine phosphokinase; indirect, direct, and total bilirubin; cholesterol, low-density lipoprotein lipase, carbon dioxide, glucose, lactacte dehydrogenase, calcium, chloride, and sodium. We hypothesize that these differences can be attributed to differences in water quality, habitat, ambient temperature, diet, sex, recent transport or shipment, and genetic background. However, testing that hypothesis is beyond the scope of the current study. In addition, clinical chemistry and hematologic reference range values Xenopus laevis are quite distinct from those for other species and are most consistent with the only values published for another fully aquatic amphibian, the Eastern hellbender (Cryptobranchus alleganiensis).

Mate calling behavior of male South African clawed frogs (Xenopus laevis) is suppressed by the antiandrogenic endocrine disrupting compound flutamide.

Several environmental pollutants have been identified as antiandrogenic endocrine disrupting chemicals (EDC), with flutamide (FLU) being a model compound for this type of action. Despite impacts of EDC interfering with sexual differentiation and reproduction in amphibians, established information about suggested effects on sexual behavior is still lacking. In this study adult male Xenopus laevis were injected with human chorionic gonadotropin (hCG) to initiate mate calling behavior. After one day hCG-stimulated frogs were treated via aqueous exposure over three days without and with FLU at concentrations of 10(-8) and 10(-6) M in comparison to untreated frogs. Androgen controlled mate calling behavior was recorded during the 12h dark period. At the end of exposure circulating levels of testosterone (T) and 17beta-estradiol (E2) were determined and furthermore gene expression was measured concerning reproductive biomarkers such as hypophysial luteinizing hormone (LH), follicle-stimulating hormone (FSH), testicular aromatase (ARO), 5alpha reductase type 1 (SRD5alpha1) and 5alpha reductase type 2 (SRD5alpha2). Both concentrations of FLU caused a significant decrease in calling activity starting at the second day of exposure. HCG injected positive controls had elevated levels of T compared to negative control frogs while in parallel treatment with FLU did not affect significantly the hCG elevated sex steroid levels. Furthermore, hCG treatment led to significantly decreased levels of gene expression for ARO and SRD5alpha2 but no impacts were detected on LH, FSH or SRD5alpha1 mRNA levels compared to negative controls. In summary, the behavioral parameter mate calling is the most sensitive biomarker detecting antiandrogenic modes of action in this challenge-experiment indicating that this non-invasive method could markedly contribute for sensitive assessment of antiandrogenic EDC.

The pharmacokinetics of enrofloxacin in adult African clawed frogs (Xenopus laevis).

Pharmacokinetics of enrofloxacin, a fluoroquinolone antibiotic, was determined in adult female Xenopus laevis after single-dose administration (10 mg/kg) by intramuscular or subcutaneous injection. Frogs were evaluated at various time points until 8 h after injection. Plasma was analyzed for antibiotic concentration levels by HPLC. We computed pharmacokinetic parameters by using noncompartmental analysis of the pooled concentrations (naive pooled samples). After intramuscular administration of enrofloxacin, the half-life was 5.32 h, concentration maximum was 10.85 µg/mL, distribution volume was 841.96 mL/kg, and area under the time-concentration curve was 57.59 µg×h/mL; after subcutaneous administration these parameters were 4.08 h, 9.76 µg/mL, 915.85 mL/kg, and 47.42 µg×h/mL, respectively. According to plasma pharmacokinetics, Xenopus seem to metabolize enrofloxacin in a manner similar to mammals: low levels of the enrofloxacin metabolite, ciprofloxacin, were detected in the frogs' habitat water and plasma. At necropsy, there were no gross or histologic signs of toxicity after single-dose administration; toxicity was not evaluated for repeated dosing. The plasma concentrations reached levels considered effective against common aquatic pathogens and suggest that a single, once-daily dose would be a reasonable regimen to consider when treating sick frogs. The treatment of sick frogs should be based on specific microbiologic identification of the pathogen and on antibiotic susceptibility testing.

Programming neuroendocrine stress axis activity by exposure to glucocorticoids during postembryonic development of the frog, Xenopus laevis.

Exposure to elevated glucocorticoids during early mammalian development can have profound, long-term consequences for health and disease. However, it is not known whether such actions occur in nonmammalian species, and if they do, whether the molecular physiological mechanisms are evolutionarily conserved. We investigated the effects of dietary restriction, which elevates endogenous corticosterone (CORT), or exposure to exogenous CORT added to the aquarium water of Xenopus laevis tadpoles on later-life measures of growth, feeding behavior, and neuroendocrine stress axis activity. Dietary restriction of prometamorphic tadpoles reduced body size at metamorphosis, but juvenile frogs increased food intake, showed catch-up growth through 21 d after metamorphosis, and had elevated whole-body CORT content compared with controls. Dietary restriction causes increased CORT in tadpoles, so to mimic this increase, we treated tadpoles with 100 nm CORT or vehicle for 5 or 10 d and then reared juvenile frogs to 2 months after metamorphosis. Treatment with CORT decreased body weight at metamorphosis, but juvenile frogs showed catch-up growth and had elevated basal plasma (CORT). Immunohistochemical analysis showed that CORT exposure as a tadpole led to decreased glucocorticoid receptor immunoreactivity in brain regions involved with stress axis regulation and in the anterior pituitary gland of juvenile frogs. The elevated CORT in juvenile frogs, which could result from decreased negative feedback owing to down-regulation of glucocorticoid receptor, may drive the hyperphagic response. Taken together, our findings suggest that long-term, stable phenotypic changes in response to elevated glucocorticoids early in life are an ancient and conserved feature of the vertebrate lineage.

Feminizing/demasculinizing effects of polychlorinated biphenyls on the secondary sexual development of Xenopus laevis.

We have previously demonstrated that polychlorinated biphenyls (PCBs) have caused phenotypic feminization/demasculinization of gonadal development in Xenopus laevis. Whether PCBs affect secondary sexual development has remained unknown. In this study, X. laevis tadpoles were exposed to Aroclor1254 and PCB(3) from stage 46/47 (system of Nieuwkoop and Faber) for up to 1 month postmetamorphosis. After 24 months postmetamorphosis, the degree of secondary sexual development was examined. Male oviducts were observed in some of the PCB-exposed male frogs, but not in control males. These male oviducts had not completely developed in histological structure when compared with mature female oviducts. Larynx weight and width of PCB-exposed males were significantly less than those of control males. Laryngeal histology showed that PCBs inhibited cartilaginous and muscular development of male frogs, i.e. elastic cartilages had not completely developed and laryngeal muscle fibers were smaller. In a further study on adult male frogs, a decrease in serum testosterone level was found in PCB-exposed frogs compared with controls, but serum estradiol level was not significantly affected. Our study suggests that PCBs can cause phenotypic feminization/demasculinization of male genital ducts and larynges, and these effects may, in part, result from the decrease in serum testosterone level in X. laevis.

ADMP2 is essential for primitive blood and heart development in Xenopus.

We describe here the cloning of a new member of the TGF-beta family with similarity to the anti-dorsalizing morphogenetic proteins (ADMPs). This new gene, ADMP2, is expressed in a broad band of mesendoderm cells that appear to include the progenitors of the endoderm and the ventral mesoderm. Antisense morpholino oligonucleotide knockdown of ADMP2 results in near-complete disruption of primitive blood and heart development, while the development of other mesoderm derivatives, including pronephros, muscle and lateral plate is not disrupted. Moreover, the development of the primitive blood in ADMP2 knockdown embryos cannot be rescued by BMP. These results suggests that ADMP2 plays an early role in specifying presumptive ventral mesoderm in the leading edge mesoderm, and that ADMP2 activity may be necessary to respond to BMP signaling in the context of ventral mesoderm induction.

Plasma concentrations of estradiol and testosterone, gonadal aromatase activity and ultrastructure of the testis in Xenopus laevis exposed to estradiol or atrazine.

The ultrastructure of testicular cells of adult male African clawed frogs (Xenopus laevis) exposed to either estradiol (0.1 microg/L) or 2-chloro-4-ethylamino-6-isopropyl-amino-s-triazine (atrazine; 10 or 100 microg/L) was examined by electron microscopy and compared to plasma concentrations of the steroid hormones, testosterone (T) and estradiol (E2), testicular aromatase activity and gonad growth expressed as the gonado-somatic index (GSI). Exposure to E2 caused significant changes both at the sub-cellular and biochemical levels. Exposure to E2 resulted in significantly fewer sperm cells, inhibition of meiotic division of germ cells, more lipid droplets that are storage compartments for the sex steroid hormone precursor cholesterol, and lesser plasma T concentrations. Although not statistically significant, frogs exposed to E2 had slightly smaller GSI values. These results may be indicative of an inhibition of gonad growth and disrupted germ cell development by E2. Concentrations of E2 in plasma were greater in frogs exposed to E2 in water. Exposure to neither concentration of atrazine caused effects on germ cell development, testicular aromatase activity or plasma hormone concentrations. These results suggest that atrazine does not affect testicular function. In contrast, exposure of male X. laevis to E2 led to sub-cellular events that are indicative of disruption of testicular development, and demasculinization processes (decrease of androgen hormone titers). These results indicate that atrazine does not cause responses that are similar to those caused by exposure to E2.