Mode of sexual differentiation and its influence on the relative sensitivity of the fathead minnow and zebrafish in the fish sexual development test (original) (raw)
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A Model Fish System to Test Chemical Effects on Sexual Differentiation and Development
A model system was characterized which may be used as an in vivo screen for effects of chemicals or environmental mixtures on sexual differentiation and development of reproductive organs and gametes. We evaluated the effects of a model environmental estrogen, ethinyl estradiol (EE2), on the d-rR strain of medaka, Oryzias latipes, using a nano-injection exposure. Gonad histopathology indicated that a single injection of 0.5-2.5 ng EE2/egg can cause phenotypic sex-reversal of genetic males to females. Sex-reversals could be detected as early as seven days post-hatch. Sex-reversed males had female-typical duct development and the secondary sex characteristics we measured were generally consistent with phenotype, with the exception of a few EE2-exposed XX and XY females which possessed ambiguous anal fins. Using discriminant analysis, we determined that the presence or absence of the secondary sex characteristic, a dorsal fin notch, was a very reliable indicator of gonadal sex. No inst...
Aquatic Toxicology, 2000
A model system was characterized which may be used as an in 6i6o screen for effects of chemicals or environmental mixtures on sexual differentiation and development of reproductive organs and gametes. We evaluated the effects of a model environmental estrogen, ethinyl estradiol (EE 2 ), on the d-rR strain of medaka, Oryzias latipes, using a nano-injection exposure. Gonad histopathology indicated that a single injection of 0.5 -2.5 ng EE 2 /egg can cause phenotypic sex-reversal of genetic males to females. Sex-reversals could be detected as early as 7 days post-hatch. Sex-reversed males had female-typical duct development and the secondary sex characteristics we measured were generally consistent with phenotype, with the exception of a few EE 2 -exposed XX and XY females which possessed ambiguous anal fins. Using discriminant analysis, we determined that the presence or absence of the secondary sex characteristic, a dorsal fin notch, was a very reliable indicator of gonadal sex. No instances of gonadal intersexes were observed. Ethinyl estradiol also appeared to reduce growth but not condition (weight-at-length) and exposed XX females appeared to have a higher incidence of atretic follicles relative to controls. Our results suggest that estrogenic chemicals may influence sexual differentiation and development and that the medaka model is well suited to assessing these effects.
Aquatic Toxicology, 2006
The Endocrine Modulators Study Group (EMSG) of the European Chemical Industry has proposed an extended fish early-life stage (ELS) test based on OECD test guideline 210 in combination with a fish pair-breeding reproduction study as a possible alternative for fish full life cycle testing. In this paper the androgen methyldihydrotestosterone (MDHT) was tested in an extended ELS test with fathead minnow supplementary to such a test with the weak estrogen 4-tert-pentylphenol (4TPP). Main endpoints were secondary sexual characteristics (SSC), plasma vitellogenin (VTG) induction and gonadal development. Early blastula embryos were exposed to 0, 0.10, 0.32 and 1.0 g MDHT l −1 for up to 114 days post-hatch (dph). A batch of fish exposed to 1.0 g l −1 was transferred to clean water after 30 or 63 dph for the remainder of the study. Ethinylestradiol (EE2) was included as estrogenic reference substance at 0.01 g l −1 . Exposure to MDHT had no significant effect on hatching success or survival, but significantly increased the condition factor of fish exposed for 63 and 114 dph (up to 150% of the control). At 63 dph MDHT exposure induced appearance of tubercles on the snout (a male SSC) of more than 80% of fish. Compared to the controls, plasma VTG was not detectable or significantly lower in fish exposed to MDHT at 0.10 g/l, but not significantly affected at higher MDHT concentrations. Both lower levels of MDHT significantly inhibited the development of female gonads as of 30 dph. Fish exposed to MDHT at 0.32 and 1.0 g l −1 showed higher incidences of mixed sex gonads (10-25%) and smaller testes or dysplasia of gonadal tissue. Dysplasia was present in 80% of the fish continuously exposed to 1.0 g l −1 up to 114 dph, but reversible when fish were transferred to dilution water. Results indicate that suppression of ovarian development was the most sensitive endpoint for MDHT exposure after 30 dph. Other endpoints (e.g., growth and SSC) required exposure during at least up to 63 dph to yield a significant effect. Androgenic effects on VTG production required even longer exposure, i.e., until sufficient number of females had matured.
Environmental Health Perspectives, 2000
Fish sexl development is sensitive to exogenous hormone manipulation, and salmonids have been used ensivy as environmental sentinels and models for biomedical research. We simulated maternal transfer of contaminants by microinjecting rainbow troiut (Oncorhynchw mykiss) and chinook salmon (Oncorbyneuhs tsbau"ynba) embryos. Fish were reared for 6 months and sexed, and gonads were removed for histology and measurement of in vitro steroid production. Analysis of fat samples showed that dichlorodiphenylethylene (DDE) levels, o,p'-DDE and pp'-DDE isomers, were elevated 6 months after treatment. A preliminary study showed an increased ratio of males to females after treatment with 80 mg/Lkg and 160 mgLkg of the xenoestrogen o,-DDE One fish treated with 160 mg/kg o,p'-DDE had gonads with cells typical of both males and females. A follow-up study, using more fish and excluding the highly toxic 160 mg/kg o,p'-DDE dose, showed no effect on sex ratio or gonadal histology. Embryonic exposure of monosex male trout, monosex female trout, and mixed se salmon to oap-DDE, pp'-DDE, mix of DDE isomers, and ocylphenol failed to alter sexual development. We observed no treatment-dependent changes in in vitro gonadal steroid production in any experiments. Trout exposed in ovo and reared to maturity spawned successfuilly. These results suggest that mortality attributable to the xenoestrogens op'-DDE, chlordecone, and octyphenol, and the antiandrogen pp'-DDE, is likely to occur before the appearance of sulbde changes in xal development. Because trout appeared to be sensitive to endocrine disruption, we cannot dismiss the threat ofheaviy contaminated sites or complex mixtues to normal sexual development of salmonids or other aquatic organisms.
2011
Teleost fish are unique among vertebrates in that phenotypic sex or onset of sex inversion can be easily manipulated by hormonal treatments. In recent years, researchers have begun reporting concentrations of synthetic and natural hormones in the environment. Although concentrations are very low (in the parts per trillion to low parts per billion), they are still of concern because of the high potency of synthetic hormones and the enhanced susceptibility of teleost fishes, especially early life stages, to hormonal exposures. In this review, we will focus on sex differentiation in teleost fishes and how these processes in fish early life stages may be impacted by environmental hormones which are known to contaminate aquatic environments. We will start by reviewing information on sources and concentrations of hormones in the environment and continue by summarizing the state of knowledge of sex differentiation in teleost gonochoristic fishes, including information on genes involved (e.g. cyp19, dmrt1, sox9 and foxl2). We will end our review with a summary of studies that have examined the effects of androgens and estrogens on fish sex differentiation after exposure of fish embryos and larvae and with ideas for future research.
The aim of the present study was to investigate if the effects of the androgen, dihydrotestosterone (DHT) on the sexual development in juvenile Murray rainbowfish (Melanotaenia fluviatilis) are canceled out by the anti-androgen, flutamide. Fish (60 days post hatch) were exposed to 250 ng/L of DHT, 25 g/L of flutamide (Flu-low), 250 g/L of flutamide (Flu-high), DHT + Flu low and DHT + Flu high. After 35 days of exposure, lengths and weights of the fish were measured and the condition factor (CF) calculated; vitellogenin (VTG) concentrations were measured in tail tissue; sex steroid hormones (17-estradiol [E2] and 11-keto testosterone [11-KT]) were measured in the head tissue and abdominal regions were used in histological investigation of the gonads. Treatment with DHT reduced the body-length of both male and female fish, an effect which was canceled out by low and high concentrations of flutamide. However, flutamide (low or high) could not nullify the DHT-induced reduction in the CF in either sex. The E2 levels were reduced only in female fish after exposure to DHT but returned to normal after treatment with Flu-high. DHT increased the levels of 11-KT and decreased the E2/11-KT ratio in both sexes. Flu-high, but not Flu-low, could nullify these effects. Both DHT and flutamide (low or high) induced VTG production and this effect persisted when both chemicals were co-administered. Treatment with DHT did not affect gonadal cell development in the testes. However, the female fish treated with DHT contained ovaries in early-vitellogenic stage in comparison to the pre-vitellogenic ovaries in control fish. Co-treatment with flutamide (low or high) resulted in oocyte atresia. The results from the present study suggest that treatment with Flu-high could cancel out DHT-induced effects only on the hormonal profile and body-length in both male and female fish. Juvenile fish co-treated with DHT and flutamide (low or high) had high VTG levels and low CF. In addition, the ovaries in female fish were atretic. These data represent potential adverse effects on the ability of the fish to reproduce successfully.
Aquatic Toxicology, 2006
An extended early-life stage test (based on OECD test guideline 210) was developed to allow the evaluation of a weak environmental oestrogen, 4-tert-pentyphenol (4TPP), on sexual differentiation and gonadal development. Fathead minnow (Pimephales promelas) embryos were exposed to three concentrations of 4TPP (56, 180 and 560 g l −1 ) in a flow-through system, at 25 ± 1 • C, for <107 days post-hatch (dph). In addition, some embryos were exposed to 180 g 4TPP l −1 until 30 or 60 dph, after which they were exposed to dilution water only until 107 dph. At 30, 60 and 107 dph fish were evaluated for growth and gonadal development (via histology), and at 107 dph fish were also evaluated for secondary sexual characteristics (SSC), gonadosomatic index (GSI) and plasma vitellogenin (VTG). There were no effects of 4TPP on hatching success or survival, however, there was a delay in the time taken for embryos to hatch (560 g 4TPP l −1 ). No treatment-related effects were observed on fish growth, with the exception of at 107 dph when the condition factor in female fish was reduced in all 4TPP continuous exposure treatments. Plasma VTG was only elevated in female fish exposed to 180 g 4TPP l −1 and inhibition of gonadal growth (GSI) occurred only in females exposed to 560 g 4TPP l −1 . Histological examination of the gonads revealed delays and disruption in male sexual differentiation and development (180 g 4TPP l −1 ) and no testicular tissue was observed in any fish exposed to 560 g 4TPP l −1 . Mixed gonads (predominately testes with a scattering of primary oocytes) were present in fish exposed to all doses of 180 g 4TPP l −1 at 107 dph. Feminisation of the reproductive ducts (formation of an ovarian like cavity) occurred in the testis of all males exposed to 180 g l −1 , regardless of length of 4TPP exposure. Results indicate that the period of 30-60 dph appears to be the sensitive window for disruption of formation of the reproductive duct and this effect is not reversible when the fish are transferred to dilution water. The data also show that this integrative test is suitable for the detection of a weak environmental oestrogen and comparisons of these results with that of a fish full life-cycle, in medaka, indicate that this test could be a suitable surrogate for a fish full life-cycle. (G.H. Panter). through a variety of mechanisms of action and as a result this has given impetus for the development of screening assays and tests for the detection of EACs, notably in aquatic species. In Europe, the EMSG developed an aquatic research programme to assess the effects of EACs on fish. Within this programme robust and sensitive fish screening assays have already been developed for the evaluation of a range of EACs, using the fathead minnow (Pimephales promelas) . However, although such screening assays will identify the potential endocrine activity of chemicals, further chronic testing will be required to provide more detailed information 0166-445X/$ -see front matter
Aquatic toxicology (Amsterdam, Netherlands), 2015
Sexual disruption in wild fish has been linked to the contamination of river systems with steroid oestrogens, including the pharmaceutical 17␣-ethinylestradiol, originating from domestic wastewaters. As analytical chemistry has advanced, more compounds derived from the human use of pharmaceuticals have been identified in the environment and questions have arisen as to whether these additional pharmaceuticals may also impact sexual disruption in fish. Indeed, pharmaceutical anti-androgens have been shown to induce such effects under laboratory conditions. These are of particular interest since antiandrogenic biological activity has been identified in the aquatic environment and is potentially implicated in sexual disruption alone and in combination with steroid oestrogens. Consequently, predictive modelling was employed to determine the concentrations of two anti-androgenic human pharmaceuticals, bicalutamide and cyproterone acetate, in UK sewage effluents and river catchments and their combined impacts on sexual disruption were then assessed in two fish models. Crucially, fish were also exposed to the anti-androgens in combination with steroid oestrogens to determine whether they had any additional impact on oestrogen induced feminisation. Modelling predicted that the anti-androgenic pharmaceuticals were likely to be widespread in UK river catchments. However, their concentrations were not sufficient to induce significant responses in plasma vitellogenin concentrations, secondary sexual characteristics or gross indices in male fathead minnow or intersex in Japanese medaka alone or in combination with steroid oestrogens. However, environmentally relevant mixtures of oestrone, 17-oestradiol and 17␣-ethinylestradiol did induce vitellogenin and intersex, supporting their role in sexual disruption in wild fish populations. Unexpectedly, a male dominated sex ratio (100% in controls) was induced in medaka and the potential cause and implications are briefly discussed, highlighting the potential of non-chemical modes of action on this endpoint.
Critical Reviews in Toxicology, 2012
Endocrine disrupting chemicals (EDCs) enter aquatic ecosystems through discharged effluents, mainly from wastewater treatment works and diffuse run off from land, and affect a wide range of aquatic biota, including fish. Evidence for altered physiology in fish as a consequence of endocrine disruption is global, with some of the most widely reported effects on sexual development and function. In recent years research has shown that fish behaviors can also be affected by EDCs which potentially has wide implications for individual fitness and population level outcomes. This review presents a critical assessment on reported effects of EDCs on behavior in fish, focusing on behaviors associated with reproduction. We investigate commonalities and differences in sexual behaviors between fish species most commonly applied in ecotoxicology, drawing out common principles for impacts of EDCs and then reviewing the evidence for, and implications of, disruptions of these behaviors after exposures to EDCs. In an analysis of the reported effects of exposure to the estrogen, ethinylestradiol, we show that life-stage at exposure is a key factor determining behavioral responses of affected populations. EDC-induced changes in behavior occur at similar concentrations as for established biomarker responses (e.g. vitellogenin induction for estrogens), indicating behavior is equally sensitive (and non-invasive) as an indicator of EDC exposure. Adopting behaviors in fish as indicators of chemical exposure and effects, however, still has many technical and interpretation challenges and there is very little information available on how behaviors under laboratory conditions equate with those occurring in wild populations.
Journal of Applied Toxicology, 2021
Under the Organisation for Economic Cooperation 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 ARβ 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.