Levonorgestrel causes feminization and dose-dependent masculinization in medaka fish (Oryzias latipes): Endocrine-disruption activity and its correlation with sex reversal.

The effect of a synthetic progestin, levonorgestrel (LNG), on the sex of exposed embryos was examined in medaka fish (Oryzias latipes). The aims of this study are to clarify the dual effect of LNG on sex and the correlation with its androgenic/estrogenic potential in medaka. LNG exposure causes significant dose-dependent masculinization (0.1-100 µg/L), whereas a decrease in the masculinization ratio is observed at 100 µg/L. LNG also causes significant feminization at 1-100 µg/L, but not in a dose-dependent manner. Exposure of estrogen-responsive gene (choriogeninH-EGFP) transgenic embryos to 100 µg/L LNG produced significant fluorescent signals in hatched fry. In vitro transcriptional assays indicated that LNG at 10-7-10-5 M induced significant activity for estrogen receptor (ESR)2a and ESR2b, but not for ESR1. In pre-self-feeding fry at 5 days post hatching (dph), 1-100 µg/L LNG caused a significant increase in the mRNA of choriogeninH, irrespective of genetic sex. Moreover, LNG (10-10-10-5 M) also caused a significant increase in the transcriptional activity of androgen receptor (AR) α and ARß in vitro, and 0.1 µg/L LNG significantly increased the mRNA levels of a testis-differentiation initiation factor, gonadal soma-derived factor (gsdf), as an androgen-upregulated and estrogen-downregulated gene, in 5 dph XX fry to levels similar to those in the control XY fry. However, 100 and 10 µg/L LNG suppressed or did not induce gsdf mRNA expression in XY and XX fry, respectively. Together, these findings show that LNG exerts estrogenic and androgenic activities in different concentration ranges, which correlate with the ratio of LNG-induced sex reversal. These results suggest for the first time, that medaka exposure to LNG can induce masculinization and feminization, based on the balance between androgenic and estrogenic activities, and the protocol applied in this study represents an alternative to the traditional animal model used to screen for endocrine-disrupting potential.

Adverse effects of thyroid-hormone-disrupting chemicals 6-propyl-2-thiouracil and tetrabromobisphenol A on Japanese medaka (Oryzias latipes).

Thyroid-hormone-disrupting chemicals are increasingly attracting attention because of their potential harmful effects on animal health, including on fishes. Here, we investigated the effects of exposure to the thyroid-hormone-disrupting chemicals 6-propyl-2-thiouracil (PTU) and tetrabromobisphenol A (TBBPA) on swim bladder inflation, eye development, growth, swimming performance, and the expression of thyroid-related genes in Japanese medaka (Oryzias latipes). PTU exposure resulted in reductions in eye size, growth, and swim bladder inflation, and these effects led to poorer swimming performance. These phenotypic effects were accompanied by increased expression of the thyroid-stimulating hormone subunit beta (tshß) paralog tshß-like, but there were no significant changes in expression for tshß, deiodinase 1 (dio1), deiodinase 2 (dio2), and thyroid hormone receptor alpha (trα) and beta (trß). For PTU exposure, we identified the key event (swim bladder inflation reduction) and an adverse outcome (swimming performance reduction). No significant effects from TBBPA exposure were seen on swim bladder inflation, eye development, growth, or swimming performance. However, expression of tshß-like and tshß (significantly enhanced) and trα and trß (significantly reduced) were affected by TBBPA exposure albeit not in dose-dependent manners. There were no effects of TBBPA on the expression of dio1 and dio2. We thus show that the two thyroid-hormone-disrupting chemicals PTU and TBBPA differ in their effect profiles with comparable effects on the studied phenotypes and thyroid-related gene expression to those reported in zebrafish.

Stress Fractures of the First Rib Related to Soft Tennis, Associated with the Tennis Ground Stroke.

Stress fractures of the first rib are uncommon and thought to be associated with overhead-throwing athletes. Soft tennis is similar to regular tennis but uses a much softer rubber ball. In the current report, a 14-year-old girl suffered from shoulder girdle pain, especially at the end of her tennis ground stroke. Plain radiographs showed overgrowth of bone with a fracture line on the first rib, and a diagnosis of stress fracture was made. She was advised to amend her stroke form to reduce force to the shoulder and was able to continue sports activity without pain 10 months after the appearance of her symptoms and before confirmation of bone healing. The current case is not associated with overhead-throwing, but possibly with repetitive exercises of her tennis ground strokes. Conservative medical follow-up with proper sport-specific professional advice allows continuation of the sport.

Summary of 17 chemicals evaluated by OECD TG229 using Japanese Medaka, Oryzias latipes in EXTEND 2016.

In June 2016, the Ministry of the Environment of Japan announced a program "EXTEND2016" on the implementation of testing and assessment for endocrine active chemicals, consisting of a two-tiered strategy. The aim of the Tier 1 screening and the Tier 2 testing is to identify the impacts on the endocrine system and to characterize the adverse effects to aquatic animals by endocrine disrupting chemicals detected in the aquatic environment in Japan. For the consistent assessment of the effects on reproduction associated with estrogenic, anti-estrogenic, androgenic, and/or anti-androgenic activities of chemicals throughout Tier 1 screening to Tier 2 testing, a unified test species, Japanese medaka (Oryzias latipes), has been used. For Tier 1 screening, the in vivo Fish Short-Term Reproduction Assay (OECD test guideline No. 229) was conducted for 17 chemicals that were nominated based on the results of environmental monitoring, existing knowledge obtained from a literature survey, and positive results in reporter gene assays using the estrogen receptor of Japanese medaka. In the 17 assays using Japanese medaka, adverse effects on reproduction (i.e., reduction in fecundity and/or fertility) were suggested for 10 chemicals, and a significant increase of hepatic vitellogenin in males, indicating estrogenic (estrogen receptor agonistic) potency, was found for eight chemicals at the concentrations in which no overt toxicity was observed. Based on these results, and the frequency and the concentrations detected in the Japanese environment, estrone, 4-nonylphenol (branched isomers), 4-tert-octylphenol, triphenyl phosphate, and bisphenol A were considered as high priority candidate substances for the Tier 2 testing.

Effects of synthetic sex steroid hormone exposures on gonadal sex differentiation and dynamics of a male-related gene, Gonadal soma-derived factor (Gsdf) and an estrogen up-regulated gene, Choriogenine-H (ChgH) gene expression in the euryhaline Javafish medaka, Oryzias javanicus, based on genetic sexes.

To clarify the basal aspects of sexual development in Javafish medaka, Oryzias javanicus (ZZ/ZW), a model marine species for ecotoxicity testing, we examined the details of gonadal sex differentiation and exogenous sex hormone-dependent sex reversals using genetic sex-linked DNA markers. Sex differences in germ cell numbers were observed at 5 days post hatching (dph), in which there was a significant increase in the germ cells of ZW. In ZW, diplotene oocytes and the ovarian cavity appeared at approximately 10, and 30 dph, respectively. In ZZ, spermatogonial proliferation was observed at approximately 20 dph. A ZZ-dominant expression of Gonadal soma-derived factor (Gsdf) mRNA was detected before hatching. The exposure of embryos to 17α-ethinylestradiol (EE2; 0.1, 1, 10 ng/mL) did not cause sex reversals in most cases. However, EE2 exposures led to significant Choriogenin-H (ChgH) mRNA expression, an estrogen up-regulated gene, in all fry; these exposures did not suppress Gsdf expression in ZZ fry. The exposure of embryos to 17α-methyltestosterone (MT; 0.1, 1, 10 ng/mL) caused sex reversals but only at low frequencies in ZW and ZZ fish. Although the 10 ng/mL MT exposure was accompanied by induction of significant Gsdf expression in ZW fry, induction of ChgH expression was also observed in several fry. Together, the present study indicates for the first time that male-dominant sexual dimorphic expression of Gsdf precedes the first morphological sex difference, i.e., the sex difference in germ cell number, and results strongly suggest that exogenous sex hormone-dependent sex reversal is not induced easily in O. javanicus.

Summary of reference chemicals evaluated by the fish short-term reproduction assay, OECD TG229, using Japanese Medaka, Oryzias latipes.

Under the Organisation for Economic Co-operation and Development (OECD), the Ministry of the Environment of Japan (MOE) added Japanese medaka (Oryzias latipes) to the test guideline fish short-term reproduction assay (FSTRA) developed by the United States Environmental Protection Agency (US EPA) using fathead minnow (Pimephales promelas). The FSTRA was designed to detect endocrine disrupting effects of chemicals interacting with the hypothalamic-pituitary-gonadal axis (HPG axis) such as agonists or antagonists on the estrogen receptor (Esr) and/or the androgen receptor (AR) and steroidogenesis inhibitors. We conducted the FSTRA with Japanese medaka, in accordance with OECD test guideline number 229 (TG229), for 16 chemicals including four Esr agonists, two Esr antagonists, three AR agonists, two AR antagonists, two steroidogenesis inhibitors, two progesterone receptor agonists, and a negative substance, and evaluated the usability and the validity of the FSTRA (TG229) protocol. In addition, in vitro reporter gene assays (RGAs) using Esr1 and ARß of Japanese medaka were performed for the 16 chemicals, to support the interpretation of the in vivo effects observed in the FSTRA. In the present study, all the test chemicals, except an antiandrogenic chemical and a weak Esr agonist, significantly reduced the reproductive status of the test fish, that is, fecundity or fertility, at concentrations where no overt toxicity was observed. Moreover, vitellogenin (VTG) induction in males and formation of secondary sex characteristics (SSC), papillary processes on the anal fin, in females was sensitive endpoints to Esr and AR agonistic effects, respectively, and might be indicators of the effect concentrations in long-term exposure. Overall, it is suggested that the in vivo FSTRA supported by in vitro RGA data can adequately detect effects on the test fish, O. latipes, and probably identify the mode of action (MOA) of the chemicals tested.

Inter- and Intraspecific Variation in Sex Hormone-Induced Sex-Reversal in Medaka, Oryzias latipes and Oryzias sakaizumii.

We compared sex-reversal ratios induced by 17α-methyltestosterone (MT) and 17ß-estradiol (E2) exposure in two inbred medaka strains: Hd-rR derived from Oryzias latipes and HNI-II from O. sakaizumii. All MT exposures (0.2-25 ng mL-1) induced complete XX sex-reversal in HNI-II. Although MT exposure at 0.2 ng mL-1 induced XX sex-reversal at > 95% in Hd-rR, other concentrations tested caused XX sex-reversal at lower frequencies (<50%). MT exposure at 1, 5, and 25 ng mL-1 induced XY sex-reversal in Hd-rR, but not in HNI-II. In Hd-rR, E2 exposure induced XY sex-reversal at > 10 ng mL-1, and in all fish feminization occurred 500 ng mL-1. In HNI-II, E2 induced XY sex-reversal at 50 and 250 ng mL-1, but only at rates below 20%. To clarify whether the strain differences in sex hormone-induced sex-reversal are characteristic of each species, we examined the effects of MT and E2 exposure on sex differentiation in five and two additional strains or wild stocks/populations of O. latipes and O. sakaizumii, respectively. MT exposure induced low XX and high XY sex-reversal rates in O. latipes, except in the Shizuoka population, but the trend was reversed in O. sakaizumii. Furthermore, E2-induced XY sex-reversal rates varied intraspecifically in O. latipes. Our results demonstrated that sensitivity to MT and E2 varied within O. latipes species. To evaluate the ecological impacts of environmental chemicals using medaka, it is important to define not only the species, but the strains, stocks, and populations to obtain accurate results.

Estrogen alters gonadal soma-derived factor (Gsdf)/Foxl2 expression levels in the testes associated with testis-ova differentiation in adult medaka, Oryzias latipes.

Testis-ova differentiation in sexually mature male medaka (Oryzias latipes) is easily induced by estrogenic chemicals, indicating that spermatogonia persist in sexual bipotentiality, even in mature testes in medaka. By contrast, the effects of estrogen on testicular somatic cells associated with testis-ova differentiation in medaka remain unclear. In this study, we focused on the dynamics of sex-related genes (Gsdf, Dmrt1, and Foxl2) expressed in Sertoli cells in the mature testes of adult medaka during estrogen-induced testis-ova differentiation. When mature male medaka were exposed to estradiol benzoate (EB; 800ng/L), testis-ova first appeared after EB treatment for 14days (observed as the first oocytes of the leptotene-zygotene stage). However, the testis remained structurally unchanged, even after EB treatment for 28days. Although Foxl2 is a female-specific sex gene, EB treatment for 7days induced Foxl2/FOXL2 expression in all Sertoli cell-enclosed spermatogonia before testis-ova first appeared; however, Foxl2 was not detected in somatic cells in control testes. Conversely, Sertoli-cell-specific Gsdf mRNA expression levels significantly decreased after EB treatment for 14days, and no changes were observed in DMRT1 localization following EB treatment, whereas Dmrt1 mRNA levels increased significantly. Furthermore, after EB exposure, FOXl2 and DMRT1 were co-localized in Sertoli cells during testis-ova differentiation, although FOXL2 localization was undetectable in Sertoli-cell-enclosed apoptotic testis-ova, whereas DMRT1 remained localized in Sertoli cells. These results indicated for the first time that based on the expression of female-specific sex genes, feminization of Sertoli cells precedes testis-ova differentiation induced by estrogen in mature testes in medaka; however, complete feminization of Sertoli cells was not induced in this study. Additionally, it is suggested strongly that Foxl2 and Gsdf expression constitute potential molecular markers for evaluating the effects of estrogenic chemicals on testicular somatic cells associated with estrogen-induced testis-ova differentiation in mature male medaka.

Summary of the development the US Environmental Protection Agency's Medaka Extended One Generation Reproduction Test (MEOGRT) using data from 9 multigenerational medaka tests.

In response to various legislative mandates, the US Environmental Protection Agency (USEPA) formed its Endocrine Disruptor Screening Program (EDSP), which in turn, formed the basis of a tiered testing strategy to determine the potential of pesticides, commercial chemicals, and environmental contaminants to disrupt the endocrine system. The first tier of tests is intended to detect the potential for endocrine disruption mediated through estrogen, androgen, or thyroid pathways, whereas the second tier is intended to further characterize the effects on these pathways and to establish a dose-response relationship for adverse effects. One of these tier 2 tests, the Medaka Extended One Generation Reproduction Test (MEOGRT), was developed by the USEPA for the EDSP and, in collaboration with the Japanese Ministry of the Environment, for the Guidelines for the Testing of Chemicals of the Organisation for Economic Co-operation and Development (OECD). The MEOGRT protocol was iteratively modified based on knowledge gained after the successful completion of 9 tests with variations in test protocols. The present study describes both the final MEOGRT protocol that has been published by the USEPA and the OECD, and the iterations that provided valuable insights into nuances of the protocol. The various tests include exposure to 17ß-estradiol, 4-t-octylphenol, o,p'- dichlorodiphenyltrichloroethane, 4-chloro-3-methylphenol, tamoxifen, 17ß-trenbolone, vinclozolin, and prochloraz. Environ Toxicol Chem 2017;36:3387-3403. Published 2017 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

Development of the Larval Amphibian Growth and Development Assay: Effects of benzophenone-2 exposure in Xenopus laevis from embryo to juvenile.

The Larval Amphibian Growth and Development Assay (LAGDA) is a globally harmonized chemical testing guideline developed by the U.S. Environmental Protection Agency in collaboration with Japan's Ministry of Environment to support risk assessment. The assay is employed as a higher tiered approach to evaluate effects of chronic chemical exposure throughout multiple life stages in a model amphibian species, Xenopus laevis. To evaluate the utility of the initial LAGDA design, the assay was performed using a mixed mode of action endocrine disrupting chemical, benzophenone-2 (BP-2). X. laevis embryos were exposed in flow-through conditions to 0, 1.5, 3.0 or 6.0 mg l-1 BP-2 until 2 months post-metamorphosis. Overt toxicity was evident throughout the exposure period in the 6.0 mg l-1 treatment due to elevated mortality rates and observed liver and kidney pathologies. Concentration-dependent increases in severity of thyroid follicular cell hypertrophy and hyperplasia occurred in larval tadpoles indicating BP-2-induced impacts on the thyroid axis. Additionally, gonads were impacted in all treatments with some genetic males showing both testis and ovary tissues (1.5 mg l-1 ) and 100% of the genetic males in the 3.0 and 6.0 mg l-1 treatments experiencing complete male-to-female sex reversal. Concentration-dependent vitellogenin induction occurred in both genders with associated accumulations of protein in the livers, kidneys and gonads, which was likely vitellogenin and other estrogen-responsive yolk proteins. This is the first study that demonstrates the endocrine effects of this mixed mode of action chemical in an amphibian species and demonstrates the utility of the LAGDA design for supporting chemical risk assessment. Copyright © 2016 John Wiley & Sons, Ltd.

Comparative Developmental Staging of Female and Male Water Fleas Daphnia pulex and Daphnia magna During Embryogenesis.

The freshwater crustacean genus Daphnia has been used extensively in ecological, developmental and ecotoxicological studies. Daphnids produce only female offspring by parthenogenesis under favorable conditions, but in response to various unfavorable conditions and external stimuli, they produce male offspring. Although we reported that exogenous exposure to juvenile hormones and their analogs can induce male offspring even under female-producing conditions, we recently established a male induction system in the Daphnia pulex WTN6 strain simply by changing day-length. This male and female induction system is suitable for understanding the innate mechanisms of sexual dimorphic development in daphnids. Embryogenesis has been described as a normal plate (developmental staging) in various daphnid species; however, all studies have mainly focused on female development. Here, we describe the developmental staging of both sexes during embryogenesis in two representative daphnids, D. pulex and D. magna, based on microscopic time-course observations. Our findings provide the first detailed insights into male embryogenesis in both species, and contribute to the elucidation of the mechanisms underlying sexual differentiation in daphnids.

Establishment of transactivation assay systems using fish, amphibian, reptilian and human thyroid hormone receptors.

Thyroid hormones are essential for the regulation of a wide range of biological processes associated with normal development and metabolism in vertebrates. For the screening of chemicals with a potential thyroid hormone and anti-thyroid hormone activities, we have established transient transactivation assay systems using thyroid hormone receptors (TRα and TRß) from three frog species (Xenopus laevis, Silurana tropicalis and Rana rugosa), a fish (Oryzias latipes), an alligator (Alligator mississippiensis) and a human (Homo sapiens). In all species examined, similar transcriptional activities were found for triiodothyronine (T3 : 10(-11) M in TRα and 10(-10) M in TRß) and thyroxine (T4 : 10(-9) M in TRα and 10(-8) M in TRß). Analogs of thyroid hormone (3,5,3',-triiodothyroacetic acid and 3,3',5,5'-tetraiodothyroacetic acid) exhibited weaker activity, requiring 10-fold higher concentrations for induction of activity when compared with T3 and T4 . These results provide support for the usefulness of in vitro screening assay systems as part of an approach to test chemicals for potential thyroid hormone receptor activity. In addition, we observed that T3 -stimulated transcriptional activity of the O. latipes TRα was inhibited by 10(-5) M tetrabromobisphenol A (TBBPA). In contrast, TR antagonist activities on TRα were not encountered in other species, even with TBBPA concentrations at 10(-5) M. In vitro transactivation assay systems using TRs from various species can be used for the screening of chemicals with thyroid-receptor agonist and antagonist activities. They also can be used for studies that examine evolutionary differences among species in the potency of TR activation.

Developmental disorders and altered gene expression in the tropical clawed frog (Silurana tropicalis) exposed to 17α-ethinylestradiol.

Several endocrine-disrupting chemicals with estrogenic activity can affect sexual development and reproduction in aquatic wildlife. The occurrence of oocytes in the testis (testis-ova) is one reproductive disorder and can be used as a valid endpoint when studying disruptive effects of estrogenic chemicals. To elucidate the molecular basis of testis-ova induction, we conducted gene expression analysis in the gonads of Silurana tropicalis exposed to 0, 3, 10 and 30 ng l(-1) 17α-ethinylestradiol (EE2) from 2 days after fertilization to the juvenile stage (14 weeks after fertilization). The frequencies of testis-ova induction or male to female sex-reversal of the gonads increased in an EE2 dose-dependent manner. Microarray analysis showed that expressions of a large number of genes were significantly changed by EE2 exposure. Genes including egg envelope composition (zp4, zpax, zpc, zp3.2 and egg cortical granule lectin), 42S particle genes (42Sp50, 42Sp43 and 42Sp48) and regulation of female germ cells (figla) are associated with the testis-ova and sex-reversal situation in the gonads. Of those, expression of zpc and 42Sp50 genes is associated with testis-ova. Thus, we propose that these genes are useful biomarkers for toxicological research in amphibians developmentally exposed to estrogenic chemicals.

Development of enzyme immunoassay for detection of DDT.

Dichlorodiphenyltrichloroethane (DDT) is one of the persistent organic pollutants (POPs) widely found in the environment and in the general population. In this study, a direct competitive enzyme immunoassay (EIA) has been developed for the quantitative analysis of DDT. To generate a specific polyclonal antibody for EIA, p, p'-DDT was conjugated to porcine thyroglobulin for rabbit immunization. At optimized EIA conditions, the standard curves ranged from 0.137 to 100 ng/mL with the quantification limit of 0.41 ng/mL. The coefficients of variation (CV%) were 5.42-10.53% for intra-assay and 6.04-7.26% for inter-assay. Cross-reactivities with DDT metabolites (DDTs, including o, p'-DDT, p, p'-DDD, o, p'-DDD, p, p'-DDE, o, p'-DDE, p, p'-dichlorobenzophenone (DCBP), o, p'-DCBP) were investigated. The polyclonal antibody showed relatively low and/or no cross-reactivity with these compounds, and the assay was seen to be highly selective for p, p'-DDT. Moreover, the DDTs could be ranked by their reactivity: DDT > DDD > DDE > DCBP. In addition, the characterization of the polyclonal antibody indicated that the antiserum possesses a high specificity for p, p'-isomers. The results indicated that the developed EIA using this antibody could be a convenient and supplemental analytical tool for monitoring DDT.