Joseph J. Korte

Aspects of basic reproductive biology and endocrinology in the fathead minnow (Pimephales promelas)

The fathead minnow (Pimephales promelas) has been proposed as a model species for assessing the adverse effects of endocrine-disrupting chemicals (EDCs) on reproduction and development. The purpose of these studies was to develop baseline reproductive biology and endocrinology data for this species to support interpretation of tests with potential EDCs. Pairs of reproductively-active fathead minnows (n=70) were evaluated with respect to reproductive cyclicity in terms of spawning interval and fecundity. The mode and mean (+/-SE) spawning intervals for the fish in this study were 3.0 and 3.7+/-0.1 days, respectively. The mean number of eggs produced per spawn was 85+/-2.8. Animals were sacrificed at periodic intervals during the established spawning cycle and measurements made of gonadal condition (gonadosomatic index [GSI], histopathology) and plasma concentrations of vitellogenin and sex steroids (beta-estradiol, testosterone, 11-ketotestosterone). The GSI in females varied significantly as a function of spawning interval, with the largest values occurring day 2 post-spawn, just prior to the interval of maximum spawning activity. Plasma beta-estradiol concentrations in females also varied significantly relative to peak values in the GSI and spawning activity. Vitellogenin concentrations in the female, and male GSI and steroid concentrations did not vary significantly relative to position in the spawning cycle. Concentrations of beta-estradiol in females and 11-ketotestosterone in males were positively correlated with testosterone concentrations.

Metamorphic inhibition of Xenopus laevis by sodium perchlorate: effects on development and thyroid histology.

The perchlorate anion inhibits thyroid hormone (TH) synthesis via inhibition of the sodium-iodide symporter. It is, therefore, a good model chemical to aid in the development of a bioassay to screen chemicals for affects on thyroid function. Xenopus laevis larvae were exposed to sodium perchlorate during metamorphosis, a period of TH-dependent development, in two experiments. In the first experiment, stage 51 and 54 larvae were exposed for 14 d to 16, 63, 250, 1,000, and 4,000 microg perchlorate/ L. In the second experiment, stage 51 larvae were exposed throughout metamorphosis to 8, 16, 32, 63, and 125 microg perchlorate/L. Metamorphic development and thyroid histology were the primary endpoints examined. Metamorphosis was retarded significantly in the first study at concentrations of 250 microg/L and higher, but histological effects were observed at 16 microg/L. In the second study, metamorphosis was delayed by 125 microg/L and thyroid size was increased significantly at 63 microg/L. These studies demonstrate that inhibition of metamorphosis readily can be detected using an abbreviated protocol. However, thyroid gland effects occur at concentrations below those required to elicit developmental delay, demonstrating the sensitivity of this endpoint and suggesting that thyroidal compensation is sufficient to promote normal development until perchlorate reaches critical concentrations.

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 enzymes 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.

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.

Comparison of ELISAs for detecting vitellogenin in the fathead minnow (Pimephales promelas).

Measurement of vitellogenin (VTG) concentrations in the fathead minnow (Pimephales promelas) is currently being considered and evaluated for screening of endocrine active substances. One of the proposed methods, an enzyme-linked immunosorbent assay (ELISA) based on VTG from carp (Cyprinus carpio), was recently evaluated in an inter-laboratory ring test using whole body homogenates from juvenile fathead minnows. The objective of the current study was to compare the performance of three different ELISAs for measuring fathead minnow VTG: (1) a heterologous carp VTG (cVTG) ELISA used in the ring test, (2) a homologous fathead minnow VTG (fVTG) ELISA, and (3) a hybrid ELISA with the antibody developed for cVTG, but using fVTG for coating the plates and preparing standard curves. VTG was measured in whole body homogenates from juvenile fathead minnows exposed to 17alpha-ethynylestradiol (EE(2); 10 ng/l) and whole body homogenates and plasma from adult fathead minnows exposed to 17beta-estradiol (E(2); 5 mg/kg; i.p.). The cVTG assay showed lower specificity for fathead minnow VTG in whole body homogenates and plasma from treated fish, compared to the fVTG assay. VTG concentrations in juvenile fathead minnow homogenates from the EE(2)-exposed group were approximately 50-fold higher when measured using the fVTG method compared to the cVTG method. Use of the homologous fVTG in the hybrid cVTG assay yielded VTG concentrations in the range of the fVTG assay but the low specificity persisted. The homologous fVTG assay is recommended to achieve accurate quantification of VTG levels in fathead minnows.

Early temporal effects of three thyroid hormone synthesis inhibitors in Xenopus laevis

Thyroid axis disruption is an important consideration when evaluating risks associated with chemicals. Bioassay methods that include thyroid-related endpoints have been developed in a variety of species, including amphibians, whose metamorphic development is thyroid hormone (TH)-dependent. Inhibition of TH synthesis in these species leads to developmental delay, and assays designed to capture these effects take several weeks to complete. In an effort to develop a shorter term approach, the early responses of various endpoints were evaluated in Xenopus laevis throughout 8d of exposure to three TH synthesis inhibitors: methimazole (100mg/L), 6-propylthiouracil (6-PTU) (20mg/L), and perchlorate (4 mg/L). Endpoints included thyroid gland histology and cell numbers, circulating TH concentrations, and thyroidal TH and associated iodo-compounds. Thyroidal 3,5-diodo-L-tyrosine (DIT) and thyroxine (T4) were significantly reduced from day 2 onward by all three chemicals, while 3-monoiodo-L-tyrosine (MIT) was significantly reduced by methimazole and perchlorate, but not by 6-PTU. These reductions were the earliest indicators of TH synthesis inhibition. Histological effects were apparent on day 4 and became more exaggerated through day 8. However, reductions in circulating T4 and increases in thyroid gland cell numbers were not apparent until day 6. Reductions of thyroidal MIT, DIT, and T4 and circulating T4 are indicative of inhibitory effects of the chemicals on TH synthesis. Changes in thyroid histology and cell number represent compensatory effects modulated by circulating TSH. These observations establish a basis for the development of short term amphibian-based methods to evaluate thyroid axis effects using a suite of diagnostic endpoints.

Characterization of thyroid hormone transporter expression during tissue-specific metamorphic events in Xenopus tropicalis.

Thyroid hormone (TH) induces the dramatic morphological and physiological changes that together comprise amphibian metamorphosis. TH-responsive tissues vary widely with developmental timing of TH-induced changes. How larval tadpole tissues are able to employ distinct metamorphic programs in a developmental stage- and TH-dependent manner is still unknown. Recently, several proteins capable of transporting TH have been identified. TH action and metabolism occurs primarily intracellularly, highlighting the importance of TH transporters. We examined the hypothesis that TH transporter expression and tissue distribution play an important role in mediating TH-induced metamorphic events. Xenopus tropicalis homologs for known TH transporting OATP, MCT and LAT family proteins were identified and gene specific qRT-PCR primers were developed. Total RNA was extracted from tissues representing three unique developmental fates including: growth/differentiation (hind limb), death/resorption (gill, tail) and remodeling (brain, liver, kidney). For growing and resorbing tissues, results showed the general trend of low initial expression levels of MCT8 and MCT10 transporters, followed by a several-fold increase of expression as the tissue undergoes TH-dependent metamorphic changes. The expression pattern in remodeling tissues was less uniform: a general decrease in transporter expression was observed in the liver, while the kidney and brain exhibited a range of expression patterns for several TH transporters. Collectively, these developmental expression patterns are consistent with TH transporting proteins playing a role in the effects of TH in peripheral tissues.

Inhibition of the thyroid hormone pathway in Xenopus laevis by 2-mercaptobenzothiazole

Determining the effects of chemicals on the thyroid system is an important aspect of evaluating chemical safety from an endocrine disrupter perspective. Since there are numerous chemicals to test and limited resources, prioritizing chemicals for subsequent in vivo testing is critical. 2-Mercaptobenzothiazole (MBT), a high production volume chemical, was tested and shown to inhibit thyroid peroxidase (TPO) enzyme activity in vitro, a key enzyme necessary for the synthesis of thyroid hormone. To determine the thyroid disrupting activity of MBT in vivo, Xenopus laevis larvae were exposed using 7- and 21-day protocols. The 7-day protocol used 18-357 μg/L MBT concentrations and evaluated: metamorphic development, thyroid histology, circulating T4, circulating thyroid stimulating hormone, thyroidal sodium-iodide symporter gene expression, and thyroidal T4, T3, and related iodo-amino acids. The 21-day protocol used 23-435 μg/L MBT concentrations and evaluated metamorphic development and thyroid histology. Both protocols demonstrated that MBT is a thyroid disrupting chemical at the lowest concentrations tested. These studies complement the in vitro study used to identify MBT as a high priority for in vivo testing, supporting the utility/predictive potential of a tiered approach to testing chemicals for TPO activity inhibition. The 7-day study, with more comprehensive, sensitive, and diagnostic endpoints, provides information at intermediate biological levels that enables linking various endpoints in a robust and integrated pathway for thyroid hormone disruption associated with TPO inhibition.

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 17β-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 17β-trenbolone exposure results in effects down to 78 ng/L, illustrating potential effects from exposure to androgenic compounds in anurans.

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-β 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.